Journal of Chemical Ecology, Vol. 12, No. 8, 1986

ROLE OF CHEMICAL SUBSTANCES FROM FISH HOSTS IN HATCHING A N D HOST-FINDING IN MONOGENEANS

G . C . KEARN School of Biological Sciences University of East Anglia Norwich NR4 7TJ, U.K. (Received October 1, 1985; accepted December 6, 1985) Abstract--Hatching responses to chemical stimuli appear to have evolved independently in different kinds of monogenean skin and gill parasites of fishes, particularly in those parasites associated with bottom-dwelling hosts. Some monogeneans, such as Entobdella soleae, have two hatching strategies, responding readily to host skin mucus but hatching spontaneously in small numbers in the absence of the host. Other monogeneans, such as Acanthocotyle lobianchi, have abandoned spontaneous hatching and rely entirely on a "sit-and-wait" strategy, but improvements in the speed of hatching provide opportunities to take advantage of brief periods of contact between the eggs and the host. This has led to the loss of ciliated epidermal cells and to the inability to swim. Comparison of the eggs and hatching responses of two unrelated monogeneans, Leptocotyle minor and Hexabothrium appendiculaturn, which share the same dogfish host, reveals evidence of convergence. Small, stable molecules such as urea, excreted by the host, have been implicated as hatching stimulants in monogeneans. There is evidence that host recognition in E. soleae is by chemoperception but, in contrast with the lack of specificity of the chemical hatching stimuli, this appears to be of a specific nature. Key Words--Monogeneans, fish parasites, chemical hatching factors, hostfinding. M o s t m o n o g e n e a n s are parasites o f the skin and gills o f fishes and display strict host specificity. In m o s t m o n o g e n e a n s the e g g s l e a v e the host and d e v e l o p on or in the b o t t o m s e d i m e n t ( K e a m , 1986). T h e e g g s h a v e shells o f tanned protein and a ciliated larva or o n c o m i r a c i d i u m d e v e l o p s within t h e m o v e r a period o f t i m e that usually falls w i t h i n the range o f 1 0 - 4 0 days. It is this ciliated larva that has the task o f finding the fish host, there b e i n g no i n t e r m e d i a t e host. 1651 0098-0331/86/0800-1651 $05.00/0 9 1986 Plenum Publishing Corporation

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The fish hosts of monogeneans have diversified greatly; they occupy many different kinds of habitat and display a variety of behavior pattens. It is perhaps not surprising that the eggs and larvae of monogeneans have responded to this diversity by adaptations that enhance their chances of infecting their specific hosts. Fishes advertize their presence in a variety of ways. As they swim, their movements disturb the surrounding water, and they may cast shadows on the bottom sediment. It has already been established that the eggs of certain monogeneans hatch when mechanically disturbed, e.g., Microcotyle salpae (see Ktari, 1969), and larvae emerge when the eggs of Entobdella diadema are plunged into darkness (Kearn, 1982). Fishes are also the source of chemical substances in their skin or gill secretions and in their urine or feces, and there is evidence that the eggs of several monogeneans are stimulated to hatch by such chemical cues (Table 1). The first evidence that monogenean larvae might respond to chemical hatching factors of host origin was presented by Euzet and Raibaut (1960). They collected and incubated eggs of a polyopisthocotylean monogenean, Squalonchocotyle torpedinis, from the gills of an electric ray, Torpedo marmorata. They observed that eggs kept in seawater, uncontaminated by the host, hatched only in small numbers but, in the presence of either an electric ray or isolated gills from such a ray, a large number of larvae emerged. Similar evidence for a chemical hatching stimulus was reported by Ktari (1969) in the gill-parasitic polyopisthocotylean Microcotyle salpae. A significant feature of the freshly hatched larvae of both S. torpedinis and M. salpae is that these larvae lack ciliated epidermal cells (see below). More detailed knowledge of the role of hatching factors from the host has emerged from a study of the skin-parasitic capsalid monogenean Entobdella soleae. Its bottom-dwelling, flat-fish host, the common sole (Solea solea), survives well in aquaria and the life cycle of the parasite continues in these aquaria, providing a supply of adult parasites and eggs for experimental work. The larva of E. soleae is ciliated, and it has been established that hatching occurs in the absence of the host (Keam, 1973). When eggs are exposed to the natural cycle of illumination or to an artificial light regime, e.g., 12 : 12 lightdark, hatching is rhythmical, most larvae emerging on each successive day during the first few hours after dawn or after the artificial illumination is switched on. However, when sole body mucus is added to fully developed eggs at any time during the illumination cycle, hatching is greatly enhanced, indicating that sole mucus contains a potent hatching stimulant (Keam, 1974). It has been shown by Kearn (1975) that hatching is preceded by ciliary activity on the part of the fully developed oncomiracidium; this activity begins spontaneously in the absence of the host or as a result of stimulation by host mucus. The beating cilia produce rotation of the larva about its longitudinal axis, the rotating head of the larva occupying the cower of the tetrahedral egg

