Parasitol Res (1993) 79:416-420

Parasitology Research 9 Springer-Verlag 1993

Physico-chemical conditions necessary for the in vitro excystment of Zygocotyle lunata (Trematoda:Paramphistomatidae) S.W.B. Irwin 1, Marian B. O'Kane 1, B. Fried 2 1 Department of Biological and Biomedical Sciences, University of Ulster, Shore Rd., Newtownabbey, Co. Antrim, Northern Ireland, BT37 OQB 2 Department of Biology, Lafayette College, Easton, Pennsylvania 18042, USA Received: 15 December 1992/Accepted: 15 March 1993

Abstract. Various physico-chemical factors associated with the in vitro excystment o f metacercariae o f Zygocotyle lunata were studied. Metacercariae could excyst with or w i t h o u t the presence o f trypsin or bile salts, a l t h o u g h the rate o f excystation was slower in the absence o f both. C r u d e ox-bile extract p r o m o t e d faster excystment t h a n did m o r e refined s o d i u m taurocholate. P r e t r e a t m e n t o f cysts with pepsin also resulted in faster excystment rates. O p t i m a l excystation o c c u r r e d at 4 0 ~ and between p H 5.5 a n d 7.5. The b r o a d p H a n d t e m p e r a t u r e ranges and the non-specific enzyme/bile salt requirements for excystation were correlated with the wide range o f vertebrate hosts used by this trematode.

This investigation was particularly a p p r o p r i a t e for Zygocotyle lunata, as Willey (1941) has stated that adults occur naturally in various hosts such as deer, cows, sheep, rats a n d n u m e r o u s waterfowl. The ability o f this t r e m a t o d e to establish in digestive systems with widely differing physiological characteristics w o u l d indicate t h a t metacercarial excystment m i g h t be induced by various non-specific factors. Fried et al. (1978) studied the in vivo a n d in vitro excystation o f Z. lunata metacercariae a n d p r o v i d e d some histochemical observations on the cyst. R o b b i n s et al. (1979) p r o v i d e d additional light m i c r o s c o p y and some transmission electron m i c r o s c o p y studies o f the excysted metacercariae. B o t h o f these studies used a complex three-step excystment p r o c e d u r e including an acid-pepsin pretreatment, the r e d u c t a n t s o d i u m dithionite a n d a final t r e a t m e n t in a t r y p s i n - s o d i u m glycocholate m e d i u m at an alkaline pH. Irwin et al. (1984) rep o r t e d a simple one-step p r o c e d u r e to excyst the metacercariae o f Himasthla leptosoma (Echinostomatidae), and in the present study a n u m b e r o f aspects o f that process were varied a n d applied to the metacercariae

Correspondence to: S.W.B. Irwin

o f Z. lunata so as to examine the range o f physical and chemical requirements for excystment in Z. lunata.

Materials and methods Cercariae of Zygocotyle lunata were obtained from naturally infected planorbid snails, Helisoma trivolvis (see Fried 1970). When the cercariae were released from the snails, they were allowed to encyst on cellophane and were then stored at 4~ in saline for up to 2 months prior to their use in these experiments. An artificial medium developed by Irwin et al. (1984) was used in the excystment experiments. It consisted of 5 ml of bicarbonate saline [0.8% (w/v) sodium, chloride and 1.5% (w/v) sodium bicarbonate] containing 0,8% (w/v) sodium taurocholate and 0.3% (w/v) trypsin to which an equal amount of 0,02 M hydrochloric acid containing 0.8% (w/v) L-cysteine had been added just before its use. This medium, referred to as standard medium, was freshly prepared prior to each incubation experiment. Incubations were carried out in a watch glass placed in a water bath at 40~ C. Cysts were removed from the cellophane and placed in the medium. The percentage of organisms excysted was recorded at 5-min intervals for up to 120 rain after transfer to the test medium. The activity of flukes within the medium was also noted at each interval with the aid of a binocular light microscope. Only fully emerged organisms were scored. Ten cysts were used for each experimental treatment and three replicates of each experiment were carried out. To determine the most effective environment for the excystment of Z. lunata, the physico-chemical conditions were varied as follows: 1. Combinations of bile salts (Na taurocholate) and trypsin in the excystment medium were varied from the original 0.8% Na taurocholate: 0.3% trypsin concentration. The other concentrations used were as follows (all concentrations are stated as the weight-tovolume percentage, of Na taurocholate to trypsin): 0.4:0.15, 1.6:0.6, 2.4:0.9, 0.8:0, 0:0.3 and 0:0. 2. Sodium cholate crude ox-bile extract (Sigma S-9875) was used instead of the 40% crude Na taurocholate (Sigma T-0750) normally used. The latter was known to contain glycocholic, cholic, deoxycholic and other bile salts. 3. The effect of pepsin on excystment was investigated, firstly by carrying out the excystment procedure in a 2% solution of pepsin at 40~ C and then by treating the cysts in a 2% solution of pepsin for 20 min prior to the normal excystment protocol. 4. Thermal effects were examined by incubating cysts in the excystment medium over a temperature range of 25~ ~ C. This was

