Opisthorchis viverrini: Finding and recognition of the fish host by the cercariae

EXPERIMENTAL

PARASITOLOGY

Opisthorchis

71, 422-43 1 (I!%@

viverrini:

Finding and Recognition the Cercariae

W. HAAS, M. GRANZER,AND~. Institat

fiir

Zoologie I der Universitiit, *Department of Microbiology,

of the Fish Host by

R. BROCKELMAN*

Staudtstrasse 5, D-8520 Erlangen, Federal Faculty of Science, Mahidol University, Bangkok 10400. Thailand.

Republic of Germany; Rama Vi Road,

and

HAAS, W., GRANZER, M., AND BROCKELMAN. C. R. 1990. Opisthorchis viverrini: Finding Parasitology 71, 422431. and recognition of the fish host by the cercariae. Experimental The cercaria of Opisthorchis viverrini finds and recognizes its fish host by using at least four steps of behavioral patterns. (1) Dispersal and selection of plant-free water microhabitat are achieved by intermittent swimming behavior with positive phototactic orientation. (2) Attachment to the host is stimulated by water currents and a hydrophilic component of fish skin surface which has a molecular weight of more than 30,000. This component is sensitive to digestion with hyaluronidase and seems to be a glycosaminoglycan other than hyaluronic acid and chondroitin sulfates. (3) Remaining on the host’s surface is induced by an unknown chemical component of fish skin surface mucus. (4) Penetration into the host is triggered by a hydrophilic component of fish skin surface of a molecular weight of more than 30,000. This host signal has a proteinaceus character as it is sensitive to proteinase digestion but not to hyaluronidase and glycosidases. The requirement of 0. viverrini cercarix for complete glycosaminoglycans and proteins as signals in host identification may have the advantage that the numerous small molecules in mud and decaying materials in the water cannot interfere with host-finding. c IWO Academic press. hc. INDEX DESCRIPTORS: Opisthorchis viverrini. trematode; Liver fluke; Cercaria; Hostfinding, host recognition; Fish host; Fish skin surface. mucus; Swimming behavior; Attachment; Penetration; Host signals; Glycosaminoglycans: Proteins.

The human liver fluke, Opisrhorchis wiverrini, is one of the most important parasitic helminths in northeastern Thailand (Sadun 1955; Wykoff et al. 1965). In highly endemic areas the prevalence in humans reaches more than 90% (Upatham et al. 1982, 1984, 1985; Brockelman et al. 1987; Sithithaworn et al. 1988) and in the fish intermediate hosts prevalences of up to 97% at high infection intensities were found (Vichasri et al. 1982; Tesana ef al. 1985). But despite the high infection rates in human and fish host, the snail intermediate host, Birhynia siamensis, shows very low infection rates. In highly endemic waters in Khon Kaen Province Brockelman et al. (1986) found only 0.11% of 30,000 adult 422 0014-4894&O $3.00 Copyright G 1990 by Academic Press. Inc. All rights of reproduction in any form resewed.

snails to be infected and only 0.38% of 14,800 adult snails collected during this study shed 0. viverrini cercariae. This suggests that successful transmission to the fish host may require highly efficient mechanisms for finding and recognition of the fish by the cercariae. Thus far, studies on the host-finding behavior of the cercariae of 0. viverrini are lacking, and it is even not clear whether aquatic animals other than the few known cyprinoid fish species (Vichasri er al. 1982) are infected. The finding and recognition of a fish host by a cercaria or any other parasite has never been analyzed completely (Haas 1988, Haas and Ostrowski de Nudez 1988). We report here on the host-finding and host identification mechanisms of the cercariae of 0. viverrini. The cercariae identify their fish hosts with a

Opisthorchis

vilBerrini: CERCARIAL HOST-FINDING

particular sequence of behavioral steps during which they respond to different environmental and host signals. MATERIAL

