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Molting in a parasitic nematode, Phocanema decipiens—VI The mode of action of insect juvenile hormone and farnesyl methyl ether
International Journal for Parasitology, 1971, VoL 1, pp. 61-66. Pergamon Press. Printed in Great Britain
MOLTING IN A PARASITIC NEMATODE, PHOCANEMA DECIPIENSmVI THE MODE OF ACTION OF INSECT JUVENILE HORMONE AND FARNESYL METHYL ETHER K. G. D A V E Y Institute of Parasitology, McGill University, Macdonald College, P.Q. Canada (Received 25 August 1970) Abslract
DAVEYK. G., 1971. Molting in a parasitic nematode, Phocanema decipiens--VI. The mode of action of insect juvenile hormone and farnesyl methyl ether. International Journal for Parasitology, 1: 61-66. Both a synthetic preparation of insect juvenile hormone and farnesyl methyl ether inhibited ecdysis but left cuticle formation almost unaffected when present in the complete in vitro medium used to culture Phocanema through its final molt. When the worms were allowed to grow for 2½ days in 0.9 % NaCI, where ecdysis does not normally occur, and then exposed to the compound, normal ecdysis occurred in many of the worms. In a similar experiment, the worms were examined for neurosecretory activity, which could be detected as little as 12 h after the addition of the compounds to the saline cultures. It is concluded that the compounds exert their effect by stimulating the neuroendocrine system of the nematode, which in turn brings about the production and release of the molting fluid. INDEX KEY WORDS: Phocanema decipiens; nematode; insect; hormone; juvenile hormone; molting; neurosecretion. INTRODUCTION EARLIER work from this laboratory has demonstrated that the final molt in vitro of the cod-worm, Phocanema ( = Terranova = Porrocaecum) decipiens is accompanied by a cycle o f neurosecretion in some of the fells of the ventral ganglion (Davey, 1966). In the absence o f these cells the nematodes produce a normal adult cuticle (Davey, 1966). On the other hand, if the worms are reared in 0.9 ~o NaCI instead of in the complex medium, they form a normal new cuticle, but fail to ecdyse. In this case the neurosecretory cells are also inactive (Davey & Kan, 1967, 1968). Coincident with ecdysis, the excretory cell goes through a cycle of production and release of leucine aminopeptidase (Davey & Kan, 1967, 1968), which Rogers (1965) has shown to be an important constituent of the molting fluid in trichonstrongyles. This cycle of leucine aminopepfidase production is abolished when the worms are cultured in 0.9 % NaC1. Finally, excretory cells isolated in vitro will show an increased content o f leucine aminopeptidase when exposed to extracts o f heads containing active neurosecretory cells (Davey & Kan, 1968). These results have led us to conclude that the complete medium contains elements which stimulate the neuroseeretory cells to release a hormone which in turn brings about the production by and release from the excretory cell o f the molting fluid. Molting fluid in the space between the old and new cuticles digests the old cuticle in a characteristic way, leading to ecdysis. Meerovitch was the first to demonstrate that substances with juvenile hormone activity interfered with development in nematodes. His earliest work (1965) dealt with the effect o f the sesquiterpene farnesol on the development of Trichinella spiralis in vitro and showed that molting was delayed. More recently (Shanta & Meerovitch, 1970), these results have been extended to include the more potent farnesyl methyl ether (FME). Both farnesol and F M E 61
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inhibit molting at a concentration of 10-%1. Inhibition of the development of the male copulatory appendages could be detected at concentrations of F M E as low as 10-TM of F M E and 10-4M of farnesol. These effects are not restricted to animal parasites. Johnson & Viglierchio (1970) have reported bizarre abnormalities in the development of Heterodera schactii, the sugar beet nematode, after exposure to high concentrations of F M E and farnesyl diethylamine. These abnormalities include supernumerary molts, failure to ecdyse, neoplastic growth of the testes, and the development of a recognizable worm within the body of a third stage larva. These studies have left unanswered the possible mode of action of these compounds. The present paper reports the results of experiments which throw some light on this problem. MATERIALS AND METHODS The worms were collected as last stage larvae, stored and cultured in the modified medium of Townsley et al. 0963) as discussed in earlier publications (Davey, 1965). The only additional modification was the use of disposable plastic culture flasks in place of petri dishes. The FME, a gift of Roche Pharmaceuticals, Montreal, Canada, was all trans farnesyl methyl ether. The synthetic juvenile hormone (JH), a gift of Ayerst Laboratories, Montreal, Canada, consisted of a mixture of 8 geometrical isomers of methyl 10, 11-epoxy-7-ethyl 3, 11, dimethyl-2,6-tridecadienoate. All of these isomers possess some juvenile hormone activity (R/Sller & Dahm, 1968 ; Wigglesworth, 1969), and the four most active account for 60 per cent of the mixture (information supplied by Dr. A. J. Manson, Ayerst Laboratories). Because the stability of these compounds was uncertain, their biological activity on insects was determined using