Hexabothriid polyopisthocotylean Hexabothriid polyopisthocotylean Mazocraeidean polyopisthocotylean Microbothriid

Acanthocotylid daetylogyridean Acanthocotylid dactylogyridean Capsalid dactylogyridean Uncertain

Squalonchocotyle torpedinis Hexabothrium appendiculatum Microcotyle salpae

Acanthocotyle lobianchi Acanthocotyle greeni Entobdella soleae

Pseudodactylogyrus bini

Leptocotyle minor

Relationship

Species

Gills

Skin

Anguilla japonica

Raja clavata Solea solea

Skin

Skin

Scyliorhinus canicula Raja spp.

Torpedo marmorata Scyliorhinus canicula Box salpa

Host

Skin

Gills

Gills

Gills

Microhabitat

Ciliated

Ciliated

Unciliated

Unciliated

Ciliated

Unciliated

Ciliated

Unciliated

Oncomiracidium

Chart and Wu, 1984

Macdonald and Llewellyn, 1980 Kearn, 1974

Macdonald, 1974

Whittington (in progress)

Ktari, 1969

Whittington (in progress)

Euzet and Raibaut, 1960

Author

Table 1. MONOGENEANS REPORTED TO RESPOND TO CHEMICAL HATCHING FACTORS FROM THE HOST

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where the lid or operculum is situated. A considerable body of evidence suggests that erosion of the cement attaching the operculum to the rest of the shell is brought about by secretory material from gland cells in the head region of the larva. The interval between the commencement of rotation and the escape of the larva from the egg may be as short as 4-5 rain. Thus, E. soleae appears to have alternative hatching strategies depending on whether or not there are soles in the vicinity of the fully developed eggs of the parasite. If there is no host in the vicinity, hatching takes place soon after dawn, but relatively small numbers of larvae emerge. Natural selection might be expected to favor spontaneous hatching of the parasite during this period of the day because the sole host rests on the bottom during the hours of daylight and slow-moving oncomiracidia seem more likely to successfully locate and attach themselves to an inactive host than to one that is actively swimming. Prolonged close contact is likely to occur between the host and the eggs of the parasite; the host spends long periods of time resting on the sea bottom and the eggs of the parasite are attached to sand grains hy adhesive material on a long appendage (Kearn, 1963a,b). In these circumstances, the ability of the larvae to respond at any time of day to chemical hatching factors from the host will enhance the survival of the parasite. If the response of the larva of E. soleae to chemical hatching factors from the host is to be effective in ensuring host invasion, it is important that the larvae are infective immediately after hatching. This has been tested by placing fully developed eggs on the upper surface of a sole. Kearn (1981) observed that some freshly hatched larvae attached themselves by the sticky areas on the head region before the posterior, hook-beating attachment organ or haptor had been withdrawn from the shell. After emergence, the haptor was then attached to the skin and the sticky areas were released and this was followed, within about 30 sec, by the loss of all the ciliated epidermal ceils of the larva. Thus the oncomiracidia of E. soleae appear to be infective immediately after hatching, and there is no requirement for an initial period of free-swimming before infection takes place. There appears to have been a further development in the monogenean Acanthocotyle lobianchi, a skin parasite of rays (Raja spp.). The eggs of this parasite, like those of E. soleae, have adhesive material on their appendages and stick to sand grains. Macdonald (1974) discovered that these eggs fail to hatch spontaneously when kept in seawater free of contamination by host mucus. Nevertheless, the larvae are able to survive within the egg for as long as 80 days at 13~ When the eggs are treated with ray mucus, hatching occurs but, unlike E. soIeae, emergence of the larva takes place with great rapidity (within a few seconds). The larva responds by greatly extending its body and in so doing pushes off the operculum; the opercular cement is already weak in