417

[Z

100

100

80

~. 80 Z ~. 60

60

X

ra~

40

40

20

20

I

20

30

I

I

~

I

9

40 50 60 TIME(MINS) []

I

70

Na Taurocholate 9

Na choleate

0

I

10

I

20 m

I

I

I

I

30 40 50 60 TIME(MINS)

70

UNTREATED CYSTS TREATED CYSTS

Fig. 1. Effect of different 0.8% bile salts on the rate and percentage of excystment of metacercariae for a 60-rain period in excystment medium at 40~ C

Fig. 2. Effect of pepsin pretreatment for 20 rain on the rate and percentage of excystment of metacercariae for a 60-rain period in excystment medium at 40~ C

carried out by varying the temperature of the water bath and monitoring it periodically using a thermometer. 5. The effect of pH on excystment was monitored from pH 4.5 to 8.5 by the addition of 1 M H2SO4 or I M NaOH.

gave 7 0 % - 8 0 % excystment of metacercariae within 60 rain. In the presence of either trypsin or bile salts in the excystment medium, the rate and percentage o f excystment remained the same. When both trypsin and bile salts were omitted from the excystment medium the rate of excystment was slower, taking approximately 70 rain for 70% excystment. Therefore, changes in trypsin and bile salt concentration did not have any significant effect on the rate or percentage of excystment of Z. lunata. However, in high concentrations of trypsin and bile salts (i.e. when the concentration of both was tripled) and in the absence of trypsin and bile salts, the larvae did not survive long, curling up and dying within 20 min of emergence. When sodium cholate crude ox-bile extract was used instead of the usual Na taurocholate, 100% excystment was achieved as compared with the 7 0 % - 8 0 % obtained using Na taurocholate. The rate of excystment was also increased substantially, with the first larva emerging after only 25 rain. Contractile movements of the metacercariae were extremely vigorous throughout the process. Figure 1 shows the effect of the Na cholate crude ox-bile extract as compared with the Na taurocholate normally used in the excysting medium at 40 ~ C. A 2% solution o f pepsin at 40 ~ C did not promote excystment. However, when cysts were treated in pepsin for 20 min at 40 ~ C prior to treatment in the usual excystment medium the rate of excystment was noticeably increased. The first larva emerged after 20 rain and the last one came out after 40 min. However, the percentage o f excystment was the usual 7 0 % - 8 0 % . Figure 2 shows the effect of pepsin pretreatment on excystment.

Results Juvenile organisms in cysts that had been removed from cellophane at 20 ~ C were not visibly active, but following 10 rain incubation at 4 0 ~ in standard medium they started to develop undulating body motions. After approximately 20 rain the movements o f the newly excysted metacercariae became more pronounced. Activity took the form of rotations within the cyst and attempts to stretch and uncurl; as a result the anterior end o f each metacercaria was forced against its cyst wall. The initially inflexible cyst wall became increasingly pliant, and after about 25 min o f intense activity the first larva emerged through an escape hatch in the flat side, anterior end first. Continuation of the extension and contraction behaviour ensured that the remainder of the body was freed withifl about 35 rain. After approximately 60 min incubation in the excystment medium, 7 0 % - 8 0 % o f the organisms had emerged. The freed metacercariae continued their contractile movements for up to 60 min after excystment. All combinations of trypsin and bile salts in the excystment medium at 40 ~ C produced successful excystment o f Zygocotyle lunata. Halving, doubling and even tripling the amounts of both ingredients consistently

418

[,. Z

100

100

8O

80 Z

r,r,,t [,,,

60

60

40

40

2O

20

0

m

20

v

9

40

m.

60

0

!

100

80

9

--

35 (oC) 40 (oC)

ol I !

!

I

I

I

!

!

3.5 4.5 5.5 6.5 7.5 8.5 9.5 pH

TIME(MINS) 30 (oC)

tel

Fig. 5. Percentage of metacercarial excystment achieved at different pH values

Fig. 3. Effect of various temperatures on the percentage of excystation in excystment medium for 100 min

100 oo

[- 80 Z [.., 60 40

t'r

20

0

!

