Parasitology (1975), 71, 211-228 With 5figuresin the text

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Hatching rhythms in three species of Diclidophora (Monogenea) with observations on host behaviour SHEILA MACDONALD School of Biological Sciences, University of East Anglia, Norwich, NEi 7TJ, U.K. {Received 10 February 1975) SUMMARY

Eggs of three species of Diclidophora were incubated in alternating 12 h periods of light and darkness at 13 °C. Eggs of D. merlangi collected at Arbroath hatched during the illumination period with most larvae being recovered in the first 4-6 h; some evidence of a seasonal difference in hatching of these eggs was found. Eggs of D. merlangi collected at Plymouth hatched with a peak of larval recovery in the 2 h period before the light came on. Eggs of D. luscae hatched over 'dusk' while those of D. denticulata hatched after the light was switched off. Neither mechanical disturbance nor the proximity of host tissue caused hatching in D. merlangi or D. luscae. Observations on the behaviour of the host fishes suggest that the hatching rhythms are adapted to specific host behaviour patterns. INTRODUCTION

The oncomiracidia of those monogeneans that have been studied have a short free-swimming life in which to make contact with their specific fish hosts (Paperna, 1963; Kearn, 1971). Since most marine monogenean eggs come to rest on the sea bottom and since active fish swim at 60-300 times the speed of an oncomiracidium (Llewellyn, 1972) it might be expected that selection pressure would favour hatching at times when the host is resting or swimming slowly near the bottom. One way in which this may be brought about is by response to a physical or chemical stimulus from the host. Bovet (1967) found that hatching of Diplozoon paradoxum was enhanced by water turbulence, a physical stimulus provided by a nearby swimming host. Other monogenean eggs are known to respond to chemical stimuli. Euzet & Raibaut (1960) found that host mucus would elicit hatching in Squalonchocotyle torpedinis and such a response has also been reported in Microcotyle salpae by Ktari (1969), in Acanthocotyle lobianchi by Macdonald (1974) and by Kearn (1974a) in Entobdella soleae. Selection pressure has also resulted in the evolution of circadian hatching rhythms related to the lighting cycle such that hatching by parasites may be related to characteristic behaviour patterns of the fish hosts. Kearn (1973) found that light, while not essential for the hatching of eggs of E. soleae, does affect the timing of larval emergence. He showed that if eggs of E. soleae were incubated in alternating 12 h periods of light and darkness a hatching pattern developed with

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the majority of larvae emerging in the first 4 h of each illumination period. Kearn was able to show that the hatching pattern had a strong endogenous component and pointed out that it could have survival value for the parasite since the host fish, Solea solea, lies inactive on the sea bed during the day, so providing a stationary target for the invading larvae. In relation to the hatching behaviour of E. soleae it may be significant that the oncomiracidium is equipped with two pairs of pigmented eyes (Kearn, 1963; Kearn & Baker, 1973) and may also have ciliary photoreceptors (Lyons, 1972). Most work on hatching rhythms in monogeneans has been done on skinparasitic monogeneans from bottom-living flatfish. An isolated observation by Zeller (quoted by Bovet, 1967), in which he claims that hatching ofDiplozoon took place at 05.00 h, is the only indication that hatching rhythms may occur in gillparasitic polyopisthocotylinean monogeneans. Because of the lack of knowledge about hatching in gill parasites it was decided to make a study of hatching in three closely related parasites of the genus Diclidophora which at that time were reported to be common parasites of the gills of round-bodied gadoid fishes. A further point of interest is that the oncomiracidia of these species have no pigmented eyes (Llewellyn, 1957) in contrast to entobdellid monogeneans and polyopisthocotylineans such as Diplozoon. The three species studied here were Diclidophora merlangi (Kuhn, in Nordm., 1832) from the gills of the whiting Merlangius merlangus (L.), D. luscae (van Beneden & Hesse, 1864) from the pouting Trisopterus luscus (L.) and D. denticulate/, (Olsson, 1876) from the coalfishPollachius virens (L.).