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anticipation of the thrust of the larva. Thus, A. lobianchi has speeded up the hatching process in response to a chemical hatching stimulus and appears to rely entirely on a "sit-and-wait" strategy, having abandoned the ability to fall back on spontaneous hatching as a "last-ditch" means of locating the host. Because of the adoption of this strategy, there is no longer a requirement for free-swimming and ciliated epidermal cells are absent. The host must virtually make contact with the eggs if infection is to take place, but the speed of hatching ensures that the larva can take advantage of exceedingly brief periods of contact. A similar evolutionary pathway may have led to the absence of ciliated cells in the larvae of S. torpedinis and M. salpae (see above). Parasitologists working at the laboratory of the Marine Biological Association of the United Kingdom at Plymouth, England, have encountered the eggs of some other monogeneans that, apparently, fail to hatch spontaneously. Whittington (work in progress) has found that hatching in the skin parasite Leptocotyle minor and the gill parasite Hexabothrium appendiculatum, both from the dogfish Scyliorhinus canicula, resembles hatching in A. lobianchi. Eggs of these parasites fail to hatch spontaneously in the absence of the host but survive for at least 80 days; the opercular cement is weakened in readiness for hatching, which is rapid and stimulated by seawater from a tank containing dogfish. Whittington has observed that the eggs of L. minor and H. appendiculatum are small, with exceptionally long and slender appendages lacking adhesive droplets. These small eggs are readily kept in suspension even by weak convection currents, and the slender appendage makes a contribution to their slow rate of sedimentation because eggs sink faster if the appendage is experimentally removed. Thus the appendages may serve to keep the eggs in suspension, like the silk threads produced by small aerial spiders. This has led Whittington to suggest that currents produced by dogfish foraging near the bottom may lift resting eggs off the bottom; these eggs may then drift sufficiently close to swimming dogfish for hatching to be stimulated by chemical substances from the host's body. The retention of the ciliated cells and the ability of the larvae of L. minor and H. appendiculatum to swim, may reflect the fact that drifting eggs are unlikely to make intimate contact with the bodies of moving hosts. Whittington has suggested that the striking similarity between the shapes and sizes of the eggs and the hatching behavior of these unrelated parasites (L. minor is a microbothriid and 1-1. appendiculatum is a polyopisthocotylean) may be an example of convergent evolution that has arisen because of the exertion of similar selection pressures on the two parasites by the same host. In contrast with this apparent convergence between dogfish parasites, the skin parasite A. lobianchi and the gill parasite Rajonchocotyle emarginata, both of which inhabit Raja spp., appear to have evolved different strategies for host