the Rhodnius bioassay of Wigglesworth 0969). The sample of F M E gave a score of l0 on the Wigglesworth scale at 5-8 nl. per insect and JH at 4.8 nl. per insect. These tests were performed immediately after the experiments described in this paper. The compounds were added after the medium had been sterilised. These materials are only very slightly soluble in aqueous media. At the concentrations employed here, they readily formed suspensions even in 0.9 ~ NaC1. For convenience the concentrations are quoted in molarities. These must be regarded as approximate figures arrived at by assuming a specific gravity of 0.9 (Wigglesworth, 1969). Cuticular structure was investigated using the osmium-ethyl gallate technique of Wigglesworth 0957) as described earlier (Davey, 1965). The neurosecretory cells were stained by the method of Cameron & Steele (1959). EXPERIMENTS AND RESULTS The effects of F M E and J H on the molting cycle In these experiments the worms were held overnight at 5°C in 0.9 ~ saline containing the mixture of antibiotics as well as the appropriate concentration of either FME or JH. The controls were exposed to the same conditions, but without added JH or FME. They were transferred, six to a flask, to the complete medium which also contained FME or JH at 37°C. The medium was changed daily and the worms fixed on the sixth or seventh day for sectioning. F M E was used at 10-aM and 10-6M. JH was used at 10-aM, 10-4M, 10-6M and 10-TM. Of the total of 18 control worms, 16 survived to the end of the experiment. All of these produced normal adult cuticle, and all ecdysed.
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MOLTINGINPhocanema decipiens
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Six worms were exposed to 10-sM JH. All died by the end of the first day. Six worms exposed to 10-7~ JH all survived, produced normal adult cuticle and eedysed in the normal way. The picture presented by worms exposed to 10-3M FME (12 worms, 11 survivors), 10-6M FME (12 worms, 10 survivors), 10-4M JH (6 worms, 6 survivors) or 10-6M JI-I (12 worms, 12 survivors)was essentially similar. Certain developmental anomalies were clear. Firstly, none of the survivors had ecdysed by the end of the experiment, in spite of the fact that all of the controls had done so. Secondly, although all of the treated worms which survived produced an adult cuticle which was complete with respect to the number and arrangement of the layers, two sorts of disruption could be discerned. The outermost layer of adult cuticle, the cortex, had a feathery appearance along its periphery (Fig. 1). There were also anomalies associated with the junction between the hypodermis and cuticle. Frequently, there were gaps (Figs. 1, 2) indicating that the cuticle was not so firmly attached as in the controls (Fig. 3). In other sections (Fig. 4), the hypodermis appeared to have invaded the cuticle. In the controls, the border between hypodermis and cuticle is always well defined (Figs. 3 and 5). A third result of applying FME or JH was the appearance of abundant osmiophilic droplets in the cytoplasm of the muscle cells (Fig. 4). These are much less apparent in sections of control worms (Fig. 5). Some observations of particular interest were made on the living cultures. During the first twelve hours in culture, many of the treated worms could be observed to expel an opalescent, milky material from what was judged to be the excretory pore. This region was also sometimes swollen, and the excretory cell was particularly prominent in some of the treated worms. The effect of F M E and J H on ecdysis An explanation of the failure to ecdyse and the premature activity of the excretory gland involves the stimulation of the process of ecdysis by the applied compounds. In order to examine this possibility, worms were cultured in 0.9 ~ NaCI for 2½ days before adding the compounds. By 2½ days most of the worms will have completed two of the three primary layers which make up the adult cuticle. It is at this stage that the old cuticle separates from the developing adult cuticle (Davey, 1965). Normally, of course, worms reared in 0.9 NaCI will not ecdyse (Davey & Kan, 1968). At 2½ days, the saline was replaced with saline containing JH or FME at 10-6rn. The fluid in the flasks was changed daily until the end of the seventh day when the worms were examined under a dissecting microscope for the presence or absence of a sheath. By dissecting off the innermost cuticle and examining it as a whole mount under the phase contrast microscope, it was possible to determine whether the cuticle was adult or larval by the presence or absence of the fibre layer (Davey, 1965). Of 12 control worms 10 survived. All produced adult cuticles and all remained ensheathed within the larval cuticle. Of 13 animals treated with JH, 11 survived. All of these produced adult cuticle and 5 remained ensheathed while 6 had ecdysed. Of 12 treated with FME, 10 survived and produced adult cuticle. Eight of these had ecdysed. Apparently, then, FME and JH are able to bring about ecdysis in some worms from which the environmental stimulus, normally present in the complete medium, has been withheld. The effect of F M E and J H on the neurosecretory system In order to determine whether the compounds exerted their effect through the neuroendocrine system, the last experiment was~repeated, using FME or JH at 10 -6 or 10-SM.
