Journal of Chemical Ecology, Vol. 12, No. 8, 1986
CHEMICAL COMMUNICATION IN HERMAPHRODITIC DIGENETIC TREMATODES
B E R N A R D FRIED Department of Biology Lafayette College Easton, Pennsylvania 18042
(Received October 1, 1985; accepted December 17, 1985) Abstract--Adult hermaphroditic digenetic trematodes show a tendency to pair or aggregate in vivo, on the chick chorioallantois, and in vitro. Intraspecific pairing studies on Echinostoma revolutum, Leucochloridiomorpha constantiae, and Amblosoma suwaense are reviewed. Lipophilic excretorysecretory products are involved in chemical communicationin hermaphroditic digeneans. Free sterols are involved in chemical attraction in L. constantiae and E. revolutum and sterol esters play a similar role in A. suwaense. In vitro pairing between E. revolutum and various other digenean species suggest that interspecificpairing occurs in Digenea and that nonspecificfactors are involved. Key Words--Platyhelminths, Trematoda, hermaphroditic digeneans, intraspecific pairing, interspecific pairing, behavior, chemical communication, pheromones, lipids, sterols.
INTRODUCTION
Except for the schistosomes and some didymozoids, digeneans are hermaphroditic. This paper examines studies on chemical communication in adult hermaphroditic digeneans. Some information on this topic has been presented in Nollen's (1983) review on sexual reproduction in parasitic flatworms, and Bone's (1982) review on reproductive chemical communication in platyhelminths. Digenetic trematodes have complex life cycles and various larval stages. The first intermediate host in the digenean cycle is usually a specific snail, in which intramolluscan stages develop. In monoecious digeneans, cercariae released from sporocysts or rediae encyst on a substratum or in a second inter1659 0098-0331/86/0800-1659505.00/0
9 1986 Plenum Publishing Corporation
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mediate invertebrate or vertebrate host. The encysted metacercariae (cysts) are infective to the definitive vertebrate host and are usually acquired by the feeding habits of the host. In the laboratory, cysts are administered per os to experimental definitive hosts and metacercariae usually excyst in the intestine. Excysted metacercariae establish in more or less specific sites in the intestine or its ramifications. How excysted flukes locate specific sites is not well understood, and this subject has been reviewed by Ulmer (1971) and Kemp and Devine (1982). Laboratory studies on chemical communication have used monoecious digeneans that have little or no economic significance, i.e., Echinostoma revo-
lutum, Zygocotyle lunata, Haematoloechus medioplexus, Leucochloridiomorpha constantiae, and Amblosoma suwaense. In the future, representative fasciolids may provide interesting models for chemical communication studies. Interference with chemical communication in fasciolids could provide a means for biological control of these important veterinary and medical trematodes.
PHEROMONES Chemical communication in animals is mediated by pheromones, The parasitology literature indicates that helminthologists use the term essentially as defined by Karlson and Luscher (1959), i.e., pheromones are substances emitted by an organism which can elicit a specific behavioral or developmental reaction in another of the same or closely related species. Both primer and releaser pheromones have been described for behavioral activities associated with digeneans (Bone, 1982). Primers cause physiological changes without immediate accompanying behavioral responses and influence behavior through hormonal changes. Releasers cause an immediate change in the behavior of the recipient and act directly on the recipient's nervous system (Wilson and Bossert, 1962). Other pheromones described for insects and arachnids, i.e., alarm, trail, marking, have not been examined in trematodes. The term excretory-secretory products or simply ES products has been used in trematode chemoattraction studies (Fried and Shapiro, 1975; Fried et al., 1980a, 1983; Bennett and Fried, 1983). This term refers to excretions and secretions released by trematodes either in vivo or into an in vitro cultivation or maintenance medium (Fried, 1978). Some of these ES products have pheromonal information. Because of the small size of most digeneans, it is difficult to associate the release of ES products with a particular system (von Brand, 1973; see Source of Pheromones, below). ES products have also been described in trematode immunology studies where they serve as a source of antigens (Rotmans et al., 1981; sandeman and Howell, 1981; Irving and Howell, 1982).