AND METHODS

Parasites. 0. \Bit,errini-infected Bithynia siamensis were collected near Khon Kaen, NE Thailand, in waterholes and a natural pond close to a small village whose inhabitants were highly infected with 0. sliverrini. The snails with 0. viverrini infection were kept in aerated aquaria with mud as substrate and various water plants from the collecting site. Cercariae were collected by attracting them with illumination to one corner and pipetting them into beakers. S~~~imming and attachment hehu\,ior. Cercarial swimming behavior was observed in a Plexiglas cuvet (described by Haas 1976)through a horizontal dissecting microscope at 28-30°C. Experimental conditions for attachment experiments were the same as developed for Acnnthostomum bruuni cercariae by Haas and Ostrowski de Nudez (1988). Briefly. test tissues or substances incorporated into agar were coated on one end of a vertically fixed glass rod 170mm long. 6 mm diameter) and offered to the cercariae 20 mm above the bottom of the cuvet. Some attachment experiments were carried out by offering the dissolved test substrates through membrane filters (Millipore HABG, pore size 0.45 p.m) which were glued to the lower end of a vertically fixed glass tube. The substrates were illuminated through the glass rod or tube from above with a cold light source (KL 1500,Schott. Wiesbaden. FRGj at an irradiance of 5. I mW/cm’. The positively phototactic cercariae swam against the illuminated substrate and their attachment behavior was registered. Shadow stimuli were produced by placing a black plastic sheet for I set between the cold light source and the glass rod. Light intensity was increased by removing the sheet from the light beam. All irradiante changes reported in this paper were between I .2 mW/cm’ (light) and 0.02 mWlcm’ (dark). Water turbulence stimuli were created by moving a steel wire (0. I mm diameter) horizontally 3 mm below the lower surface of the glass rod. The responses of the cercariae after contact with a substrate were similar to those described for A. brcwni cercariae and the attachment responses were quantified as described by Haas and Ostrowski de Nufiez (1988). Repeated attachment to the substrate served as the criterion for effectiveness of agar substrates in stimulating attachment. In this response the cercaria stops swimming and attaches 2-5 times with the oral sucker. With animal tissue substrates, the cercariae often remained on the substrate after the first attachment. Therefore, a single attachment, where the cercaria remained at least 3 set on the substrate was also selected as a criterion. All cercariae which were at-

423

tached to the substrate for at least 3 set were considered as remaining on the substrate. Penetration experiments. Agar substrates containing animal tissue extracts or pure chemicals were poured into flat-bottomed wells (7 mm diameter) of Falcon 3072 tissue culture plates, 50 ~1 per well, and allowed to solidify. A suspension of 30-150 cercariae in 20 pl water was added to each well and the wells were sealed with adhesive plastic tape. After l-3 h of incubation at 30-35°C. the percentage of penetration, mortality, and tail shedding was determined using a dissecting microscope with dark-field illumination. Test substrates. Mucus from fish skin surface (Chcmna striatus or Carcrssius auratus) was obtained by gently rubbing the surface of a living or freshly killed fish. Snail mucus (Achutina fulica and Indopkmorbis exustus) was collected by stimulating the snails with an electric current (I.5 V. dc). The snails then withdrew into their shells while excreting mucus and haemolymph. Mucus samples were pooled and homogenized with an Ultra Turrax emulsifier (Type T 25, shaft 8N. IKA-Werk. Staufen. FRG) and stored at - 20°C. Fish tins and gills and frog skin were used for attachment experiments. To stop the activity of cells in tissues of freshly killed animals. the tissues were placed in plastic bags. sealed. and immersed in liquid nitrogen for 5 min. Lipophilic and hydrophilic fish skin surface extracts were prepared according to Folch et al. (1957) and processed as described in Haas and Ostrowski de Nudez (1988). Skin extracts. mucus. and pure chemicals were integrated into agar-solution (agar for electrophoresis. analytical grade. Serva, Heidelberg, FRG) with a final agar concentration of I% for the attachment experiments and 0.15% for the penetration experiments. Therefore. the agar-solution and the substrates (except mucus) were prepared in double concentration and each substrate was mixed with an equal volume of agar-solution at 50°C. The pH was adjusted to 6.5 (attachment experiments) or 7.0 (penetration experiments) with an equal amount of phosphate buffer (7-25 mM) for each experimental series. Modifications offish skin extract. Enzymatic digestions of both fish mucus and hydrophilic fish skin surface extract were carried out at 37°C in an incubationshaker. The mucus proved to be a more suitable substrate because of the higher cercarial penetration rate. The enzymes were purchased from Sigma, Deisenhofen, FRG. unless otherwise stated. Gentamicin (100 pg/ml) was added as a bacteriostat. The different incubation conditions modified the stimulating activity of the substrates considerably. Therefore three control substrates had to be run under identical incubation conditions, namely enzyme without substrate, substrate without enzyme (“mucus similarly treated”. i.e. positive control), and buffer only.