MATERIALS AND METHODS

Sources of infected fish

Because of previous unpublished reports that D. merlangi was common on whiting caught in the English Channel parasites were initially collected at Plymouth. The infection rate in the area, however, was found to be low and has remained at 1-5 % amongst both inshore and offshore fishes for the last three years (1971-3). Whiting from the inshore waters off the north-east Coast of Scotland were found to be more heavily infected (about 60 % in 1973-4) and so the majority of experiments was performed using parasites from this region. Fish 20-35 cm (fish measured to tip of tail) were landed at Arbroath 80 km south of Aberdeen. Specimens of D. luscae were obtained from Plymouth pouting where the parasite is still common; infected hosts varied in length from 20 to 25 cm. D. denticulata was collected from coalfish 40-50 cm long landed at Fraserburgh (80 km north of Aberdeen) and from fish 25-40 cm in length from Robin Hood's Bay, Yorkshire. The incidence of D. denticulata on coalfish off the Yorkshire coast has dropped from over 50 % in 1970-1 to 8-10% in 1974 (Mr G. Boxshall, personal communication). The incidence of this species in Scottish waters was not measured accurately since the worms had to be collected quickly during the public auction of fish but approximately 50 % were found to be infected.

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Collection and maintainance of eggs Parasites were sent to Norwich in vacuum flasks of cooled, filtered sea water and continued to lay eggs for 3-7 days after removal from their hosts (at 10-13 °C). The eggs comprising each batch were laid within a 3-day period. The batches of eggs were cultured in ' Perspex' dishes (9 mm internal diameter) each fitted with a perspex handle and immersed in glass crystallizing vessels (5 cm diameter) containing filtered Plymouth sea water. The vessels were covered to prevent evaporation and stored in a refrigerated incubator housed in a constant temperature room. Both room and incubator were maintained at the same temperature to avoid temperature changes when opening the incubator door. Environment for incubation Attempts were made to simulate natural conditions in the experimental environment for incubation. The eggs of all three species were assumed to fall to the sea bed (see below). Eggs were incubated at 13 °C as this temperature falls within the 6-16 °C range of bottom temperatures in areas of the North Sea where parasites were collected (International Council for the Exploration of the Sea, 1965) and within the 10-14 °C range found at 50 m in the English Channel (Armstrong & Butler, 1962). Llewellyn (1957) found that eggs of D. merlangi failed to develop fully at temperatures of 8 °C and below. All experiments were run at 13 °C for purposes of comparison. Ideally each batch of eggs should have been incubated at a temperature specific to locality and season together with controls at a standard temperature but this was not possible because of lack of sufficient material and because of practical difficulties. The eggs were exposed to light from a fluorescent tube and the spectral quality and intensity of illumination were adjusted in order to simulate, as closely as possible, conditions on the sea bed. Since the host fishes can be found at depths similar to those at which soles normally live (Lythgoe, 1971), lighting of the same intensity and spectrum as that used by Kearn (1973) was employed and this work should be consulted for full details of the apparatus used. Briefly, the light from the fluorescent tube passed through a bath of saturated copper sulphate solution which produced a quality of light similar to that at 50 m depth. The copper sulphate solution also acted as a heat filter. The intensity of light reaching the eggs was then reduced to between 60 and 100 nW/cm2 by five No. 55 ' Cinemoid' neutral density filters mounted below the copper sulphate bath. Batches of eggs were incubated in alternating 12 h periods of light and darkness (LD 12:12); the light came on at 09.00 h (GMT) and was switched off at 21.00 h by means of a time switch. Recovery of free-swimming larvae The oncomiracidia are ciliated and, on hatching, swim freely in the crystallizing vessel. The number which emerged during any particular time interval was assessed by transferring the perspex dish containing the eggs to a crystallizing vessel of fresh sea water, adding a few drops of 10 % formaldehyde to the water in the vessel 14

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Fig. 1. Recovery of larvae of Diclidophora merlangi at 2 h-intervals from eggs collected from Arbroath whiting in (A) June 1973 and (B) October 1973. LD 12:12 at 13 °C. Panels indicate periods of light (stippled) and darkness (black).

containing the larvae, and counting the larvae. Some of the oncomiracidia sank to the bottom of the vessel but many were trapped in the surface film. It seemed possible that in some cases a few larvae were transferred from one crystallizing vessel to another. To test this the perspex dish was, on three occasions, transferred to fresh sea water twice in rapid succession. The majority of larvae was recovered after the first transfer and the number carried over to the second vessel was found to be small. During the experiments, once hatching began, larvae were recovered and counted every 2 h (e.g. 09.00 h, 11.00 h, etc.). RESULTS (1) Fate of eggs