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The head ends of worms were fixed 16 h or 48 h after the addition of the compounds and examined for neurosecretory cells. Abundant evidence of neurosecretion was found in all treated worms. The cells in the ventral ganglion which are thought to be involved in ecdysis are clearly stained (Fig. 6). While it is difficult to make quantitative comparisons, the impression was gained that there was more stainable material in the cell bodies and axones than had previously been recognized in normal worms. For example the presence of fuchsinophilic material in axones of the ventral nerve cord was particularly obvious (Fig. 7). Control worms, which had been cultured in 0.9 ~o NaC1 alone, contained no neurosecretory material. In order to obtain adequate fixation, it is necessary to remove the old cuticle from the head of the worm. An opaque material was frequently observed between the two cuticles o f treated worms just behind the lips. Since this rather viscous material usually adhered to the larval cuticle it was convenient to process it for the histochemical test for leucine aminopeptidase employed in earlier studies (Davey & Kan, 1967, 1968). The material proved to be positive for the enzyme. DISCUSSION It is the principal conclusion of this study that the most obvious effects of JH and F M E - inhibition of ecdysis--stem from the ability of these compounds to stimulate the neurosecretory system of the worms. When the compounds are present from the beginning of ecdysis, they stimulate the neurosecretory cells to secrete, leading to a premature activation of the excretory cell. The molting fluid is, therefore, passed out of the excretory pore before the new cuticle is sufficiently formed to prevent its escape. On the other hand, the application of the compounds to worms which have deposited new cuticle, but which are destined not to ecdyse by virtue of being reared in 0.9 ~ NaC1, leads to their ecdysis by stimulation of the neurosecretory cells and the consequent release of the molting fluid into the space between the cuticles. These effects are not held to indicate a possible physiological effect for the compounds. The concentrations required--10-6M or better--are certainly above the physiological range of concentrations normally associated with endocrine activity. Furthermore, the effect of juvenile hormone and its mimics on neurosecretory cells may be non-specific. Thus, other work in this laboratory suggests that large doses of topically applied F M E may be able to stimulate neurosecretory cells in the insect Rhodnius prolixus (Pratt & Davey, 1969). There is also the suggestion in the present work that the degree of neurosecretory activity was greater than in normal worms. What the mechanism of this stimulation might be is not clear. The materials may be acting directly on the neurosecretory system or at the level of the sense organs. The fact that Baumann (1968) has demonstrated that high concentrations of mimics of juvenile hormone bring about depolarization of insect salivary gland membrane potentials is of some interest in this respect. Shanta & Meerovitch (1970) have suggested that the possibility of these compounds being produced by nematodes should be investigated. In view of the wide distribution among living organisms of substances with juvenile hormone activity (Williams, Moorhead & Pulis, 1959; Schneiderman & Gilbert, 1958; Highman & Hill, 1969), it would scarcely be surprising if nematodes were found to contain similar materials. Until the endocrine control of development in nematodes has a more extensive experimental basis, it is probably best to regard the introduction of these materials as simply one more stressful factor in the environment, which, as I have suggested elsewhere (Davey, 1963, 1964), can lead to a release of endocrines.
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MOLTINGIN Phocanema decipiens
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Whether the effects on the reproductive system noted by other workers (Johnson & Viglierchio, 1970; Shanta & Meerovitch, 1970) are also mediated by the neurosecretory system is difficult to say. Preliminary studies in this laboratory have shown that the neurosecretory cells of Phocanema became active again about two weeks after ecdysis, when development of the reproductive structures is proceeding apace. The other effects of adding JH or F M E to the medium are less dear. Thus, the feathery appearance of the outermost layer of the new cuticle may be a result of the partial inhibition of its formation by the materials. Since the nematodes are only feeding during the period from 12 to 30 h after being put in culture (Davey, 1969), it is possible that the compounds are only present internally during the early stages of cuticle formation. The apparent failure of the new cuticle to remain attached to the hypodermis may be an artifact of fixation, but it is difficult to see why such an artifact should not be equally obvious in untreated controls. It may result from the continued growth of the new cuticle while the total growth of the nematode is restricted by the presence of the old cuticle. On the other hand, these effects have never been noticed in sections of worms placed in 0.9 ~o NaC1 without added F M E or JH. In these worms, of course, ecdysis does not take place. The failure of attachment may result from an interference, direct or indirect, with the as yet undescribed morphogenetic systems involved in the attachment of the cuticle to the hypodermis. The apparent invasion of the cuticle by the hypodermis may be another aspect of this failure. The appearance o f the osmiophilic droplets may simply be an indication that the diet in the treated animals is richer in lipid by virtue of the addition of F M E or JH. On the other hand, it may signal an increased mobility of lipid, which is known to vary during the molting cycle in any case (Kan & Davey, 1968). Acknowledgements--I am indebted to Mr. G. F. Webster for his skilled and careful assistance, and to Mr. A. F. Mclnnes, Department of Fisheries, Sydney, Nova Scotia, who supplies the infected cod fillets. This research is supported by the National Research Council of Canada.