424

HAAS, GRANZER, AND BROCKELMAN

Separation of the substrates according to molecular weight was performed by centrifugal ultrafiltration using Centricon Kits (Amicon, Witten, FRG) with molecular cutoffs of 30,000 and 10,000. The filtrate was used as obtained whereas the residue was washed twice with distilled water to completely remove the smaller molecules. Polyanionic glycosaminoglycans were precipitated with Alcian blue (8 GS Standard Fluka, FRG) by methods modified after Whitehead (1978); 100 mg Alcian blue was dissolved in 100 ml 0.2 M sodiumacetate buffer (pH 5.8) containing 50 mM MgCI, x 2 H,O; 80 ul of this solution was added to 320 )I] of fish mucus and incubated for 2 h at 28°C. After centrifugation the supernatant was collected. EIecfric voltage grudienr. To create an electric gradient at the agar surface, chlorinated silver electrodes were fixed at a vertically mounted glass tube (Fig. 1). The stimulating electrodes were connected over two variable resistors to a I .5-V battery. The actual gradient was monitored with a digital microvoltmeter connected with a recording electrode and the electrode in the agar. RESULTS

Swimming behavior. 0. viverrini cercariae show a typical intermittent swimming mode. An active phase, in which a cercaria actively swims towards a light source, alternates with a passive phase in which the cercaria sinks. The positive phototactic be----50mm

---

--S&R

H

Chlorinated

H

lnsuloted

s11ver w,,e

silver electrode

0 R

Aw Recording

electrode

S

Stlmulatlng

electrode

FIG. 1. Experimental device to create a voltage gradient at an agar substrate. The voltage gradient is applied with the stimulating electrodes (S) and monitored with the recording electrodes (R).

havior results in an accumulation of the cercariae close to the water surface. Cercarial swimming behavior was modified by switching the light intensity between 0.02 and 1.2 mW/cm2. An increase in light intensity during the passive phase had no influence on the duration of the passive phase (15.8 set, control 14.7 set, means of 50 observations) but it prolonged the subsequent active phase. Cercariae swam an average of 1.8 mm without light stimulation, but 6.6 mm with stimulation (means of 50 observations). Dark stimuli applied during the active phase promptly inhibited swimming movements, but stimulated the start of a swimming movement when given during the passive phase. The length of these swimming movements was shortened, however, when the dark stimulus was extended. Swimming movements started by a OS-set shadow stimulus had a length of 6.0 * 1.9 mm, similar to spontaneous swimming movements (5.9 + 2.2 mm), whereas those triggered by a permanent decrease of light intensity were only 1.4 t 0.9 mm long (N = 50). The cercariae adapted to shadow stimuli and did not respond to repeated shadowing. Dark stimuli (1 set) applied in the passive phase stimulated swimming in 22% of the cercariae when the stimulus interval was 30 sec. Higher percentages of cercariae responded when the interval was extended. Results from 40 stimulations revealed 38% for 1-min, 58% for 2-min, and 69% for 5-min intervals. When shadow-stimulated cercariae touched a substrate, they did not show more attachment responses than the nonstimulated ones (12.3% attachments, control 15.7%, percentages of 300 contacts each, P = 0.08). Water turbulence applied during the passive phase also stimulated swimming movements. When such cercariae touched a substrate they tended to attach to it (38.7% attachments, control 15.7%, percentages of 300 contacts each, P < 0.001). Attachment and remaining. When spon-

Opisthorchis viverrini: CERCARIAL HOST-FINDING taneously swimming cercariae encountered fish skin surface or gills they tended to attach to these substrates. The responses to the endemic fish host Puntius leiacanthus were similar to those to C. auratus or Ch. striatus. The cercariae readily attached to living fins and gills of fish and to frog skin surface, but not to the inside of frog skin (Table I). They remained and crept on living fish fins and gills and some penetrated these substrates. But the fish substrates lost a great deal of their effectiveness in stimulating attachment and remaining when they were modified by killing the cells. Cercariae could distinguish between living and dead fish skin. This was probably not the result of electrical properties of the fish skin. as an electrical voltage gradient (50 mV/mm) had no effect on cercarial attachment to fish skin surface mucus substrates: The rate of repeated attachments increased insignificantly from 30.6% to 35.3% when the mucus was positively charged and to 31.8% when negatively charged (600 con-

425

tacts each, P = 0.09 and 0.5). Also the remaining rate was not altered by the voltage gradient: 20.3%) at positively charged and 20.0% at negatively charged mucus, control 15.8% (600 contacts each, P = 0.07 and 0.1). Carbon dioxide and bicarbonate content were also not decisive, as freshly buffered NaHCO, solutions (10 mM) did not stimulate more attachment responses to fish skin surface mucus (Table II A). As the pH of the skin surface of fish (Ch. striates and C. auratus) increased from 6.5-7.5 to 7.5-8.0 after death. we investigated the effect of the pH of fish skin mucus on cercarial attachment (Table II B). The effectiveness of the mucus in stimulating attachment decreased with increasing pH. This could have been responsible for the weaker attachment response to dead fish fins and gills. The host signal which stimulates attachment was a component of fish mucus. Mucus from the snails A. firlica and 1. esustus had no effect. The signal proved to be a

TABLE 1

Responses of O~~isrhorchis

t,ir,errini

Cercariae

.4ttachments” tci of contacts)

Puntitrs Fin Gill Agar

/~iacc~rA~s

(control)

after

Spontaneous

Contact

with

Animal

Remaining (% of attachments)