20

I

I

I

I

I

25 30 35 40 45 TEMPERATURE (oC)

Fig. 4. Percentage of excystation achieved in standard excystment medium at various temperatures

The rate and percentage of excystment increased with temperature from a low threshold level of approximately 30 ~ C to an optimal temperature of about 40 ~ C; temperatures above this level caused larvae to die before they emerged (Figs. 3, 4). During incubations at 25 ~ C, no movement was observed and no excystment occurred. At about 30 ~ C the larvae began to show a little movement within the cyst. The first larva emerged after

70 rain of incubation in the excystment medium. Only 30% excystment was achieved, and on emerging the larvae subsequently became sluggish and died within 15 rain. At a temperature of about 35 ~ C the encysted larvae showed more movement and the first larva emerged after 55 rain. Excystment of 50% was achieved and larvae remained active for about 60 min after emergence. At 40 ~ C the optimal rate and percentage of excystment occurred. Contractile movements of the larvae were vigorous throughout the excystment process. The first larva emerged after 35 min incubation in the excystment medium; 80% excystment was achieved and free metacercariae remained active for about 60 rain. On an increase in temperature to 45 ~ C the encysted larvae were active after 25 min incubation in the excystment medium, but after approximately 45 min the activity ceased and no excystment occurred. The larvae appeared to have died within their cysts. Experiments on the effect of different p H values on excystment showed that 70% excystment occurred over a range from p H 5.5 to 7.5. Excystment below p H 5.5 fell markedly, with only 10% excystment being observed at p H 4.5 and none being noted at p H 3.5. At a p H of above 7.5 excystment also dropped, with 30% excystment being recorded at p H 8.5 and no excystment occurring at p H 9.5. The effect of p H on the percentage of excystment is shown in Fig. 5.

Discussion

Zygocotyle lunata is similar to many other digenetic trematodes such as Microphallus abortivus (Saville and Irwin 1991), Parapronocephalum symmetricum (Irwin et al. 1989), Echinostorna liei and E. revolutum (Fried and Emi-

419 li 1988) in that they all excyst in a trypsin/bile salt medium at approximately 40 ~ C. Indeed, Z. lunata is not unique in its ability to excyst in the absence o f enzymes or bile salts, as Dunn et al. (1990) demonstrated that Probolocoryphe uca excysted in Hanks' balanced salt saline at 40 ~ C. Z. lunata and P. uca show a low degree of host specificity, as P. uca adults are found not only in a wide variety o f shore birds but also in various fish species. Bock (1986) showed that nearly 100% excystment could be observed in Plagiorchis species I without trypsin or bile salts, although as was the case in Z. lunata, the speed of the process was greatly reduced in their absence. Plagiorchis species I is also a parasite of birds. Pepsin pretreatment has been found to be a prerequisite for the excystation of some metacercariae such as Sphaeridiotrema globulus (Macy et al. 1968) and Apatemon minor (Kearn et al. 1989). Other researchers such as Schroeder et al. (1981) and Kirschner and Bacha (1980) have shown improved excystment rates after acidified pepsin pretreatment in Neascuspyriformis and Himasthla quissetensis, respectively. T h o m p s o n and Halton (1982) suggested that the similar improved rates o f excystation observed in Cotylurus variegatus after pepsin pretreatment were due to digestion o f extraneous host tissue from the cysts. This could not have been the case for Z. lunata, as the metacercarial cysts were devoid of host tissue yet pepsin pretreatment clearly speeded up the rate o f excystment. Although Macy et al. (1968) found that the addition of sodium taurocholate to their excystment medium for S. globulus had no effect on the results, the present study, like that of T h o m p s o n and Halton (1982), demonstrated that bile salts were an important ingredient that influenced the rate o f metacercarial excystment. The study of T h o m p s o n and Halton (1982) and the present investigation compared the effect of crude and refined bile salts and found that the crude form promoted more rapid excystment. This finding has been attributed to the presence of traces o f other bile salts or acids in the crude extract. Z. lunata was capable o f successfully excysting in the absence of trypsin, and although trypsin has often been included in excystment procedures, it has seldom been shown to be essential to the process. D u n n et al. (1990) demonstrated that P. uca excysted successfully in the absence o f trypsin, whereas the presence o f this enzyme was a critical factor in the excystment of another microphallid, Gynaecotyla adunca. It must be noted that Z. lunata larval activity and rate o f excystment was greatly reduced in the absence of trypsin and/or bile salts. It is possible that these two factors have a synergistic effect on one another, as Howell (1970) reported that excystment o f Echinoparyphium serratum occurred in a trypsin bile salt medium and that enzyme treatment alone was ineffectual. On the other hand, Spellman and Johnson (1987) found that there was no synergistic effect between bile salts and trypsin during the excystation of Uvulifer ambloplitis. Even though adult P. uca are found in fish, D u n n et al. (1990) stated that only 13% excysted at 22 ~ C as compared with 77.8% at 40 ~ C in Hanks' balanced salt