In the present study it was assumed that the eggs of the three species of Diclidophora fall to the sea bed although there is little direct evidence for this. I have examined the skin, gills and opercula of 100 whiting and only rarely have found eggs of D. merlangi adhering to the gill mucus of freshly killed fish. The eggs of D. merlangi are laid singly. If they are placed just below the surface of a 2 1 measuring cylinder filled with sea water at 13 °C they sink, even when the cylinder is gently rocked. Llewellyn (1962) found that eggs of Gastrocotyle trachuri sink in still water at 18 °C and his evidence suggests that, even in the turbulent waters of the sea, the eggs would reach bottom waters long before the end of their incubation period. Llewellyn also found the eggs of this parasite in the sediment from the floor of a

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Fig. 2. Summary of recoveries of larvae of Dielidophora merlangi from (A) Arbroath in June, (B) Arbroath in October, (C) Plymouth. The recoveries for each of the 12 daily 2 h-periods are summed and given as percentage recoveries. LD 12:12 at 13 °C. Panels indicate periods of light (stippled) and darkness (black).

tank containing infected fishes. In the present study over 50 freshly killed pouting were examined but no eggs of D. luscae were found attached to the host. Kearn (personal communication) has found egg bunches of D. luscae in the gravel at the bottom of an aquarium containing pouting at Lisbon. Cerfontaine (1895) claimed that the egg bunches of Dactylocotyle carbonarii (= Dielidophora denticulata) adhered to the gills of the coalfish but Frankland (1955) searched for, but never found, developing eggs of D. denticulata attached to any part of the host. (2) D. merlangi (a) Hatching in LD 12:12 Batches of eggs of D. merlangi were incubated at 13 °C in alternating 12 h periods of light and darkness. At this temperature hatching began after 30-34 days and continued for 3-4 days. Once hatching began the number of larvae recovered every 2 h was recorded. Experiments using parasites from Arbroath were carried out in June (498 larvae hatched) and in October (444 larvae). Fig. 1A shows the pattern of hatching of a single egg batch over a period of 62 h in June and Fig. 1B shows the pattern in October. A total of 942 oncomiracidia hatched during the course of 6 experiments (June + October) and of these 59 % hatched in the first 6 h 14-3

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of the illumination periods. During the periods of darkness only 15 % of all the larvae were recovered. In Fig. 2 A, B (A, June parasites; B, October parasites) the recoveries of larvae for each of the 12 daily 2 h periods have been added up for the whole of the hatching period and are given as percentage recoveries. It can be seen that in June 14-7 % of the larvae were recovered at the end of the 2 h period (19.00 h-21.00 h) before the beginning of the subjective 'night', while in October only 4-05 % were recovered in this period. In both months, therefore, hatching occurred mainly in the mornings and declined sharply during the hours of darkness but in June a second much smaller peak also occurred just before 'dusk'. Because of this second peak the pattern of hatching in June is statistically significantly different from the pattern in October (x2 = 72-51, P < 0-001). Experiments using eggs from parasites collected at Plymouth were performed in September, November and February. There was no evidence of different hatching patterns at different times of the year. Few eggs were available for study (total number of larvae recovered in all experiments was 142) and therefore daily recoveries of larvae for each of the 12 daily 2 h periods have been added together and the total percentage recovery of larvae at different times of day is given in Fig. 2C. The characteristic daily pattern of hatching for eggs from the two localities differed slightly. Hatching of eggs from Arbroath began after the light was switched on and large recoveries of larvae were made during the first 6 h of the light periods. Hatching continued throughout the illumination period and declined during the hours of darkness. Hatching of eggs collected at Plymouth also showed the same generally distinctive pattern, but with large recoveries of larvae in the final 2 h period of the subjective 'night'. (b) Effects of mechanical disturbance In view of reports by Ktari (1969) that in Microcotyle salpae mechanical disturbance provides a hatching stimulus, attention was turned to the possible role of mechanical disturbance in the hatching of D. merlangi. Samples were taken from hatching batches of eggs at 2 h intervals throughout the day and the night but there was no evidence that any larvae emerged as a result of this disturbance and the hatching patterns of these eggs were closely related to the light regime. As a further test, some of the batches of eggs were subjected to mechanical disturbance during the period between regular sampling times and also at times in the lighting regime when hatching was not expected to occur. This disturbance was effected by revolving and gently shaking the perspex egg dish in the sea water for 1 min. No larvae emerged and mechanical disturbance did not appear, therefore, to play a part in hatching in D. merlangi. (c) Effects of host shin mucus and gill tissue The percentage hatching success of eggs of D. merlangi from Arbroath was never high and of any particular batch of eggs no more than 60 % of the larvae emerged. Some eggs (about 10 %) failed to develop while the remainder developed normally