Tissues

P for attachments” (x2 test)

(dead) 18.2 SO.’ I.S

3.5 8.3 0

34.5 9.0 76.0 51.1 1.5

77,s 16.7 95.7 48.8 0

30.5 40.6 3.8 0.2

14.7 14.3 21.0 0


Carassius atirutus Fin. Fin. Gill. Gill, Agar

living dead living dead (control)


Ronu tigrinct Skin outside dead Skin outside living Skin inside living Glass (control)

Note. Percentages of 400-600 cercarial contacts (4-6 replicates) each. Living itated animals; dead tissues were incubated for 5 min in liquid nitrogen: Puntius for ? weeks. U Repeated attachments + single attachments with remaining. ’ P for difference between adjacent values.


are from freshly decapwas stored at - 20°C

leiucanthus

426

HAAS,

GRANZER,

TABLE II Attachments of Opisthorchis viverrini Cercariae Agar Containing Fish Skin Surface Components Repeated attachments (%) Test A Fish mucus (Carassius Mucus control

AND

to

P

Control

(x? test)

auratus) with carbonate

solution

13.5

1.8


9.3

2.9


0.6 Mucus + NaHCO, B pH of fish mows pH 6.5

(Cnmsrius

aurarus)

36.9

1.0


pH 7.0

13.3

3.0


pH 7.5

12.8

3.1



0.7 C Hydrophilic

and lipophilic

fish skin surface extracts

(Cyprinus corpio) Hydrophilic extract Lipophilic extract Mixture of both extracts D Ultrafiltration Crude mucus

11.9 3.3

2.6 2.6


13.2

2.6


of fish mucus (Carassius auratus) 17.5 2.1
MW > 30,000

28.4

2.1

10.001

MW < 30,000

2.5

2.1

0.5

0.4 I.2 I.0

0.6 0.6 0.6

0.1

2.9

3.5

0.4

39.9

3.5



0.5 0.3

Nore. Percentage attachments of 100&1400 cercarial contacts (l&l4 replicates). Controls were buffer substrates. Concentrations: mucus. 5m%; hydrophilic skin extract, 20 mg/ ml; lipophilic skin extract, 5 mglml; pore chemicals, I mg/ml. pH of the substrates: (A) 7.0, (C-E) 6.5 by 15-25 mM phosphate buffer. ” Chondroitin sulfate, Type A from whale cartilage (Sewa). ’ Chondroitin sulfate, Type B from porcine skin (Sigma). ( Chondroitin sulfate. Type C from shark cartilage (Sigma). ’ Mucin. Type I-S from bovine submaxillary glands (Sigma). c Hyaluronic acid from human umbilical cord ISigma)

hydrophilic component (Table II C) which had a molecular weight of more than 30,000 (Table II D). Indeed, pure chemicals with lower molecular weight, such as amino acids, carbohydrates, and electrolytes, were without any effect (data not shown), as were chondroitin sulfates, mucin, and hyaluranic acid (Table II E). The attachment stimulating component of fish mucus was

BROCKELMAN

resistant to digestion with pronase E, trypsin, neuraminidase, and chondroitinase ABC, but it was sensitive to a weak digestion with hyaluronidase (Table IV A). After attachment the cercariae remained and crept well on living fish skin. But on agar substrates they remained only when these contained dense unhomogenized fish skin surface mucus. The remaining response was stimulated by chemical properties of fish mucus, but not by the pure chemicals tested or by a combination of fish skin mucus with carbonate solutions or with an electrical dc gradient (data not shown). Penetration. The cercariae penetrated fins of freshly killed fish (C. auratus, P. leiacanthus, Poecilia reticulata), but rarely the gills. Only a few penetrations occurred into frog skin and into dead fish fins. Agar substrates were penetrated by the cercariae when they contained body-surface mucus of fish or of an aquatic snail (1. exustus), but the mucus of a terrestrial snail (A. fulica) and fish serum were without any effect on penetration (Table III A). The penetration-stimulating component of fish skin surface was hydrophilic; lipids were without any effect (Table III B). The penetration-stimulating effectiveness of fish skin mucus increased with increasing pH (Table III C). This is in contrast to the attachment stimulating effectiveness which increased with decreasing pH (Table II B). At neutral and acid pH levels nearly all penetrating cercariae shed their tails, only 36% did so at pH 7.5. Therefore, penetration at neutral pH was considered physiologically normal and all penetration experiments were carried out at pH 7.0. Pure chemicals known to occur in fish skin and related substances, such as amino acids, carbohydrates, electrolytes, urea, ammonia, mucin, hyaluronic acid, and chondroitin sulfates, did not stimulate cercarial penetration (data not shown). As determined by ultrafiltration the penetration-stimulating component of fish skin

