The in vitro cultivation of strigeid trematodes. III. Yeast as a medium constituent

EXPERIMENTAL

PARASITOLOGY

The

11,

in Vitro

Modupe

121-127

(1961)

Cultivation of Strigeid Trematodes. as a Medium Constituent

0. Williams, Department

C. Adrian of Zoology,

(Submitted

Hopkins,

The

University,

for publication,

and Glasgow,

10 February

M. Rose

III.

Yeast

Wyllie

Scotland 1961)

hutoclaved yeast extract when added to a basal medium of serum, hen-egg albumen and gluco-saline, promotes sperm and vitelline cell production in D. phoxini. Two active fractions were found in the yeast; (i) a dialyzable fraction, the effect of which can be replaced by amino acids, (ii) a non-dialyzable fraction which is resistant to hot acid and alkaline hydrolysis, and is basically charged. Part of the nondialyzable fraction appears to be a small molecular substance occurring in a bound form, since it is lost if dialysis is preceded by mild acid hydrolysis. The possibility that this substance is pyridoxine is discussed. Supplementation of the basal medium with amino acids and Be produced 705% of the development reached in yeast extract, but only if the Be was in a concentration much greater than in yeast extract.

A technique for the in vitro cultivation of the strigeid trematode, Diplostomum phoxini, was described by Bell and Hopkins (1956). Survival of the fluke for 5-7 days was obtained in synthetic media, but little development of the genitalia occurred. In vivo the flukes were found to mature within 3-4 days in a duck. Bell and Smyth (1958) using hen-egg yolk and albumen medium obtained maturation of the testes and the production of abnormal eggs, In attempts to determine the conditions and substancesactive in this medium Wyllie et al. (1960) tried to replace yolk by a chemically defined supplement, but failing this, they used extracts of natural products which could be analyzed more easily than yolk. The most effective medium used was one containing yeast extract. The object of the present work was to determine which fraction of the yeast extract was active, and discover to what extent a chemically defined supplement could replace it. MATERIAL

AND

METHODS

were not included. This method differs slightly from that used in earlier work and gives a slightly lower figure for percentage development. Media The basal medium used consisted of a balanced salt solution (BSS), 0.6% glucose (G), 255%horse serum, and 12% fresh henegg albumen. Amino acid supplement was that of Parker’s medium 703 (Healy et al., 1954). The yeast extract was the dehydrated water soluble portion of autolyzed fresh yeast (Difco) used in a concentration of 4 mg per ml of medium. The B vitamin solution used was that described by Waymouth (quoted by Paul, 1959). It was made up 40x concentrated, sterilized by filtration through sintered glass, and stored for up to 4 weeks in the dark at -15” C. Pyridoxine hydrochloride, pyridoxal hydrochloride and pyridoxamine dihydrochloride were prepared 10X concentrated and sterilized by autoclaving.

Cultivation techniques were those described Analytical Procedures by Wyllie et al. (1960). Cultures were as1. Acid hydrolysis was carried out by sessedby removing flukes at random after 6 days’ incubation at 40’ C, and examining refluxing 12% yeast extract in 1N HCl for 1 hour. The solution was then autoclaved at 25. Dead flukes, which rarely exceeded lop, 121

122

WILLIAMS,

HOPKINS,

115” C for 1.5 minutes. 1.7 ml of the yeast solution was added to 45 ml of basal medium without albumen. The pH was corrected to between 6 and 7 with 0.2 N NaOH, and the medium dispensed in 10-l 1 ml aliquots. l.O1.5 ml albumen was added to each culture and the pH corrected to 6.7. 2. Alkaline hydrolysis was carried out by autoclaving a 4% yeast extract made up in 0.1 N NaOH, for 15 minutes at 115” C. 5 ml was added to 4.5 ml of BSS + G + serum and the pH corrected to 6-7. Procedure was then as with acid hydrolyzed extract. 3. Dialysis was carried out by placing 50 ml of an autoclaved 4% solution of yeast extract in a sterilized l-inch diameter cellulose tube, and dialyzing against running water for 7 days. During this process the volume increased to about 80 ml. The original volume was regained by evaporating under reduced pressure at 40-50” C. The solution was resterilized by autoclaving and 1 ml added to each culture. 4. Combined hydrolysis and dialysis consisted of refluxing a 12% solution as in procedure 1; diluting to 4% with water, and then dialyzing, concentrating, sterilizing, and dispensing as in procedure 3. 5. Anion exchange was carried out using Dowex 1 resin in the chloride form. A 6X 1 cm column was washed with 1 N HCl to charge. Excess Cl- was washed out with distilled water until no precipitate was formed with AgNOa. 20 ml of 4% yeast extract, corrected with 0.2 N NaOH to pH 8 was passed through the column. The effluent was collected, without washing the column, and autoclaved. 1 ml was added to each culture. 6. For cation exchange, ZeoKarb 2 1.5 in the hydrogen form was used at pH 4. The resin was kept in 4 N HCI for 3 hours to convert to hydrogen form, then washed with distilled water until the effluent was free of Cl-. 20 ml of 2% yeast extract was acid hydrolyzed (procedure l), pH corrected to 4, and passed through the column. The effluent was autoclaved, pH adjusted to between 6 and 7, and 2 ml added to each culture. RESULTS