C H E M I C A L C O M M U N I C A T I O N IN H E R M A P H R O D I T I C T R E M A T O D E S
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RECEPTORS Studies on the reception of stimuli in hermaphroditic digeneans are sparse (Kemp and Devine, 1982). Scanning electron microscopy (SEM) and light level argentation studies have shown that larval and adult digeneans have various types of sensory papillae, some of which are probably chemoreceptors (Hoole and Mitchell, 1981; Fried and Fujino, 1984). However, chemoreceptors have not been unequivocally identified in monoecious digeneans, and there are no electrophysiological studies that elucidate their role in chemoreception. In insect physiology, the use of the electroantennogram has allowed for quantitative studies in sensory physiology. Until similar methodology is developed in trematode physiology, sensory studies will remain speculative. METHODOLOGY Pairing, i.e., the tendency of two monoecious digeneans to come together, and clustering, i.e., the aggregation of three or more worms have been studied in vivo, on the chick chorioallantois and in vitro (Fried and Roberts, 1972; Fried et al., 1980a, b; Fried and Alenick, 1981; Fried and Robinson, 1981). For in vivo studies on Echinostoma revolutum, experimental hosts were fed metacercarial cysts (Fried and Weaver, 1969); in brachylaimid studies, free (unencysted) metacercariae were inoculated per cloaca into domestic chicks (Fried and Harris, 1971; Fried and Schnier, 1981). At necropsy, observations were made on pairing or clustering of worms in their normal sites (Fried and Roberts, 1972; Fried and Alenick, 1981; Fried and Robinson, 1981). To eliminate host factors associated with worm migration and establishment in vivo, pairing and clustering have been studied on the chick chorioallantois of domestic chick embryos maintained in ovo or in vitro (Fried and Roberts, 1972; Fried et al., 1980b). Pairing studies that exclude organisms other than the parasites have been done in vitro in 3.5-, 5.0-, and 8.5-cm-diameter plastic Petri dishes usually maintained in an incubator or on a slide warmer at 35-41 ~ (Fried and Roberts, 1972; Fried et al., 1980a; Fried and Robinson, 1981; Fried and Pallone, 1984). In this design, a 1% agar substratum is prepared in the dish, since worms move better on the agar than the plastic. A sterile Locke's (Paul, 1975) solution overlay is usually used, and worms migrate at the agar-Locke's interface. In this nonbarrier design, worms show a maximal tendency to pair. However, this design does not exclude worm thigmotaxis, i.e., worm-to-worm contact pairing. In the nonbarrier design worms are usually started 2 cm from each other and the tendency of one worm to attract the other is examined at various intervals up to 24 hr.
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Worms in contact with or within 5 mm of each other are considered paired (Fried and Roberts, 1972; Fried et al., 1980a; Fried and Pallone, 1984). This criterion for pairing has also been used by Perkins and Fried (1982) in studies on free-living planarians in the genus Dugesia. Both Dugesia and the digeneans often show noncontact pairing, i.e., worms come within 1-2 mm of each other, do not make contact, and then retreat to about 5 mm from each other. In nonbarrier studies initial starting distance is a factor in subsequent pairing. E. revolutum started more than 8 cm apart did not pair, whereas those started less than 8 cm did (Fried and Pallone, 1984). Other factors involved in E. revolutum pairing are temperature and the overlay medium. More pairing occurred at 39~ than 35 ~ and an overlay of sterile Locke's was better than the defined medium NCTC 135 (Fried and Pallone, 1984). In our nonbarrier studies with monoecious digeneans and planarians, a single worm did not pair with a dead worm or an inert object. Attraction of a single worm toward either a filter paper disk or silica gel square containing worm homogenate or ES products has been studied in Petri dish cultures (Fried and Gioscia, 1976; Fried, et al., 1980a; Fried and Robinson, 1981). These studies begin by placing the worm equidistant between the experimentally impregnated disk or square and its control. Migration toward or contact with the experimental disk or square is considered attracted. In barrier designs, one worm is isolated from the other, thus eliminating the possibility of worm thigmotaxis. Such designs have been used to study heterosexual and homosexual chemical attraction in schistosomes maintained in polycarbonate linear chambers (Imperia et al., 1980; Eveland et al., 