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but remained alive within their shells without hatching, occasionally for up to 80 days before dying. Many of these larvae developed clamps and the refringent droplets, characteristically abundant in the body of newly hatched larvae, gradually disappeared. These observations will be discussed more fully in relation to work on the morphology of the oncomiracidium (Macdonald, in preparation). In view of the low overall hatching success it seemed that light, while certainly influencing the timing of larval emergence, might not be the sole factor affecting hatching. Whiting mucus, scraped from the skin of a captive fish was added to a batch of eggs about 34 days old (hatching begins after 30—34 days at 13 °C) and also to a batch of eggs 60 days old including many larvae which had developed clamps in their shells. Mucus did not cause hatching. Fresh whiting gill tissue was added to eggs of similar ages but this did not elicit hatching. (d) Longevity of the oncomiracidium When oncomiracidia of D. merlangi (maximum 2 h old) were kept in sealed perspex containers filled with filtered sea water at 13 °C it was found that they could swim actively for 12—15 h. At 18 °C they swam actively for a minimum of 9 h, lost their cilia after 24 h and died within 30 h of hatching. The free-swimming period of the larva is, therefore, short. (3) Behaviour of the whiting (Merlangius merlangus) Kearn (1973) suggested that the pattern of hatching found in Entobdella soleae is related to the daily activity of the host. It seems possible that the hatching rhythm in D. merlangi might be related in a similar way to the behaviour patterns of its host. The whiting is of considerable commercial importance in Britain and certain aspects of its biology, e.g. feeding ecology, have been closely studied. Steven (1930), from a study of gut contents, concluded that whiting fed both on and off the bottom. Knudsen (1968), working with fish in Danish waters, found that the diet consisted largely of Crangon spp., Gammarus sp. mysids and clupeoids. Nagabushanam (1964) studying the food of whiting in Manx waters found that larger whiting were active predators preferring a fish diet but that at certain times slow moving or burrowing, benthic prey was also taken. Some work has been carried out specifically on vertical migration of this species (Bagenal, 1958; Parrish, Blaxter & Hall, 1964; Woodhead, 1964; Bailey, 1975) but the findings are based largely on trawl haul analysis and the results are conflicting. Trawl haul analysis as a method of sampling has been criticised by Jones (1956), and Saetersdal (1967), in his review of gadoid behaviour, lists the limitations of this type of analysis. Parrish et al. (1964) stress the importance of the behaviour of the fish in relation to the gear, e.g. fish may be more likely to see and so avoid or escape from a trawl in daylight than in darkness. They found from trawling experiments that average catches of whiting were greater at night than in daylight while Bagenal (1958) had found that the actual numbers of whiting landed in the daytime were higher than at night. Bagenal found no direct evidence for vertical

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migration using echo-location equipment. Woodhead (1964), trawling from dawn to dusk over a 3-day period, reported that the average length of the whiting caught tended to increase during the course of the day such that the smallest fish were caught in highest numbers in the first haul after sunrise. Hislop (personal communication), while hand-lining for whiting, noticed that the fish started to rise off the bottom towards dusk and could be taken near the surface during darkness, apparently returning to the bottom at day-break. Bailey (1975), again basing his results on trawl haul analysis, suggested that while 0-group whiting spent all their time in midwaters older fish migrated vertically. In view of the confusion in the literature and the insistence of authors that trawl haul analysis may give misleading results it seemed desirable to find another method of investigating possible vertical migratory behaviour by whiting. Initially attempts were made to catch whiting using vertical lines baited at regular intervals from sea bed to surface but lack of available boats for night work and strong tidal currents rendered these attempts unsuccessful. Finally experiments were performed using equipment housed at the M.A.F.F. Laboratory at Lowestoft. The apparatus consisted of a tube 6 m high with a diameter of 58 cm which had been built especially for work on vertical migration. The tube was filled with continuously circulating filtered sea water and the water temperature at the time of the experiment (October) was 12 °C. Six port-holes of 38 cm diameter were sited down one side of the tube at intervals of 53 cm. A skylight was situated immediately above the open top of the tube and the port-holes were covered with black curtaining so that a gradient of natural day light was formed through the water column. A false bottom was inserted to enable recovery of the fish after the experiment, and during observations this lay at the level of the bottom of the lowest port-hole. Nine whiting measuring 20-30 cm in length were captured by hand-lining off Lowestoft and were immediately transferred to the tube. These fish fell within the same length range as those from which parasites had been collected. After 20 h acclimatization time observations began. The fish were not offered food during the experiment. The positions of the fish in the tube were plotted each hour over a 53 h period and in addition observations were made every half hour over dawn and dusk periods. The fish were counted by shining a torch covered with No. 6 Primary Red 'Cinemoid' filter through each port-hole for as short a time as possible. It had been found in previous tests that whiting showed no detectable response to red light whereas they were attracted by white light from an uncovered torch. An attempt was made to estimate at each port-hole the number of fish in the cylinder of water ranging from 40 cm above the centre of each port-hole to 40 cm below it. I t was not always possible to determine the positions of all 9 fish at each observation time because the port-holes were built on one side of the tube only and so if a fish lay between port-holes on the same side as the observer it could not be seen. In spite of the inadequacies of the tube for observing fish some clear behavioural trends emerged. The results are shown in Fig. 3. The majority of the fish rose off the bottom at sunset (17.30 h) and descended again at sunrise (07.30 h). This activity pattern continued throughout the 53 h of study. On the second day, i.e.