The level of development reached by flukes cultured for 6 days at 40” C in various

AND

WYLLIE

media is shown in Tables I and II. The criteria used to assess development are the presence or absence of certain genital structures. The percentage of flukes in which the testes could be seen to consist of discrete follicles is shown in column 5, Table I. Since very good cultures often have a few flukes in which little development has occurred, variations above 90 are of little significance and are not given. This may be the result of a small but variable number of young metacercariae being included in the cultures. No investigation, however, has been carried out to determine the age at which D. phoxini is capable of maturing, or whether partially developed metacercariae can survive without developing at 40” C. Columns 6, 7, and 8 show respectively the percentage of flukes in which: active sperm can be seen in the male ducts; characteristic large vitelline cells filled with yolk globules are visible; a yolk reservoir is present. This last gives some idea of the quantity of vitellaria, as a yolk reservoir is normally not formed if the vitelline cells are very few. The figures in parentheses have been calculated to indicate the percentage loss in activity of the various yeast supplements in promoting maturation. For example, in medium 8a in which the yeast extract was acid hydrolyzed before use, 28% of the flukes developed active sperm, compared with 65% in medium 8; the difference of 37 shows a loss in activity of 57%. Similarly development of genitalia in flukes in all other media (8a-f) derived from medium 8 is expressed as a percentage change. In calculating the difference in media derived from medium 9, which differs from the medium 8 series only in that an amino acid supplement was added (column 3, Table I), a correction factor has been used. This was necessary because some sperm and vitellaria are formed without yeast if amino acids are added (medium 7). Since the figures in parentheses show changes in the activity of the yeast, it was necessary to subtract the results of medium 7 from medium 9 to arrive at the proportion of development that could be attributed to the yeast. Thus, in medium 9a, active sperm occurred in 64%

THE

ifi

lih’0

CULTIVATION

OF

TABLE The Level

STRIGEID

TREMATODES.

I

of Development Reached by D. phoxini after Cultivation for 6 Days at 40” C in a Basal Supplemented by Various Fractions of Yeast Extract & Amino Acids

Medium No.

Supplement

9 8

Yeast

7 6

None

extract

9a Acid

hydrolyzed

Active sperm %

+ -

300/12 250/10

90 90

70 65

65 56

56 44

+ -

125/s -0

68 24

10 0

5 0

3 0

+

125/s

90

64

90

C-10) 28 (-57)

%

+

9b hydrolyzed

Y.E.

8b

-

125/S 7513 75/3

90 75

(& 32

+ Dialyzed

150/6

90

Y.E.

-

SC

40 Dialyzed fraction of acid hydrolyzed Y.E.

8d

+

7513

32

Effluent from anion exchange pH8

+

7513

90

Se

-

75/3

0

90

Effluent of acid hydrolyzed Y.E. from cation exchange pH4

a Quoted from Wyllie of development reached

et al. (1960). by the flukes

--

150/6

40

The figures in parentheses in the medium compared

of the flukes compared with 70% in the untreated extract (medium 9). The loss in activity due to hydrolysis of the yeast is therefore 6/60th (not 6/70th), since 10% of the flukes could be expected to form active sperm in the absence of any form of yeast (medium 7j . Similarly there was a fall of 7% in the number of flukes producing vitellaria, which indicates a loss of 7/60th, i.e., 12%) in the ability of acid hydrolyzed yeast to promote vitelline development. Since considerable variation occurs in the same medium and slight difference in conditions, such as the thickness of the albumen may cause a large number of flukes to form,

show with

(-ii) (-lOi)

64

C-2)

54 (4)

28 (---so)

5 (-92)

,-ii)

0 (-100)

37 C-55) 24

56 (0)

40

C--Q)

(-63) 8f

68 ($23) 40 (-9)

44

(-100) 9e

75 (f17) 40 71

(43) 7513

(-ii)

($10) 16 C-71)

81

-

40

(+8) (&

52

(k-8)

C-29)

7s

75/3

70

58

C-12)

C-29)

(-51)

9c

Vitellaria %

Yolk reservoir

No. of flukes /cultures

-

Alkaline

Follicular testes

Medium

Amino acid

Y.E.