1982, 1983). The barrier usually consists of a chimney made of dialysis tubing (Imperia et al., 1980) or a dialysis sac (Fried et al., 1980a). Single Echinostoma revolutum adults migrated significantly toward a dialysis sac containing other E. revolutum (Fried et al., 1980a). In these designs worms isolated in the sac are designated "releasers" and those outside, "migrators" (Belosevic and Dick, 1980; Fried and Wilson, 1981a). In a recent study, E. revolutum pheromonal dosage was examined in a barrier design where either two, four, or six releasers were isolated in a cylindrical stainless steel screen (40-mesh) chimney and a single migrator was allowed to move toward or away from the barrier (Figure 1). The results showed that four and six releasers were better at attracting a single migrator than two releasers (Figure 2) (Fried and Wilson, 1981a). A recent study has allowed us to track E. revolutum adults in an 0.8 agarDulbecco's modified Eagle's medium supplemented with 10 % fetal calf serum. Worms survived up to 48 hr in the medium and left distinct acetabular tracks in the agar. Some worms showed directed movement toward each other, suggesting that chemotaxis was involved (Fried and Vates, 1984). In most studies statistical significance of pairing and chemoattraction data at each time period were determined using either the chi-square test or file two-
1663
CHEMICAL COMMUNICATION IN HERMAPHRODITIC TREMATODES
C
LUT
A
'
0
' I
U
' I
3.5cm > FIG. 1. Petri dish chamber used in pheromonal studies. C = chimney; A = attraction zone; O = neutral zone; U = unattraction zone. Reprinted from Fried and Wilson (1981a) with permission of the Helminthological Society of Washington.
CHEMICAL COMMUNICATION IN HERMAPHRODITIC DIGENETIC TREMATODES
B E R N A R D FRIED Department of Biology Lafayette College Easton, Pennsylvania 18042
(Received October 1, 1985; accepted December 17, 1985) Abstract--Adult hermaphroditic digenetic trematodes show a tendency to pair or aggregate in vivo, on the chick chorioallantois, and in vitro. Intraspecific pairing studies on Echinostoma revolutum, Leucochloridiomorpha constantiae, and Amblosoma suwaense are reviewed. Lipophilic excretorysecretory products are involved in chemical communicationin hermaphroditic digeneans. Free sterols are involved in chemical attraction in L. constantiae and E. revolutum and sterol esters play a similar role in A. suwaense. In vitro pairing between E. revolutum and various other digenean species suggest that interspecificpairing occurs in Digenea and that nonspecificfactors are involved. Key Words--Platyhelminths, Trematoda, hermaphroditic digeneans, intraspecific pairing, interspecific pairing, behavior, chemical communication, pheromones, lipids, sterols.
INTRODUCTION
Except for the schistosomes and some didymozoids, digeneans are hermaphroditic. This paper examines studies on chemical communication in adult hermaphroditic digeneans. Some information on this topic has been presented in Nollen's (1983) review on sexual reproduction in parasitic flatworms, and Bone's (1982) review on reproductive chemical communication in platyhelminths. Digenetic trematodes have complex life cycles and various larval stages. The first intermediate host in the digenean cycle is usually a specific snail, in which intramolluscan stages develop. In monoecious digeneans, cercariae released from sporocysts or rediae encyst on a substratum or in a second inter1659 0098-0331/86/0800-1659505.00/0
9 1986 Plenum Publishing Corporation
1660
FRIED
mediate invertebrate or vertebrate host. The encysted metacercariae (cysts) are infective to the definitive vertebrate host and are usually acquired by the feeding habits of the host. In the laboratory, cysts are administered per os to experimental definitive hosts and metacercariae usually excyst in the intestine. Excysted metacercariae establish in more or less specific sites in the intestine or its ramifications. How excysted flukes locate specific sites is not well understood, and this subject has been reviewed by Ulmer (1971) and Kemp and Devine (1982). Laboratory studies on chemical communication have used monoecious digeneans that have little or no economic significance, i.e., Echinostoma revo-
lutum, Zygocotyle lunata, Haematoloechus medioplexus, Leucochloridiomorpha constantiae, and Amblosoma suwaense. In the future, representative fasciolids may provide interesting models for chemical communication studies. Interference with chemical communication in fasciolids could provide a means for biological control of these important veterinary and medical trematodes.