REFERENCES BAtlMANN G. 1968. Zur Wirkung des Juvenilhormons; Elektrophysiologische Messangen an der Zellmembran der Speicheldruse yon Galleria mellonella. Journal of lnsect Physiology 14: 1459-1476. CAMERONM. L. & STEELE J'. E. 1959. Simplified aldehyde-fuchsin staining of the neurosecretory cells. Stain Technology 34: 265-266. DAVEYK. G. 1963. The release by enforced activity of the cardiac accelerator from the corpus cardiacum of Periplaneta americana. Journal of Insect Physiology 9: 375-381. DAVEYK. G. 1964. Neurosecretory cells in a nematode, Ascaris lumbricoides. Canadian Journal of Zoology 42: 731-734. DAVEYK. G. 1965. Molting in a parasitic nematode, Phocanema decipiens--I. Cytologicalevents. Canadian Journal of Zoology 43: 997-1003. DAVEYK. G. 1966. Neurosecretionand molting in some parasitic nematodes. American Zoolog&t 6: 243-249. DAVEYK. G. 1969. Molting in a parasitic nematode, Phocanema decipiens--V. Timing of feeding during the molting cycle.Journal of the Fisheries Research Board of Canada 26: 935-939. DAVEYK. G. & KAN S. P. 1967.An endocrine basis for ecdysis in a parasitic nematode. Nature, London 214: 737-738. DAVEVK. G. & KAN S. P. 1968. Molting in a parasitic nematode. Phocanema decipiens--IV. Ecdysis and its control. Canadian Journal of Zoology 46: 893-898. HIOHNAMK. C. & HILLL. C. 1969. The comparative endocrinology of the invertebrates. 270 pp. Edward Arnold, London. JOHNSON R. N. and VIOLIERCHIOD. R. 1970. Heterodera schactii responses to exogenous hormones. Experimental Parasitology 27, 301-309. KAN S. P. & DAVEYK. G. 1968. Molting in a parasitic nematode. Phocanema decipiens--III. The histochemistry of cuticle deposition and protein synthesis. Canadian Journal of Zoology 46: 723-727. M~EROVlTCHE. 1965. Studies on the in vitro axenic development of Trichinella spiralis---II. Preliminary experiments on the effect of farnesol, cholesterol, and an insect extract. Canadian Journal o f Zoology 43: 81-85. I.P. I/I--R
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PRATT G. E. & DAVEY K. G. 1969. The corpus allatum in mated and virgin Rhodnius. Proceedings of the Annual Meeting of the Entomological Society of Ontario 1969. In press. ROGERS W. P. 1965. The role of leucine aminopeptidase in moulting of nematode parasites. Comparatiae Biochemistry and Physiology 14: 311-321. ROLLER H. & DAHM K. H. 1968. T h e chemistry and biology of juvenile hormone. Recent Progress In Hormone Research 24: 651-680. SCHNEIDERMAN H. A. t~; GILBERT L. I. 1958. Substances with juvenile hormone activity in Crustacea. Biological Bulletin 11S: 530-535. SHANTA C. S. • MEEROVrrCH E. 1970. Specific inhibition of morphogenesis in Trichinella spiralis by insect juvenile hormone mimics. Canadian Journal of Zoology 48: 617-620. TOWNSLEY P. M., WIGHT H. G., SCOTT M. A. & HUGHES M. L. 1963. The in vitro maturation of the parasitic nematode Terranova decipiens from cod muscle. Journal of the Fisheries Research Board of Canada 20: 743-747. WILLIAMS C. M., MOORt-mAD L. V. & PULIS J. V. 1959 Juvenile hormone in thymus, human placenta and other mammalian organs. Nature, London 183: 405. WIGGLESWORTH V. ~B. 1957. The use of osmium in the fixation and staining of tissues. Proceedings of the Royal Society of London, Series B. 147: 185-199. WIGGLESWORTH V. B. 1969. Chemical structure and juvenile hormone activity. Comparative tests on Rhodnius prolixus. Journal of Insect Physiology 15: 73-94.