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invasion. Previous workers had failed to hatch the eggs of R. emarginata, but Whittington and Kearn (1986) have found that the ciliated larvae of this parasite do hatch spontaneously but have a relatively long incubation period (44-47 days at 13-14~ Moreover, these eggs fail to respond when treated with mucus from ray skin or gills. There are indications that the chemical hatching factors in the skin mucus of soles and rays are small and stable molecules (Kearn, 1974; Keam and Macdonald, 1976). Exploratory tests with potential hatching stimulants implicated urea, ammonium chloride, and arginine in the hatching of E. soleae, but the only substance tested that stimulated hatching in A. lobianchi was urea (Kearn and Macdonald, 1976). Incubation with urease was found to be a most effective way of destroying the stimulatory properties of ray mucus for A. lobianchi eggs, but similar tests with sole skin washings and eggs of E. soleae gave inconsistent results. Recent work by Whittington (in progress) suggests that extrapure urea may be less effective as a hatching stimulant for E. soleae eggs than less pure grades, and he has found that lower grade urea rarely proves as effective as sole mucus. This suggests that a trace substance present in commercial urea may stimulate hatching in the eggs of E. soleae or, perhaps more likely, that the effectiveness of urea may be enhanced by the presence of one or more other substances. This contrasts with the situation in A. lobianchi, the unhatched larvae of which respond equally readily when treated with extrapure urea or lower grade urea. Furthermore, urea appears to be just as effective as ray mucus. Preliminary observations by Whittington (work in progress) indicate that the eggs of L. minor and H. appendiculatum respond to urea in a similar way. Keam and Macdonald (1976) estimated the levels of urea in ray and sole skin mucus. The levels of urea in ray skin thucus are relatively high, providing a strong hatching signal for the eggs of A. lobianchi. The levels of urea in sole skin mucus are much lower, and this may have led to the development of sensitivity on the part of unhatched oncomiracidia of E. soleae to a combination of substances present in mucus, one component of which may be urea. Keam (1974) showed that the chemical hatching stimulus for the eggs of E. soleae is nonspecific, the eggs readily hatching if treated with skin washings from a variety of fishes including whiting (Merlangius merlangus) and rays. This lack of specificity seems at first sight to be distinctly disadvantageous since hatching might be induced by nonhost fishes or by other bottom-dwelling organisms. However, this nonspecific stimulus may not be disadvantageous if soles are locally abundant, because, in these circumstances, the hatching stimulus is more likely to be provided by a sole than by any other organism, Moreover, even if hatching were stimulated by a nonhost organism, it is most unlikely that the larvae would attach themselves to it, When oncomiracidia are presented experimentally with a choice of skin samples, either in the fbrm of

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detached scales or small pieces of skin from more than one fish species, the larvae ofE. soleae show a strong preference for sole skin, even in total darkness (Kearn, 1967). Larvae also show a preference for agar blocks which have been in contact with sole skin rather than similar blocks soaked in seawater. This indicates that chemoperception may be important in host identification and suggests that the chemical cues provided by the host are specific, in contrast with the nonspecific nature of the hatching stimulus. However, there is not yet any evidence that oncomiracidia are attracted to the sole by chemotaxis, and recognition of the host may rely on contact chemoperception. The monogeneans that are known to respond to chemical hatching factors from the host are listed in Table 1. Three points are worth stressing. First, the phenomenon appears to have arisen independently several times. Secondly, the hosts that provide the chemical hatching cues may be teleosts or elasmobranchs but are usually flat-fishes or round-bodied hosts that feed on the bottom. This is perhaps not surprising because most monogenean eggs come to rest on the bottom, and the effectiveness of a hatching response to chemical substances of host origin depends on contact or close proximity between the hosts and the fully developed eggs. Thirdly, there is a tendency for the oncomiracidia to dispense with ciliated cells. Hatching responses to chemical substances from the host of the kind described here have rarely been reported in other flatworm parasites. Shinn (1983) has presented evidence to suggest that in the parasitic turbellarian, Syndisyrinx franciscanus, the intestinal fluid of its sea-urchin host stimulates the production of a hatching fluid by the parasite. Host digestive secretions play an important role in hatching of some cestode eggs, perhaps partly by stimulating larval activity, but their main role is concerned with erosion of the egg envelopes (see, for example, Caley, 1975; Holmes and Fairweather, 1982) or the opercular cement (Kennedy, 1965). The molluscan hosts of digeneans are slow-moving, bottom-dwelling organisms, and contact between these hosts and digenean eggs seems likely to be a frequent occurrence. In similar circumstances, several monogeneans have independently acquired a hatching response to chemical substances from the host, and it is rather surprising that there appear to be no reports of a similar phenomenon in those digenean eggs that hatch in water. However, some digeneans have exploited the opportunities offered by accidental ingestion of their eggs by their molluscan hosts, hatching apparently being stimulated by host digestive enzymes and/or physicochemical conditions in the gut (Smyth and Halton, 1983). Smyth and Halton have pointed out that this area has received little attention and further study seems likely to be rewarding. Host location by digenean miracidia that hatch in water has been studied more intensively than in monogeneans (Smyth and Halton, 1983). Striking changes in the behavior of the free-swimming miracidia have been reported on