saline. Many researchers have shown that approximately 40 ~ C represents an optimal temperature for excystment in species that live as adults in homiothermic vertebrates. The range of temperature that resulted in successful excystment in Z. lunata was similar to the 21~ ~ C range of Plagiorchis species I (Bock 1986) and the 280-48 ~ C range reported for C. variegatus (Thompson and Halton 1982). The latter authors have suggested that the ability to excyst at low temperatures would avoid the time delay necessary for the host intestinal contents to equilibrate with the host body temperature and that it therefore speeds up the excystment time. Maximal excystment of Z. lunata, in c o m m o n with that o f Posthodiplostomum leonensis (Asanji and Williams 1975) and C. variegatus (Thompson and Halton 1982), occurred over a range o f p H from slightly acidic to slightly alkaline, which must correspond to the p H in various parts of the digestive tracts o f a variety o f prospective hosts. Unlike the case of the bird parasite Parorchis acanthus, of which 80% of the metacercariae tested excysted between p H 7 and 8 (Asanji and Williams 1975), hydrogen ion concentrations between p H 5.5 and 8.5 had little effect on the excystment of Z. Iunata. The broad p H and temperature ranges along with the very non-specific enzyme/bile salt requirements shown to bring about consistently high rates of metacercarial excystment in Z. lunata are likely to be present in the digestive tracts of many homiothermic vertebrates. This ability to respond positively to such a variety of intestinal conditions is likely to represent an important survival strategy for this parasite by broadening the range of its prospective hosts.

References

Asanji MF, Williams MO (1975) Studies on the excystment of trematode metacercariae in vitro. Z Parasitenkd 47:71-77 Bock D (1986) In vitro excystment of the metacercaria of Plagiorthis species I (Trematoda, Plagiorchiidae). Int J Parasitol 16:641-645

Dunn TS, Ownbey TC, Vannarath T (1990) In vitro excystment of the metacercariae of Gynaecotyla adunca and Probolocoryphe uca (Microphallidae) from the fiddler crab, Uea pugilator. Can J Zool 68 : 2376-2384 Fried B (1970) Infectivity, growth, development, excystation and transplantation of Zygocotyle lunata (Trematoda) in the chick. J Parasitol 56:44-47 Fried B, Emili S (1988) Excystation in vitro of Echinostoma liei and E. revolutum (Trematoda) metacercariae. J Parasito174:98102 Fried B, Robbins SH, Nelson PD (1978) In vivo and in vitro excystation of Zygocotyle lunata (Trematoda) metacercariae and histochemical observations on the cyst. J Parasitol 64:395-397 Howell MJ (1970) Excystment of the metacercariae of Eehinoparyphium serratum (Trematoda:Eehinostomatidae). J Helminthol 44: 35-56 Irwin SWB, McKerr G, Judge BC, Moran I (1984) Studies on metacercarial excystment in Himasthla leptosoma. Int J Parasitol 14:415-421 Irwin SWB, McShane G, Saville DH (1989) A study of metacercarial excystment in Parapronocephalurn symmetrieum (Trematoda: Notocotylidae). Parasitol Res 76: 45-49 Kearn GC, Cleveland G, Wilkins S (1989) An in vitro study of the forcible ejection of the metacercaria of the strigeid digenean

420

Apatemon (Australapatemon) minor Yamaguti, 1933 by rapid inward expansion of the cyst wall. J Helminthol 63:63-71 Kirschner K, Bacha WJ (1981) Excystment of Himasthla quissetensis (Trematoda:Echinostomatidae) metacercariae in vitro. J Parasitol 66: 263-267 Macy RW, Berntzen AK, Benz M (1968) In vitro excystation of Sphaeridiotrema globulus metacercariae, structure of cyst, and the relationship to host specificity. J Parasitol 54:28-38 Robbins SH, Hammett M, Fried B (1979) Light and transmission electron microscopical studies and amino acid analysis of the metacercarial cyst of Zygoeotyle lunata (Trematoda). Int J Parasitol 9: 257-260 Saville DH, Irwin SWB (1991) A light and electron microscope study on in vitro excystation of Microphallus abortivus (Digenea: Microphallidae) metacercariae. Parasitol Res 77: 82-85

Schroeder D J, Johnson AD, Khawla HM (1981) In vitro excystment of the black spot trematode Neaseuspyriformis Chandler, 1951 (Trematoda:Diplostomatidae). Proc Helminthol Soc Wash 48:184-189 Spellman SJ, Johnson AD (1987) In vitro excystment of the black spot trematode Uvulifer ambloplitis (Trematoda:Diplostomatidae). Int J Parasitol 17:897-902 Thompson M, Halton DW (1982) Observations on excystment in vitro of Cotylurus variegatus metacercariae (Trematoda: Strigeidae). Z Parasitenkd 68 : 201-209 Willey CH (1941) The life history and bionomics of Zygocotyle lunata (Paramphistomidae). Zoologica 26:65-88