Opisthorchis

viverrini:

CERCARIAL

was in the fraction with a molecular weight of more than 30,000 (Table III D); we therefore suspected a glycoprotein or proteoglycan as a key component. Hydrophilic fish skin extract, however, retained its stimulatTABLE III Penetration of Opisrhorchis \ir*errini Cercariae into Agar Containing Animal Skin Surface Extracts and Their Components Penetrations 1%)

Test Control

P (XZ test)

A Skin surface mucus of snails and fish: fish serum Mucus of Achotincr 0.7 0.4 0.5 fdica Mucus of In&p/anorbis e.wstus 63. I” 0.4
Lipophilic extract

1.9

2.3

0.8

26.4

2.3



0.9 Mixture of both extracts

25.7

2.3


C pH of hydrophilic fish skin surface extract (Cyprintrs

TABLE

IV

Influence of Enzymatic Degradation of Fish Surface Components on the Attachment and Penetration Responses of Opisrhorchis klerrini Cercariae Cercarial responses (D/r) Digested

Control

P

IX’ test)

A Repeated attachments: digestion of fish mucus (Chanrrct striotm and Curnssitrs utmmts) 12.5 10.9 0.2 Pronase E” Trypsinh 20.1 19.2 0.6 23.3 20.4 0.2 Neuraminidase’ 5.0 21.9 10.000
carpio)

pH 6.5

11.9h

0.6

<0.001

pH 7.0

18.3h

1.9

10.001

pH 7.5

45.06

1.4



Complete extract

17.4

0


MW > 30.000

10.5

0


2.1 0.4

0 0

0.02 0.3

0.05
E Removal of polyanionic glycosaminoglycans by Alcian-blue treatment from hydrophilic skin extract (Cvprinus

427

HOST-FINDING

carpio)

Skin extract Alcian-blue treated Skin extract similarly treated

29. I

I.0


32.5

I.0

10.001

0.3

Note. Percentage of penetrations of 160-520cercariae (7-10 replicates). Controls are buffer substrates. Concentrations: Mucus 50-66%, hydrophilic skin extracts I5 mgiml, lipophilic skin extracts I mg/ml: pH of the substrates (except Cl 7.0 by IO-30 mM phosphate buffer. ‘I Other preparations of I. exxustusmucus stimulated penetrations only poorly and some were without any effect. ’ With increasing pH more cercariae penetrated without shedding their tails. The tail shedding rate of the penetrated cercariae decreased from 93% at pH 6.5 to 51% at pH 7.0 and 36% at pH 7.5.

Note. Percentage attachments of 1000-1400cercarial contacts I l&l4 replicates): percentage penetrations of 200-800 cercariae (7-l I replicates). Controls are fish surface components similarly treated as in the digestion assay. Enzyme controls had no effect. Concentrations: mucus 5&66%: hydrophilic skin extracts I5 mg/ml. Enzymatic digestions were performed with 300 ~1 tnncus. U Protease. Type XXV (Pronase El from Streptom.vces grisews. Incubation: I20 units, pH 7.5. 2 h. h Trypsin, EC 3.4.21.4. Type IX from bovine pancreas. Incubation: 1000units, pH 7.6, I h. ’ Neuraminidase. EC 3.2.1.18, Type V from Ckxtridium perfriingens. Incubation: 2.5 units, pH 5.0. 2 h. d Hyaluronidase. EC 3.2.1.35, Type I-S from bovine testes. Incubation: 2.5 units, pH 6.0. I4 h. ’ Chondroitinase ABC. EC 4.2.2.4. from Proteus dgnris. Incubation: 5.0 units. pH 8.0. I4 h. f a-Glucosidase, EC 3.2.1.20, from brewers yeast, Type IV. Incubation: 30 units, pH 6.8, 2 h. $ B-Glucosidase. EC 3.2.1.21, from almonds. Incubation: 30 units, pH 5.0. 2 h. ‘I a-Galactosidase, EC 3.2.1.22. from Escherichia co/i. Incubation: 2.5 units. pH 6.5, I5 h. ’ B-Galactosidase, EC 3.2.1.23. from E. co/i, Type VI. Incubation: 30 units. pH 7.3, 2 h. ’ t-Fucose-dehydrogenase, EC I. I. I. 122. from porcine liver. Incubation: 2.5 units, pH 8.0, I5 h. ’ Pepsin, EC 3.4.23. I, from porcine stomach mucosa. Incubation: 1000units, pH 2.0, 2 h. “’ Proteinase K (fungal) (Bethesda Research Laboratories). EC 3.4.23. I. Incubation: 60 units, pH 7.5, 2 h. n As d, but I3 mg skin extract with 590 units in 440 pl, pH 6.0. 2 h.