MOLTING IN A PARASITIC NEMATODE, PHOCANEMA DECIPIENSmVI THE MODE OF ACTION OF INSECT JUVENILE HORMONE AND FARNESYL METHYL ETHER K. G. D A V E Y Institute of Parasitology, McGill University, Macdonald College, P.Q. Canada (Received 25 August 1970) Abslract
DAVEYK. G., 1971. Molting in a parasitic nematode, Phocanema decipiens--VI. The mode of action of insect juvenile hormone and farnesyl methyl ether. International Journal for Parasitology, 1: 61-66. Both a synthetic preparation of insect juvenile hormone and farnesyl methyl ether inhibited ecdysis but left cuticle formation almost unaffected when present in the complete in vitro medium used to culture Phocanema through its final molt. When the worms were allowed to grow for 2½ days in 0.9 % NaCI, where ecdysis does not normally occur, and then exposed to the compound, normal ecdysis occurred in many of the worms. In a similar experiment, the worms were examined for neurosecretory activity, which could be detected as little as 12 h after the addition of the compounds to the saline cultures. It is concluded that the compounds exert their effect by stimulating the neuroendocrine system of the nematode, which in turn brings about the production and release of the molting fluid. INDEX KEY WORDS: Phocanema decipiens; nematode; insect; hormone; juvenile hormone; molting; neurosecretion. INTRODUCTION EARLIER work from this laboratory has demonstrated that the final molt in vitro of the cod-worm, Phocanema ( = Terranova = Porrocaecum) decipiens is accompanied by a cycle o f neurosecretion in some of the fells of the ventral ganglion (Davey, 1966). In the absence o f these cells the nematodes produce a normal adult cuticle (Davey, 1966). On the other hand, if the worms are reared in 0.9 ~o NaCI instead of in the complex medium, they form a normal new cuticle, but fail to ecdyse. In this case the neurosecretory cells are also inactive (Davey & Kan, 1967, 1968). Coincident with ecdysis, the excretory cell goes through a cycle of production and release of leucine aminopeptidase (Davey & Kan, 1967, 1968), which Rogers (1965) has shown to be an important constituent of the molting fluid in trichonstrongyles. This cycle of leucine aminopepfidase production is abolished when the worms are cultured in 0.9 % NaC1. Finally, excretory cells isolated in vitro will show an increased content o f leucine aminopeptidase when exposed to extracts o f heads containing active neurosecretory cells (Davey & Kan, 1968). These results have led us to conclude that the complete medium contains elements which stimulate the neuroseeretory cells to release a hormone which in turn brings about the production by and release from the excretory cell o f the molting fluid. Molting fluid in the space between the old and new cuticles digests the old cuticle in a characteristic way, leading to ecdysis. Meerovitch was the first to demonstrate that substances with juvenile hormone activity interfered with development in nematodes. His earliest work (1965) dealt with the effect o f the sesquiterpene farnesol on the development of Trichinella spiralis in vitro and showed that molting was delayed. More recently (Shanta & Meerovitch, 1970), these results have been extended to include the more potent farnesyl methyl ether (FME). Both farnesol and F M E 61
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inhibit molting at a concentration of 10-%1. Inhibition of the development of the male copulatory appendages could be detected at concentrations of F M E as low as 10-TM of F M E and 10-4M of farnesol. These effects are not restricted to animal parasites. Johnson & Viglierchio (1970) have reported bizarre abnormalities in the development of Heterodera schactii, the sugar beet nematode, after exposure to high concentrations of F M E and farnesyl diethylamine. These abnormalities include supernumerary molts, failure to ecdyse, neoplastic growth of the testes, and the development of a recognizable worm within the body of a third stage larva. These studies have left unanswered the possible mode of action of these compounds. The present paper reports the results of experiments which throw some light on this problem. MATERIALS AND METHODS The worms were collected as last stage larvae, stored and cultured in the modified medium of Townsley et al. 0963) as discussed in earlier publications (Davey, 1965). The only additional modification was the use of disposable plastic culture flasks in place of petri dishes. The FME, a gift of Roche Pharmaceuticals, Montreal, Canada, was all trans farnesyl methyl ether. The synthetic juvenile hormone (JH), a gift of Ayerst Laboratories, Montreal, Canada, consisted of a mixture of 8 geometrical isomers of methyl 10, 11-epoxy-7-ethyl 3, 11, dimethyl-2,6-tridecadienoate. All of these isomers possess some juvenile hormone activity (R/Sller & Dahm, 1968 ; Wigglesworth, 1969), and the four most active account for 60 per cent of the mixture (information supplied by Dr. A. J. Manson, Ayerst Laboratories). Because the stability of these compounds was uncertain, their biological activity on insects was determined using the Rhodnius bioassay of Wigglesworth 0969). The sample of F M E gave a score of l0 on the Wigglesworth