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midway through the experiment, the natural high tide was at 10.06 h and the low tide at 16.15 h. No relationship between the behaviour of the fish in the tube and the tides was observed. (4) Relationship between hatching in D. merlangi and host behaviour As stated above eggs of D. merlangi collected at Arbroath and incubated in LD 12:12 hatched during the illumination period with large recoveries of larvae in the first 4-6 h after the beginning of the subjective ' day'; eggs of the same species collected at Plymouth also hatched during the day but the majority of larvae were recovered in the 2 h period immediately before the light came on. Since neither mechanical disturbance nor the proximity of host gill tissue or skin mucus was shown to have an effect on the hatching of the parasite it appeared that light was acting as the main cue for the rhythm. Under experimental conditions it was found that the majority of the whiting showed a marked pattern of vertical movement in relation to light; they remained close to the bottom during the day and rose to mid-waters and even to the surface at night. Not all fish tested reacted in this way; some remained close to the bottom at all times. It may have been that certain individuals adjusted less well to the change from a natural to the experimental environment, or were slightly damaged during capture. It is possible that in the natural environment only part of the whiting population undergoes vertical migration. No evidence was found of a persistent tidal rhythm but it must be emphasized that under the influence of tides in the sea the behaviour of the fish may be different. A criticism of the apparatus may be that since the diameter of the tube was small

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(58 cm) any exploratory movement by the fish would be, of necessity, vertical rather than horizontal. I t still remains clear, however, that the fish spent the daylight hours inactive near the bottom and were only active at night. Since it seems likely that the eggs of the parasite come to rest on the sea bed and that larvae have only a short free-swimming life it is important that hatching should take place at a time when the host is near the bottom. The observations on D. merlangi and its host indicate that this is so; the parasite hatches just before ' dawn' (Plymouth parasites) or soon after 'dawn' (Arbroath parasites) at the beginning of the period spent by the host on the bottom. Since the active swimming of the larvae is limited (at 13 °C) to the 12-15 h after hatching, a dawn hatching would initiate the optimum period of swimming for making contact with a host. The difference in hatching pattern found between eggs collected at Arbroath and those collected at Plymouth could perhaps be related to differences in the behaviour of whiting from the two areas. The whiting population found in British waters comprise several separate stocks (Hislop, 1972; MacKenzie, 1972). Those fish north and south of the Dogger Bank, for example, form two non-interbreeding stocks. The differences are reflected in the meristic characters, with fish in the southern North Sea having a lower vertebral count (Roessingh, 1959; Gamble, 1959), and differences are also found infinray numbers (Gamble, 1958). Williams & Prime (1966) and Hislop (1972) found that when tagged fish were released in the northern North Sea no recoveries were made from the south. Kabata (1963, 1967), using myxosporidian parasites of the gall-bladders of whiting as biological tags, found that distribution of infection demonstrated clearly that fish inhabiting the areas north of 56 °N and south of 54° N respectively, comprised two separate stocks with a small mixing zone near the Dogger Bank. It may be that two populations of whiting occur in the northern North Sea, one along the Scottish east coast and the other in the central offshore area (MacKenzie, 1972; MacKenzie & Wootten, 1973). All parasites collected at Arbroath came from inshore waters and, therefore, from the same population of whiting, but those collected at Plymouth certainly belonged to a separate stock of fish. The present study of vertical migration was made using fish caught off Lowestoft and so the results may relate more closely to findings using Plymouth worms. However, there remains an overall similarity in the hatching patterns of eggs of D. merlangi from both areas in that most larvae hatch during the day rather than at night. In June, eggs from Arbroath hatched with a peak in the first 4-6 h of the illumination period but with a second smaller peak just before the beginning of the subjective 'night'. This second peak was not obvious when eggs were tested in October of the same year. If this is a significant seasonal difference it may be related in some way to seasonal behaviour changes by the fish. Alternatively it may be an example of one of the major difficulties in using a population for the study of rhythms. It may be that within the population of D. merlangi there are at least two types of animal, one type, geared to hatch after 'dawn', and the other at 'dusk'. Such a form of balanced polymorphism may give maximum chances of host location to the population as a whole. I t could be that, by chance, a more