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the addition of snail-conditioned water, for example in Schistosoma mansoni but comparable changes in the behavior of oncomiracidia have not been reported. REFERENCES CALEY, J. 1975. In vitro hatching of the tapeworm Moniezia expansa (Cestoda: Anoplocephalidae) and some properties of the egg membranes. Z. Parasitenk. 45:335-346. CHAN, B., and Wu, B. 1984. Studies on the pathogenicity, biology and treatment of Pseudodactylogyrus for the eels in fish farms. Acta Zool. Sin. 30:173-180 (in Chinese). EUZET, L., and RAIBAtJT, A. 1960. Le d6veloppement post-larvaire de Sqaalonchocotyle torpedinis (Price 1942) (Monogenea, Hexabothrlidae). Bull. Soc. Neuchdttel. Sci. Nat. 83:101-108. HOLMES, S.D., and FAIRWEATHER,I. 1982. Hymenolepis diminuta: The mechanism of egg hatching. Parasitology 85:237-250. KEARN, G.C. 1963a. The life cycle of the monogenean Entobdella soleae, a skin parasite of the common sole. Parasitology 53:253-263. KEARN, G.C. 1963b. The egg, oncomiracidium and larval development of Entobdella soleae, a monogenean skin parasite of the common sole. Parasitology 53:435-447. KEARN, G.C. 1967. Experiments on host-finding and host-specificity in the monogenean skin parasite Entobdella soleae. Parasitology 57:585-605. KEARN, G.C. 1973. An endogenous circadian hatching rhythm in the monogenean skin parasite Entobdella soleae and its relationship to the activity rhythm of the host (Solea solea). Parasitology 66:101-122. KEARN, G.C. 1974. The effect of fish skin mucus on hatching in the monogenean parasite Entobdella soleae from the skin of the common sole, Solea solea. Parasitology 68:173-188. KEARN, G.C. 1975. The mode of hatching of the monogenean Entobdella soleae, a skin parasite of the common sole (Solea solea). Parasitology 71:419-431. KEARN, G.C. 1981. Behaviour of oncomiracidia. Parasitology 82:57-59. KEARN, G.C. 1982. Rapid hatching induced by light intensity reduction in the monogenean Entobdella diadema. J. Parasitol. 68:171-172. KEARN, G.C. 1986. The eggs of monogeneans. Adv. Parasitol. 30: In press. KEARN, G.C., and MACDONALD, S. 1976. The chemical nature of host hatching factors in the monogenean skin parasites Entobdella soleae and Acanthocotyle lobianchi. Int. J. Parasitol. 6:457-466. KENNEDY, C.R. 1965. The mode of hatching of the egg of the cestode Archigetes limnodrili (Yamaguti). Parasitology 55:18 pp. KTARI, M.H. 1969. Recherches sur l'anatomie et la biologie de Microcotyle salpae Parona et Perugia. 1890 parasite de Box salpa L. (T616ost6en). Ann. Parasitol. Hum. Comp. 44:425-440. MACDONALD,S. 1974. Host skin mucus as a hatching stimulant in Acanthocotyle lobianchi, a monogenean from the skin of Raja spp. Parasitology 68:331-338. MACDONALD,S., and LLEWELLYN,J. 1980. Reproduction in Acanthocotyle greeni n. sp. (Monogenea) from the skin ofRaja spp. at Plymouth. J. Mar. Biol. Assoc. U.K. 60:81-88. SmNN, G.L. 1983. The life history of Syndisyriraxfranciscanus, a symbiotic turbellarian from the intestine of echinoids, with observations on the mechanism of hatching. Ophelia 22:57-79. SMYTH, J.D., and HALTON, D.W. 1983. The Physiology of Trematodes. Cambridge University Press, Cambdge, U.K., London, New York. WHITTINGTON, I.D., and KEARN, G.C. 1986. Rhythmical hatching and oncomiracidial behaviour in the hexabothriid monogenean Rajonchocotyle emarginata from the gills of Raja spp. J, Mar. Biol. Assoc. U.K. 66:93-111.

Role of chemical substances from fish hosts in hatching and host-finding in monogeneans.

Hatching responses to chemical stimuli appear to have evolved independently in different kinds of monogenean skin and gill parasites of fishes, partic...
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