PHEROMONES Chemical communication in animals is mediated by pheromones, The parasitology literature indicates that helminthologists use the term essentially as defined by Karlson and Luscher (1959), i.e., pheromones are substances emitted by an organism which can elicit a specific behavioral or developmental reaction in another of the same or closely related species. Both primer and releaser pheromones have been described for behavioral activities associated with digeneans (Bone, 1982). Primers cause physiological changes without immediate accompanying behavioral responses and influence behavior through hormonal changes. Releasers cause an immediate change in the behavior of the recipient and act directly on the recipient's nervous system (Wilson and Bossert, 1962). Other pheromones described for insects and arachnids, i.e., alarm, trail, marking, have not been examined in trematodes. The term excretory-secretory products or simply ES products has been used in trematode chemoattraction studies (Fried and Shapiro, 1975; Fried et al., 1980a, 1983; Bennett and Fried, 1983). This term refers to excretions and secretions released by trematodes either in vivo or into an in vitro cultivation or maintenance medium (Fried, 1978). Some of these ES products have pheromonal information. Because of the small size of most digeneans, it is difficult to associate the release of ES products with a particular system (von Brand, 1973; see Source of Pheromones, below). ES products have also been described in trematode immunology studies where they serve as a source of antigens (Rotmans et al., 1981; sandeman and Howell, 1981; Irving and Howell, 1982).
C H E M I C A L C O M M U N I C A T I O N IN H E R M A P H R O D I T I C T R E M A T O D E S
1661
RECEPTORS Studies on the reception of stimuli in hermaphroditic digeneans are sparse (Kemp and Devine, 1982). Scanning electron microscopy (SEM) and light level argentation studies have shown that larval and adult digeneans have various types of sensory papillae, some of which are probably chemoreceptors (Hoole and Mitchell, 1981; Fried and Fujino, 1984). However, chemoreceptors have not been unequivocally identified in monoecious digeneans, and there are no electrophysiological studies that elucidate their role in chemoreception. In insect physiology, the use of the electroantennogram has allowed for quantitative studies in sensory physiology. Until similar methodology is developed in trematode physiology, sensory studies will remain speculative. METHODOLOGY Pairing, i.e., the tendency of two monoecious digeneans to come together, and clustering, i.e., the aggregation of three or more worms have been studied in vivo, on the chick chorioallantois and in vitro (Fried and Roberts, 1972; Fried et al., 1980a, b; Fried and Alenick, 1981; Fried and Robinson, 1981). For in vivo studies on Echinostoma revolutum, experimental hosts were fed metacercarial cysts (Fried and Weaver, 1969); in brachylaimid studies, free (unencysted) metacercariae were inoculated per cloaca into domestic chicks (Fried and Harris, 1971; Fried and Schnier, 1981). At necropsy, observations were made on pairing or clustering of worms in their normal sites (Fried and Roberts, 1972; Fried and Alenick, 1981; Fried and Robinson, 1981). To eliminate host factors associated with worm migration and establishment in vivo, pairing and clustering have been studied on the chick chorioallantois of domestic chick embryos maintained in ovo or in vitro (Fried and Roberts, 1972; Fried et al., 1980b). Pairing studies that exclude organisms other than the parasites have been done in vitro in 3.5-, 5.0-, and 8.5-cm-diameter plastic Petri dishes usually maintained in an incubator or on a slide warmer at 35-41 ~ (Fried and Roberts, 1972; Fried et al., 1980a; Fried and Robinson, 1981; Fried and Pallone, 1984). In this design, a 1% agar substratum is prepared in the dish, since worms move better on the agar than the plastic. A sterile Locke's (Paul, 1975) solution overlay is usually used, and worms migrate at the agar-Locke's interface. In this nonbarrier design, worms show a maximal tendency to pair. However, this design does not exclude worm thigmotaxis, i.e., worm-to-worm contact pairing. In the nonbarrier design worms are usually started 2 cm from each other and the tendency of one worm to attract the other is examined at various intervals up to 24 hr.