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Fig. 4. Recovery of larvae of Diclidophora luscae. (A) Recovery of larvae over a 3-day period. (B) The recoveries for each of the 12 daily 2 h-periods are summed and given as percentage recoveries. LD 12:12 at 13 °C. Panels indicate periods of light (stippled) and darkness (black). homogeneous group of animals was collected in October and so the presence of two types was not observed or possibly the ratio of the two types changes seasonally. (5) D. luscae (a) Hatching in LD 12:12 Batches of eggs of D. luscae were exposed to alternating 12 h periods of light and darkness at 13 °C. The lighting regime and methods of sampling were as previously described. At 13 °C hatching began after 32-36 days. Experiments were carried out in January, March, May, July and October. A characteristic daily hatching pattern emerged. Fig. 4 A shows the recovery of larvae from a single batch of eggs over a 3-day period and the combined results of 8 experiments (total of 1152 larvae) are given in Fig. 4B. Little hatching occurred until 17.00 h and the majority of larvae (78-5 %) were recovered between 17.00 h and 23.00 h after which hatching declined sharply. Most larvae, therefore, were recovered over the 'dusk' period (light off 21.00 h). There was no evidence of seasonal changes in the hatching pattern. In these experiments with D. luscae more than 90 % of all eggs tested hatched. (b) Effects of stimuli other than light No evidence was obtained to suggest that mechanical disturbance of the eggs played a role in the hatching of the larvae of D. luscae.

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Table 1. Prey species from the stomachs of 67 pouting Polychaeta Aphrodite aculeata L. ? Nereid Decapoda Galathea dispersa, Bate Portunus depurator (L.) Cardnus maenas (L). Macropodia rostrata (L.) Crangon vulgaris, Fabrieius. Palaemon serratus Pennant Pandalidae probably Pandalina brevirostris (Rathke) Teleostei Callionymwi sp. Small Gadidae (not identifiable) Bell (1853) and Plymouth Marine Fauna (1957) were used for identification of species.

The effect of the proximity of host tissue on the eggs was tested. Mucus and gill tissue from the host fish were added to sea water containing eggs 35-37 days old. Some tissue had been deep frozen and was subsequently thawed and cooled to 13 °C before being added to the eggs; some tissue was fresh. The mucus and gill tissue were placed with the eggs at times in the illumination cycle when least hatching was expected, i.e. during the early part of the light period. Hatching was not stimulated by mucus or by gill tissue from the host. (6) Behaviour of the pouting, Trisopterus luscus The pouting is of limited economic importance and its ecology has been little studied. It is considered by fishermen to be a bottom-living fish found most frequently around wrecks and rock outcrops, and, as Godsen (1880) pointed out, the presence of a barbel suggests a benthic way of life. Chevey (1929) reported that the fish are pelagic until they reach a length of 18-20 mm when they move to deeper waters, remaining within a few metres of the bottom for the rest of their lives. Steven (1930) and Wheeler (1969) dealt briefly with the diet of the species but gave no information on feeding patterns. Steven found that they feed largely on crustaceans and Wheeler reported that fish under 20 cm in length feed mainly on Crangon sp., Pandalus sp. and shore crabs while the diet of adult fish had been little studied. No information could be found on daily activity patterns of the fish. In the present study the stomach contents of 67 pouting caught off Plymouth and varying in length from 15 to 40 cm were examined. No change in the diet could be found with different size groups of fish. Table 1 gives a list of organisms found and it appeared from the study that the diet of pouting consisted largely of benthic or demersal prey. There was no difference in the diet of fish infected or uninfected with D. luscae. Experimental studies of vertical migration of pouting were not possible because all fish captured alive subsequently died after a few hours in the aquarium at