428

HAAS,

GRANZER,

ing activity after removal of polyanionic glycosaminoglycans (Table III E). Similarly, digestion with hyaluronidase and various glycosidases as well as with trypsin and pepsin did not reduce the penetration stimulating effect of fish mucus (Table IV B, C) or of hydrophilic fish skin extract (data not shown). Only digestion with pronase E and pronase K (Table IV B) eliminated the stimulating activity completely. The stimulating component therefore seems to be a protein which is not sensitive to pepsin and trypsin digestion. Hyaluronidase digestion increased the penetration-stimulating effect of fish skin surface extracts and mucus. This was not achieved by smaller cleavage products, as the active component could not be removed by ultrafiltration with a molecular cutoff of 10,000 (Table IV C). DISCUSSION

Swimming behavior. The cercaria of 0. viverrini shows a typical intermittent swim-

ming mode, as described for several other cercarial species (see Haas 1988 for a review). The swimming movements during the active phase are directed toward the source of light, usually upwards; cercariae thus tend to accumulate near the water surface. This may increase the likelihood of encounter with small cyprinoid fishes. Cercariae of other species such as Diplostomum spathaceum (Haas 1969) or Schistosoma spindale (Haas et a/. 1990) accumulate in midwater or near the water surface by the mechanism of the intermittent swimming alone without the aid of such “expensive” pigmented ocelli as occur in 0. Gverrini cercariae. Therefore, the very sensitive phototactic orientation in 0. viverrini cercariae may have a further function, for example, in the microhabitat selection. The snail hosts B. siamensis often burrow in the mud or hide in plants or underwater detritus. In such circumstances positive phototaxis may be more useful for

AND

BROCKELMAN

the cercariae in order to reach the plantfree midwater or water surface than simply negative geotaxis alone. Light and shadow stimuli influence the active and passive phases independently, in a way similar to that described for the fishpenetrating D. spathaceum cercaria (Haas 1969). As in that species, the association of these responses with host-finding is not obvious. A sudden shadow stimulus applied during the active phase inhibits active swimming in 0. viverrini cercariae and in many other cercarial species. This response might serve to prevent the fast-swimming cercariae from colliding with other obstacles. A shadow stimulus during the passive phase starts a swimming movement. This might increase the chance of contact with a fish host. But the fact that the swimming movement itself is inhibited by the same shadow stimulus and is prolonged by light stimuli (at least in D. spathaceum, Haas 1969) has no obvious advantage in terms of host-finding. Also, that shadow-stimulated cercariae did not tend to attach to miscellaneous substrates seems incompatible with a role of these responses in host-finding. But it is interesting that both species of fish infecting cercariae. 0. viverrini and D. spathaceum, although taxonomically unrelated, have evolved similar behavioral responses to light and shadow stimuli. 0. viverrini cercariae respond to water currents and touch during the passive phase by initiating long swimming movements during which they tend to attach to any substrate encountered. This behavior has also been described for the cercariae of D. spathaceum (Haas 1974a) and clearly may be attributed to host-finding. Attachment to the host. 0. viverrini cercariae attached better to living than to dead fish fins. This was perhaps due to changes in the pH of the skin surface after the death of the fish, as carbonate solutions and an electric voltage gradient similar to that in fish skin were without any effect. The attachment stimulating component of fish

Opisthorchis viverrini: CERCARIAL HOST-FINDING skin surface mucus and skin extracts had a molecular weight of more than 30,000 and was not inactivated by either protease or neuraminidase digestion. But digestion by hyaluronidase, which acts as an endoN-acetylhexosaminidase and hydrolyses the PI-4 links to glucuronic acid (Beeley 1985) inactivated the stimulating component completely. As components of fish skin surface mucus containing glycosidic pl-4 links to glucuronic acid, van de Winkel et al. (1986) identified hyaluronic acid and chondroitin sulfates. But in our study chondroitinase ABC digestion did not reduce the attachment stimulating activity of fish mucus, and neither chondroitin sulfates A. B, and C nor hyaluronic acid had any attachment stimulating effect. Thus, the attachment stimulating host signal seems to consist of glycosaminoglycans other than hyaluronic acid and chondroitin sulfates, which still have to be identified in fish mucus. Their composition may be in the same category as the unidentified hyaluronidase-sensitive glycosaminoglycans found in the gill mucus of carp (Wasserman et al. 1972). Host signals that stimulate attachment by cercariae have been identified for three fish-penetrating parasite species. CO2 + H&O, stimulate the attachment of D. spathaceum cercariae (Haas 1974a, b, 1975); CO*, H,C03, and HCO,- in combination with the pH trigger the attachment of Isthmiophoru melis cercariae (Motzel and Haas 1985); the attachment response of cercariae of A. bruuni is stimulated by glycoproteins with N-acetylneuraminic acid as the effective residue (Haas and Ostrowski de Nufiez 1988). It is surprising that the factors which trigger the attachment of 0. viverrini are very different from those of A. bruuni. These two species are very similar in their cercarial gross morphology, pipe form resting posture, swimming behavior, and attachment response. This situation is similar to that found among schistosome cercariae (Haas et al. 1990)-closely re-