1662
FRIED
Worms in contact with or within 5 mm of each other are considered paired (Fried and Roberts, 1972; Fried et al., 1980a; Fried and Pallone, 1984). This criterion for pairing has also been used by Perkins and Fried (1982) in studies on free-living planarians in the genus Dugesia. Both Dugesia and the digeneans often show noncontact pairing, i.e., worms come within 1-2 mm of each other, do not make contact, and then retreat to about 5 mm from each other. In nonbarrier studies initial starting distance is a factor in subsequent pairing. E. revolutum started more than 8 cm apart did not pair, whereas those started less than 8 cm did (Fried and Pallone, 1984). Other factors involved in E. revolutum pairing are temperature and the overlay medium. More pairing occurred at 39~ than 35 ~ and an overlay of sterile Locke's was better than the defined medium NCTC 135 (Fried and Pallone, 1984). In our nonbarrier studies with monoecious digeneans and planarians, a single worm did not pair with a dead worm or an inert object. Attraction of a single worm toward either a filter paper disk or silica gel square containing worm homogenate or ES products has been studied in Petri dish cultures (Fried and Gioscia, 1976; Fried, et al., 1980a; Fried and Robinson, 1981). These studies begin by placing the worm equidistant between the experimentally impregnated disk or square and its control. Migration toward or contact with the experimental disk or square is considered attracted. In barrier designs, one worm is isolated from the other, thus eliminating the possibility of worm thigmotaxis. Such designs have been used to study heterosexual and homosexual chemical attraction in schistosomes maintained in polycarbonate linear chambers (Imperia et al., 1980; Eveland et al., 1982, 1983). The barrier usually consists of a chimney made of dialysis tubing (Imperia et al., 1980) or a dialysis sac (Fried et al., 1980a). Single Echinostoma revolutum adults migrated significantly toward a dialysis sac containing other E. revolutum (Fried et al., 1980a). In these designs worms isolated in the sac are designated "releasers" and those outside, "migrators" (Belosevic and Dick, 1980; Fried and Wilson, 1981a). In a recent study, E. revolutum pheromonal dosage was examined in a barrier design where either two, four, or six releasers were isolated in a cylindrical stainless steel screen (40-mesh) chimney and a single migrator was allowed to move toward or away from the barrier (Figure 1). The results showed that four and six releasers were better at attracting a single migrator than two releasers (Figure 2) (Fried and Wilson, 1981a). A recent study has allowed us to track E. revolutum adults in an 0.8 agarDulbecco's modified Eagle's medium supplemented with 10 % fetal calf serum. Worms survived up to 48 hr in the medium and left distinct acetabular tracks in the agar. Some worms showed directed movement toward each other, suggesting that chemotaxis was involved (Fried and Vates, 1984). In most studies statistical significance of pairing and chemoattraction data at each time period were determined using either the chi-square test or file two-
1663
CHEMICAL COMMUNICATION IN HERMAPHRODITIC TREMATODES
C
LUT
A
'
0
' I
U
' I
3.5cm > FIG. 1. Petri dish chamber used in pheromonal studies. C = chimney; A = attraction zone; O = neutral zone; U = unattraction zone. Reprinted from Fried and Wilson (1981a) with permission of the Helminthological Society of Washington.