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Norwich. A shoal of 30 small pouting was, however, successfully transferred to a tank in the public aquarium at the Plymouth Marine Laboratory following capture in the English Channel. The fish were about 12 cm long and the tank in which they were housed was 2 m in length with a height of 1-2 m and a width of 1 m. The lights in the aquarium are gradually dimmed at dusk but are not completely extinguished at night so that it is just possible to see the fish in the tank. At daybreak the light intensity is slowly increased. The positions of the fish were recorded at regular intervals throughout the day and night for 70 h. The fish remained close to the bottom of the tank during both the day and the night. They retained a tight shoal formation until dusk when the shoal dispersed. The fish scattered but still remained near the bottom throughout the night, shoaling again 2-4 h after daybreak. Only a small amount of upward movement occurred and this was at daybreak with individual fish making short excursions to the surface before quickly returning to the shoal. The fish used in this experiment were smaller than those from which parasites were collected and the observation tank was also small. It seemed clear, however, that the fish remained close to the bottom at all times but that during daylight shoaling occurred whereas at night the shoal dispersed and the fish scattered over the bottom. (7) Relationship between hatching in D. luscae and host behaviour When eggs of D. luscae were incubated in LD 12:12 at 13 °C the majority of larvae were recovered over the 'dusk' period with 78-5% of all larvae being recovered between 17.00 h and 23.00 h. Since neither mechanical disturbance nor the proximity of host tissue appeared to influence hatching it seemed that light alone provided the cue for rhythm formation. Analysis of the gut contents of adult pouting suggested that they feed almost exclusively on benthic or demersal prey. The observations carried out at the Plymouth aquarium on the behaviour of pouting demonstrated that the fish remained close to the bottom at all times, retained a tight shoal formation during the day and scattered over the bottom at night. The eggs of D. luscae are, as previously stated, assumed to fall to the sea-bed where hatching takes place. It seems that pouting would always be open to infection since they remain close to the bottom at all times. However, if the eggs are scattered over the sea bed, e.g. by currents, it might be of value to hatch at a time when the hosts are also dispersed over the sea-bed. (8) D. denticulata Hatching in LD 12:12 Batches of eggs of D. denticulata were incubated at 13 °C in LD 12:12. At this temperature hatching occurred after 20-22 days. The pattern of larval recovery is shown in Fig. 5A, B. In the course of 2 experiments 404 larvae hatched and of these 93-6 % were recovered during the first 4 h of the dark periods. Over 90 % of all eggs collected hatched successfully.

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Pig. 5. Recovery of larvae of Diclidophora denticulate,. (A) Recovery of larvae over a 2-day period. (B) The recoveries for each of the 12 daily 2 h-periods are summed and given as percentage recoveries. LD 12:12 at 13 °C. Panels indicate periods of light (stippled) and darkness (black).

(9) Behaviour of the host, Pollachius virens According to Wheeler (1969) immature coalfish are common just offshore where they feed on crustaceans and such fishes as sand eels, gobies and herrings, while mature fish are found in mid-waters feeding almost entirely on fishes. Schmidt (1955) working in Norwegian and Icelandic waters studied the daily activity patterns of coalfish by means of trawl hauls taken over periods of 24 h with additional evidence from echo sounder observations. He found that small and middle-sized fishes, e.g. < 85 cm in length, spent the hours of darkness on the sea bottom ascending to higher waters at day break. At dawn fish from 85 to 125 cm in length descended to the bottom having spent the night in mid-waters. Wagner (1959), working on trawl haul analysis in the northern North Sea, also reported a predominance of large fish in daylight catches but he caught both large and small fish in the trawl at night. In the present study parasites were collected at Fraserburgh from fish of 40-55 cm and in Yorkshire from fish of 25-30 cm. Frankland (1955), studying D. denticulata at St Andrews, examined coalfish of about 10-40 cm. The fish from which parasites have been collected fall, therefore, within Schmidt's smaller size group and so probably spend the night on the sea bed and the day-light hours in mid-waters.