429

lated species may identify their hosts using very different strategies, even though they infect the same host genera. Remaining on the host. The stimuli for cercariae to remain on the host after attachment could be characterized only as chemical components of fish skin surface mucus. All pure chemicals tested, as well as fish mucus in combination with carbonate solutions and an electric voltage gradient, failed to stimulate the remaining response. Obviously, the cercariae remain only on surfaces which contain this mucus component in a dense layer. In S. munsoni cercariae, the remaining response is stimulated by host cues other than those that stimulate attachment, creeping on the host, and penetration (Woicke and Haas 1989). and this may be true also for 0. \i\yerrini cercariae. Penetration. The penetration stimulating host signal proved to be a hydrophilic component of fish skin surface with a molecular weight of more than 30,000. It was not sensitive to removal of carbohydrate components or to digestion by various glycosidases. pepsin, or trypsin. As it was inactivated only by pronase digestion, we consider it to be a protein, although not sensitive to pepsin or trypsin digestion. Hyaluronidase digestion of mucus, which inactivated the attachment stimulating component, increased the penetration stimulating activity of the mucus and this was not achieved by small cleavage products. This effect may have been due to the liberation of stimulating proteins from covering glycosaminoglycans. Host skin proteins also act as penetration stimuli in the dog hookworm Ancyfostomu cuninum (Granzer 1989). But in A. cuninum penetration is also stimulated by serum proteins, whereas 0. tiverrini cercariae did not respond to fish serum with penetration. That 0. viverrini cercariae use complete glycosaminoglycans and proteins for fish host recognition may be to their advantage because small molecules of mud and decaying materials in water such as amino acids,

430

HAAS,

GRANZER,

AND

sugars, and electrolytes will not be mistaken as host signals. ACKNOWLEDGMENTS

This research was supported by the Deutsche Forschungsgemeinschaft (DFG) and the Deutsche Gesellschaft fur Technische Zusammenarbeit (GTZ), and was carried out at the Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand. We are greatly indebted to Dr. Paiboon Sithithawom, University of Khon Kaen, for his essential help in providing us with infected snails. We also thank Sukanya Ahmed (Bangkok) and Elisabeth Metzger (Erlangen) for technical assistance, and the staff of the Department of Microbiology, Mahidol University, for their help.

REFERENCES BEELEY, J. G. 1985. Glycoprotein and Proteoglycan

Techniques. Elsevier Science Publishers. Amsterdam. BROCKELMAN, W. Y., UPATHAM, E. S., VIYANANT. V.. ARDSUNGNOEN. S., AND CHANTANAWAT,

R.

BROCKELMAN

mische Invasionsstimuli. International Journal for Parasitology 4, 321-330. HAAS, W. 1975. Einfluss von CO, und pH auf das Fixationsverhalten der Cercarie von Diplostomum spathaceum (Trematoda). Zeitschrift fiir Purusitenkunde 46, 53-60. HAAS, W. 1976. Die Anheftung (Fixation) der Cercarie von Schistosoma mansoni. Zeitschrift fiir Parasitenkunde 49, 63-72. HAAS. W. 1988. Host finding-A physiological effect. In “Parasitology in Focus” (H. Mehlhom, Ed.), pp. 454-464. Springer-Verlag, Heidelberg. HAAS, W.. AND OSTROWSKI DE NUAEZ, M. 1988.

Chemical signals of fish skin for the attachment response of Acnnthostomum brauni cercariae. Parasitology Research 74, 552-557. HAAS, W.. GRANZER, M., AND BROCKELMAN, C. R.

1990. Finding and recognition of the bovine host by the cercariae of Schistosoma spindale. Parasitology Research 76, 343-350. MOTZEL, W., AND HAAS, W. 1985. Studies on the attachment response of Isthmiophora melis cercariae (Trematoda: Echinostomatidae). Zeitschriftfir Parasitenkunde 71, 51%5?6. SADUN. E. H. 1955. Studies on Opisthorchis viverrini in Thailand. The American Journal of Hygiene 62,

81-115.