scale at 5-8 nl. per insect and JH at 4.8 nl. per insect. These tests were performed immediately after the experiments described in this paper. The compounds were added after the medium had been sterilised. These materials are only very slightly soluble in aqueous media. At the concentrations employed here, they readily formed suspensions even in 0.9 ~ NaC1. For convenience the concentrations are quoted in molarities. These must be regarded as approximate figures arrived at by assuming a specific gravity of 0.9 (Wigglesworth, 1969). Cuticular structure was investigated using the osmium-ethyl gallate technique of Wigglesworth 0957) as described earlier (Davey, 1965). The neurosecretory cells were stained by the method of Cameron & Steele (1959). EXPERIMENTS AND RESULTS The effects of F M E and J H on the molting cycle In these experiments the worms were held overnight at 5°C in 0.9 ~ saline containing the mixture of antibiotics as well as the appropriate concentration of either FME or JH. The controls were exposed to the same conditions, but without added JH or FME. They were transferred, six to a flask, to the complete medium which also contained FME or JH at 37°C. The medium was changed daily and the worms fixed on the sixth or seventh day for sectioning. F M E was used at 10-aM and 10-6M. JH was used at 10-aM, 10-4M, 10-6M and 10-TM. Of the total of 18 control worms, 16 survived to the end of the experiment. All of these produced normal adult cuticle, and all ecdysed.
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Six worms were exposed to 10-sM JH. All died by the end of the first day. Six worms exposed to 10-7~ JH all survived, produced normal adult cuticle and eedysed in the normal way. The picture presented by worms exposed to 10-3M FME (12 worms, 11 survivors), 10-6M FME (12 worms, 10 survivors), 10-4M JH (6 worms, 6 survivors) or 10-6M JI-I (12 worms, 12 survivors)was essentially similar. Certain developmental anomalies were clear. Firstly, none of the survivors had ecdysed by the end of the experiment, in spite of the fact that all of the controls had done so. Secondly, although all of the treated worms which survived produced an adult cuticle which was complete with respect to the number and arrangement of the layers, two sorts of disruption could be discerned. The outermost layer of adult cuticle, the cortex, had a feathery appearance along its periphery (Fig. 1). There were also anomalies associated with the junction between the hypodermis and cuticle. Frequently, there were gaps (Figs. 1, 2) indicating that the cuticle was not so firmly attached as in the controls (Fig. 3). In other sections (Fig. 4), the hypodermis appeared to have invaded the cuticle. In the controls, the border between hypodermis and cuticle is always well defined (Figs. 3 and 5). A third result of applying FME or JH was the appearance of abundant osmiophilic droplets in the cytoplasm of the muscle cells (Fig. 4). These are much less apparent in sections of control worms (Fig. 5). Some observations of particular interest were made on the living cultures. During the first twelve hours in culture, many of the treated worms could be observed to expel an opalescent, milky material from what was judged to be the excretory pore. This region was also sometimes swollen, and the excretory cell was particularly prominent in some of the treated worms. The effect of F M E and J H on ecdysis An explanation of the failure to ecdyse and the premature activity of the excretory gland involves the stimulation of the process of ecdysis by the applied compounds. In order to examine this possibility, worms were cultured in 0.9 ~ NaCI for 2½ days before adding the compounds. By 2½ days most of the worms will have completed two of the three primary layers which make up the adult cuticle. It is at this stage that the old cuticle separates from the developing adult cuticle (Davey, 1965). Normally, of course, worms reared in 0.9 NaCI will not ecdyse (Davey & Kan, 1968). At 2½ days, the saline was replaced with saline containing JH or FME at 10-6rn. The fluid in the flasks was changed daily until the end of the seventh day when the worms were examined under a dissecting microscope for the presence or absence of a sheath. By dissecting off the innermost cuticle and examining it as a whole mount under the phase contrast microscope, it was possible to determine whether the cuticle was adult or larval by the presence or absence of the fibre layer (Davey, 1965). Of 12 control worms 10 survived. All produced adult cuticles and all remained ensheathed within the larval cuticle. Of 13 animals treated with JH, 11 survived. All of these produced adult cuticle and 5 remained ensheathed while 6 had ecdysed. Of 12 treated with FME, 10 survived and produced adult cuticle. Eight of these had ecdysed. Apparently, then, FME and JH are able to bring about ecdysis in some worms from which the environmental stimulus, normally present in the complete medium, has been withheld. The effect of F M E and J H on the neurosecretory system In order to determine whether the compounds exerted their effect through the neuroendocrine system, the last experiment was~repeated, using FME or JH at 10 -6 or 10-SM.