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(10) Relationship between hatching in D. denticulata and host behaviour When eggs of D. denticulata were incubated in LD 12:12 at 13 °C the peak of hatching occurred after the light was turned off at 21.00 h. Of all eggs tested 93-6 % hatched between 21.00 h and 01.00 h, i.e. in the first 4 h of the dark period. Little is known of host behaviour but according to Schmidt (1955) smaller coalfish (those < 85 cm which includes all fish from which parasites were collected) spend the night on the sea bed and day-light hours in mid-waters. It may be that once again the hatching behaviour of the parasite is adjusted to the characteristic behaviour pattern of the fish so as to increase the chances of larvae successfully finding a host. In view of Schmidt's observations on the different behaviour of the two size groups of coalfish it would be of interest to study hatching rhythms of D. denticulata collected from fish greater than 85 cm in length, though of course it is possible that the invasion of coalfish by D. denticulata is restricted to small hosts. (It was found by Frankland (1955) that young coalfish are invaded by D. denticulata, but nothing appears to be known about the possible invasion of old hosts.)

DISCUSSION

It has been shown that in each of three species of Diclidophora there is a lighttriggered rhythmic pattern of hatching. In two of the species (D. merlangi and D. luscae) mechanical stimuli and chemical stimuli that may be provided by the presence of host mucus and gill tissue do not appear to influence hatching. The effect of such stimuli on eggs of D. denticulata was not investigated. The pattern of hatching in each species of parasite can be related to the behaviour of the respective host species. In D. merlangi hatching takes place before or soon after ' dawn' and in the present investigation the host whiting were found, under experimental conditions, to descend to bottom waters at this time. The eggs of D. luscae hatch over ' dusk' and although the host pouting were found to stay close to the sea bed at all times, shoaling was less concentrated during the hours of darkness. If it is assumed that the eggs of D. luscae are scattered over the sea bed it may be advantageous for the larvae to emerge when the hosts are also more widely distributed. Eggs of D. denticulata hatch after dark and according to Schmidt (1955) the host coalfish spend the night near the sea bottom. The survival value of hatching when the fish are near the sea bed is that in such a situation the specific hosts are most likely to come within the swimming range of the parasite larvae and so be vulnerable to infection. A corresponding situation has been shown by Kearn (1973, 19746) for some entobdellid skin parasites. Eggs of Entobdella soleae hatch at ' dawn' when the host soles come to rest on the sea bed after nocturnal swimming and those of E. hippoglossi hatch at ' dusk' when the halibut is thought to descend to the sea bottom. Thus it has been shown that in each of those situations investigated experimentally the behaviour of the monogenean larvae is adapted to certain aspects of the behaviour of the specific hosts. The egg shells of both E. soleae and E. hippoglossi, as in other monogeneans, are transparent. The larvae of both species are equipped with 2 pairs of pigmented

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eyes with lenses (Kearn, 1974c) and may also have ciliated photoreceptors (Lyons, 1972). There is no evidence, however, as yet to suggest that the pigmented eyes monitor the light and since there are no pigmented eyes in the three species of Diclidophora it would seem that they are not essential for hatching rhythm formation or synchronization. In Diclidophora spp. the light may be monitored by unpigmented photoreceptors but structures of this kind have not so far been reported in these species. Not all monogenean larvae respond to light. Acanthocotyle lobianchi, for example, will hatch only after contact with host mucus irrespective of lighting conditions (Macdonald, 1974). It seems likely, however, that hatching rhythms among monogeneans may be common phenomena, not restricted to any particular group of parasites or hosts. Such behavioural adaptations must have evolved alongside the more familiar anatomical changes, e.g. in adhesive organs, that have accompanied the development of host specificity among the monogeneans. I wish to thank Dr G. C. Kearn for invaluable help and discussion. I am grateful to the Director of the Laboratory of the Marine Biological Association at Plymouth for providing facilities and especially to Mr John Green who collected living parasites on many occasions and sent them to Norwich. I also wish to thank the Director of the M.A.F.F. Laboratory, Lowestoft for supplying equipment. Dr M. Pawson (M.A.F.F., Lowestoft) and Mr G. Boxshall (University of Leeds) kindly helped to collect material. I am much indebted to Mr Walter Cargill and his crew of the Family's Pride for regularly supplying fish from Arbroath. Thanks are due to Mr G. Cleveland for technical assistance. The work was carried out during tenure of a Science Research Council studentship.

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