1986.Field studies on the transmission of the human liver fluke, Opisthorchis viverrini, in northeast Thailand: Population changes of the snail intermediate host. International Journal for Parasitology 70,

SITHITHAWORN. P., TESANA, S., PIPITKOOL, V., KAEWKES, S., THAIKLAR, K., PAUPAIROJ, A., SRIPA. B.. AND PAIROJKUL, C. 1988. Frequency distribution of Opisthorchis viverrini in man. Proceed-

545-552. BROCKELMAN, W. Y.. UPATHAM, E. S., VIYANANT. V., AND HIRUNRAKS, A. 1987. Measurement of incidence of the human liver fluke, Opisthorchis viverrini. in northeast Thailand. Transactions of the Royal Society of Tropical Medicine and Hygiene 81, 372-335. FOLCH, J.. LEES, M., AND SLOANE STANLEY, G. H.

ings of the 14th Conference on Science and Technology of Thailand. Bangkok, 1988, 248-249.

1957. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497-509. GRANZER, M. 1989. Wirtsfindung und Wirtserkennung der infektiosen Larven des Hundehakenwurms Ancylostoma caninum. Ph D Thesis, Naturwissen-

schaftliche Fakultiiten, Universitat ErlangenNiimberg. HAAS, W. 1969. Reizphysiologische Untersuchungen an Cercarien von Diplostomum spathaceum. Zeitschrift 287.

fur

vergleichende

Physiologie

64, 254-

HAAS, W. 1974a. Analyse der Invasionsmechanismen der Cercarie von Diplostomum spathaceum I. Fixation und Penetration. International Journal for Parasitology 4, 3 1 l-3 19. HAAS, W. 1974b. Analyse der Invasionsmechanismen der Cercarie von Diplostomum spathaceum II. Che-

TESANA, S.. KAEWKES. S., SRISAWANGWONK, T., AND PHINLAOR. S. 1985. Distribution and density of Opisthorchis riverrini metacercariae in cyprinoid fish from Khon Kaen province. The Journal ofParusitology and Tropical Medicine Association of Thailand 8, 36-39. UPATHAM, E. S., VIYANANT, V., KURATHONG, S., BROCKELMAN. W. Y., MENARUCHI, A., SAOWAKONTHA, S., INTARAKHAO, C., VAJRASTHIRA, S., AND WARREN, K. S. 1982. Morbidity in relation to

intensity of infection in opisthorchiasis viverrini: Study of a community in Khon Kaen, Thailand. American Journal for Tropical Medicine and Hygiene 31, 1156-l 163. UPATHAM, E. S., VIYANANT, V., KURATHONG, S., ROJBORWONWITAYA, J., BROCKELMAN, W. Y., ARDSUNGNOEN, S., LEE, P., AND VAJRASTHIRA, S.

1984.Relationship between prevalence and intensity of Opisthorchis viverrini infection, and clinical symptoms and signs in a rural community in northeast Thailand. Bulletin of the World Health Organization 62, 45 l-461. UPATHAM, E. S., BROCKELMAN, W. Y.. VIYANANT, V., LEE, P., KAENGRAENC, R.. AND PRAYOONWI-

Opisthorchis

viverrini:

WAT, B. 1985. Incidence of endemic Opisfhorchis viverrini infection in a village in northeast Thailand. American Journai of Tropical Medicine and Hygiene 34, 903-906. VAN DE WINKEL, J. G. J., VAN KUPPEVELT. T. H. M. S. M., JANSSEN, H. M. J., AND LOCK,

R. A. C. 1986. Glycosaminoglycans in the skin mucus of rainbow trout (Salmo gairdneri). Comparative Biochemistry and Physiology 858, 473475. VICHASRI, S., VIYANANT, V., AND UPATHAM. E. S. 1982. Opisthorchis viverrini: Intensity and rates of

infection in cyprinoid fish from an endemic focus in northeast Thailand. Southeast Asian Journal of Tropical Medicine and Public Health 13, 138-141. WASSERMAN, L.. BER, A., AND ALLALOUF, D. 1972.

Acidic glycosaminoglycan composition of the gills

CERCARIAL HOST-FINDING

431

of Cyprinus carpio. Comparative Biochemistry and Physiology 42B, 669-617. WHITEHEAD, D. L. 1978. Application of a rapid method for determining glycosaminoglycans in mucus secreted by an aquatic pulmonate Biomphalaria glabrata.

Comparative

Biochemistry

and Physiol-

ogy 59A, 27-30. WOICKE. E., AND HAAS, W. 1989. Schistosoma mansoni: Stimuli der menschlichen Haut fiir die Verbleibreaktion der Cercarie. Verhandiungen der Deutschen Zoologischen Gesellschaft 82, 250. WYKOFF, D. E., HARINASUTA, C., JLJTTIJUDATA, P., AND WINN, M. M. 1965. Opisthorchis L’il*errini in

Thailand-The

life cycle and comparison with 0. of Parasitology 51, 207-214. Received 10 October 1989; accepted with revision 15 April 1990 felineus.

The Journal