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The head ends of worms were fixed 16 h or 48 h after the addition of the compounds and examined for neurosecretory cells. Abundant evidence of neurosecretion was found in all treated worms. The cells in the ventral ganglion which are thought to be involved in ecdysis are clearly stained (Fig. 6). While it is difficult to make quantitative comparisons, the impression was gained that there was more stainable material in the cell bodies and axones than had previously been recognized in normal worms. For example the presence of fuchsinophilic material in axones of the ventral nerve cord was particularly obvious (Fig. 7). Control worms, which had been cultured in 0.9 ~o NaC1 alone, contained no neurosecretory material. In order to obtain adequate fixation, it is necessary to remove the old cuticle from the head of the worm. An opaque material was frequently observed between the two cuticles o f treated worms just behind the lips. Since this rather viscous material usually adhered to the larval cuticle it was convenient to process it for the histochemical test for leucine aminopeptidase employed in earlier studies (Davey & Kan, 1967, 1968). The material proved to be positive for the enzyme. DISCUSSION It is the principal conclusion of this study that the most obvious effects of JH and F M E - inhibition of ecdysis--stem from the ability of these compounds to stimulate the neurosecretory system of the worms. When the compounds are present from the beginning of ecdysis, they stimulate the neurosecretory cells to secrete, leading to a premature activation of the excretory cell. The molting fluid is, therefore, passed out of the excretory pore before the new cuticle is sufficiently formed to prevent its escape. On the other hand, the application of the compounds to worms which have deposited new cuticle, but which are destined not to ecdyse by virtue of being reared in 0.9 ~ NaC1, leads to their ecdysis by stimulation of the neurosecretory cells and the consequent release of the molting fluid into the space between the cuticles. These effects are not held to indicate a possible physiological effect for the compounds. The concentrations required--10-6M or better--are certainly above the physiological range of concentrations normally associated with endocrine activity. Furthermore, the effect of juvenile hormone and its mimics on neurosecretory cells may be non-specific. Thus, other work in this laboratory suggests that large doses of topically applied F M E may be able to stimulate neurosecretory cells in the insect Rhodnius prolixus (Pratt & Davey, 1969). There is also the suggestion in the present work that the degree of neurosecretory activity was greater than in normal worms. What the mechanism of this stimulation might be is not clear. The materials may be acting directly on the neurosecretory system or at the level of the sense organs. The fact that Baumann (1968) has demonstrated that high concentrations of mimics of juvenile hormone bring about depolarization of insect salivary gland membrane potentials is of some interest in this respect. Shanta & Meerovitch (1970) have suggested that the possibility of these compounds being produced by nematodes should be investigated. In view of the wide distribution among living organisms of substances with juvenile hormone activity (Williams, Moorhead & Pulis, 1959; Schneiderman & Gilbert, 1958; Highman & Hill, 1969), it would scarcely be surprising if nematodes were found to contain similar materials. Until the endocrine control of development in nematodes has a more extensive experimental basis, it is probably best to regard the introduction of these materials as simply one more stressful factor in the environment, which, as I have suggested elsewhere (Davey, 1963, 1964), can lead to a release of endocrines.
I.J.P. VOL.I. 1971
MOLTINGIN Phocanema decipiens
65
Whether the effects on the reproductive system noted by other workers (Johnson & Viglierchio, 1970; Shanta & Meerovitch, 1970) are also mediated by the neurosecretory system is difficult to say. Preliminary studies in this laboratory have shown that the neurosecretory cells of Phocanema became active again about two weeks after ecdysis, when development of the reproductive structures is proceeding apace. The other effects of adding JH or F M E to the medium are less dear. Thus, the feathery appearance of the outermost layer of the new cuticle may be a result of the partial inhibition of its formation by the materials. Since the nematodes are only feeding during the period from 12 to 30 h after being put in culture (Davey, 1969), it is possible that the compounds are only present internally during the early stages of cuticle formation. The apparent failure of the new cuticle to remain attached to the hypodermis may be an artifact of fixation, but it is difficult to see why such an artifact should not be equally obvious in untreated controls. It may result from the continued growth of the new cuticle while the total growth of the nematode is restricted by the presence of the old cuticle. On the other hand, these effects have never been noticed in sections of worms placed in 0.9 ~o NaC1 without added F M E or JH. In these worms, of course, ecdysis does not take place. The failure of attachment may result from an interference, direct or indirect, with the as yet undescribed morphogenetic systems involved in the attachment of the cuticle to the hypodermis. The apparent invasion of the cuticle by the hypodermis may be another aspect of this failure. The appearance o f the osmiophilic droplets may simply be an indication that the diet in the treated animals is richer in lipid by virtue of the addition of F M E or JH. On the other hand, it may signal an increased mobility of lipid, which is known to vary during the molting cycle in any case (Kan & Davey, 1968). Acknowledgements--I am indebted to Mr. G. F. Webster for his skilled and careful assistance, and to Mr. A. F. Mclnnes, Department of Fisheries, Sydney, Nova Scotia, who supplies the infected cod fillets. This research is supported by the National Research Council of Canada.
REFERENCES BAtlMANN G. 1968. Zur Wirkung des Juvenilhormons; Elektrophysiologische Messangen an der Zellmembran der Speicheldruse yon Galleria mellonella. Journal of lnsect Physiology 14: 1459-1476. CAMERONM. L. & STEELE J'. E. 1959. Simplified aldehyde-fuchsin staining of the neurosecretory cells. Stain Technology 34: 265-266. DAVEYK. G. 1963. The release by enforced activity of the cardiac accelerator from the corpus cardiacum of Periplaneta americana. Journal of Insect Physiology 9: 375-381. DAVEYK. G. 1964. Neurosecretory cells in a nematode, Ascaris lumbricoides. Canadian Journal of Zoology 42: 731-734. DAVEYK. G. 1965. Molting in a parasitic nematode, Phocanema decipiens--I. Cytologicalevents. Canadian Journal of Zoology 43: 997-1003. DAVEYK. G. 1966. Neurosecretionand molting in some parasitic nematodes. American Zoolog&t 6: 243-249. DAVEYK. G. 1969. Molting in a parasitic nematode, Phocanema decipiens--V. Timing of feeding during the molting cycle.Journal of the Fisheries Research Board of Canada 26: 935-939. DAVEYK. G. & KAN S. P. 1967.An endocrine basis for ecdysis in a parasitic nematode. Nature, London 214: 737-738. DAVEVK. G. & KAN S. P. 1968. Molting in a parasitic nematode. Phocanema decipiens--IV. Ecdysis and its control. Canadian Journal of Zoology 46: 893-898. HIOHNAMK. C. & HILLL. C. 1969. The comparative endocrinology of the invertebrates. 270 pp. Edward Arnold, London. JOHNSON R. N. and VIOLIERCHIOD. R. 1970. Heterodera schactii responses to exogenous hormones. Experimental Parasitology 27, 301-309. KAN S. P. & DAVEYK. G. 1968. Molting in a parasitic nematode. Phocanema decipiens--III. The histochemistry of cuticle deposition and protein synthesis. Canadian Journal of Zoology 46: 723-727. M~EROVlTCHE. 1965. Studies on the in vitro axenic development of Trichinella spiralis---II. Preliminary experiments on the effect of farnesol, cholesterol, and an insect extract. Canadian Journal o f Zoology 43: 81-85. I.P. I/I--R
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PRATT G. E. & DAVEY K. G. 1969. The corpus allatum in mated and virgin Rhodnius. Proceedings of the Annual Meeting of the Entomological Society of Ontario 1969. In press. ROGERS W. P. 1965. The role of leucine aminopeptidase in moulting of nematode parasites. Comparatiae Biochemistry and Physiology 14: 311-321. ROLLER H. & DAHM K. H. 1968. T h e chemistry and biology of juvenile hormone. Recent Progress In Hormone Research 24: 651-680. SCHNEIDERMAN H. A. t~; GILBERT L. I. 1958. Substances with juvenile hormone activity in Crustacea. Biological Bulletin 11S: 530-535. SHANTA C. S. • MEEROVrrCH E. 1970. Specific inhibition of morphogenesis in Trichinella spiralis by insect juvenile hormone mimics. Canadian Journal of Zoology 48: 617-620. TOWNSLEY P. M., WIGHT H. G., SCOTT M. A. & HUGHES M. L. 1963. The in vitro maturation of the parasitic nematode Terranova decipiens from cod muscle. Journal of the Fisheries Research Board of Canada 20: 743-747. WILLIAMS C. M., MOORt-mAD L. V. & PULIS J. V. 1959 Juvenile hormone in thymus, human placenta and other mammalian organs. Nature, London 183: 405. WIGGLESWORTH V. ~B. 1957. The use of osmium in the fixation and staining of tissues. Proceedings of the Royal Society of London, Series B. 147: 185-199. WIGGLESWORTH V. B. 1969. Chemical structure and juvenile hormone activity. Comparative tests on Rhodnius prolixus. Journal of Insect Physiology 15: 73-94.