8a

9d

123

III.

24 (45)

5 (-91)

C-9;)

$% increase or decrease in the level that in medium 8 or 9 (see text).

or alternatively just fail to form, sperm and vitellaria, little significance should be attributed to differences of less than 20%. Moreover, the criteria used are purely qualitative and a fluke which produces a single bundle of active sperm is recorded as equal to one producing a dense mass. The results given for medium 6 are quoted from earlier work (Wyllie et al., 1960); active sperm and vitellaria have rarely been found in flukes cultured in this medium. However, a small number of flukes were found, as in earlier work, to produce sperm and vitellaria if amino acids were present (medium 7).

124

WILLIAMS,

HOPKINS,

TABLE The Efect

Medium No. 9 10

Supplement Yeast

extract

of Vitamin

10b 1Oc 10d

WYLLIE

II

Be on the Development

of D. phoxini

Concentration of Be in medium

No. of flukes /cultures

Active sperm %

0.1 @ml

300/U

70

Vitellaria %

Yolk reservoir % 56

65

B Vitamins

37

2 10a

AND

pdml

75/3

(-ii)

(47)

(-ii)

1 @ml

75/3

(-Z)

44 (-351

(4)

Pyridoxamine dihydrochloride

50 p/ml

75/3

48 C-37)

t-201

Pyridoxal hydrochloride

50 pg/ml

Pyridoxine hydrochloride

50 @ml

Pyridoxal hydrochloride

Figures in parentheses as in Table I. Q Supplied by Light’s, England. b Supplied by Nutritional Biochemicals

ALL 125/s

Co.,

50 G-331

53

FLUKES

(-E)

DEAD

52

C-42)

48 C-15)

Cleveland.

The yeast supplements added to the basal medium are shown in column 2 ; they were prepared as described in Methods. Table II shows the results obtained when B vitamins (medium 10) or B,J alone (media IOa-d) replaced the yeast in medium 9. Figures in parentheses were calculated as described above for media 9a-e. The BG content of the yeast extract (medium 9) was 0.1 pg per ml of medium (Difco, 1958), or possibly slightly higher, 0.3 pg per ml (Sykes, 1956). Waymouth’s B vitamin solution was used at double concentration, giving a B6 concentration of 2 pg/ml in the form of pyridoxine hydrochloride. Two preparations of pyridoxal HCl were used in medium 10~. The concentration of B. shown in column 3 is that added in the supplement; it does not include the amount present in the serum of the basal medium. DISCUSSION

Aqueous yeast extract was found to have a considerable growth promoting effect (medium 8, Table I), when added to a basal medium consisting of horse serum, hen-egg albumen and glucose saline (medium 6, Table I). In the absence of the yeast extact, sperm and vitellaria were rarely formed. Part of the beneficial effect of yeast extract

may result from the high concentration of amino acids, the importance of which was demonstrated by adding an amino acid supplement to the basal medium (medium 7). However, the results obtained with amino acids were never as good as with yeast, and the addition of yeast to amino acid supplemented media further improved development (medium 9). It was apparent therefore, that amino acids were not the only substances contributed by yeast extract which were affecting development. It should be noted that the value of yeast extract was assessed when added to a medium containing serum and albumen. Many substances present in yeast and essential for strigeid development may also occur in adequate quantities in the serum or albumen and therefore need not be included in a supplement designed to replace the yeast extract in the medium. To gain some idea of the nature and size of the molecules in the active fraction, yeast extract was subjected to a series of analytical procedures and the effect on the activity of the extract measured (media 8a-f, 9a-e, Table 1). Mild acid hydrolysis (9a) had no effect when an amino acid supplement was added, which suggests that the B vitamin components which are destroyed by boiling 1 N HCl [i.e.,

THE

it2

?ihJ

CULTIVATION

OF

pantothenic acid (Lepkovsky, 1954), Blz (Lester-Smith, 1956) and folic acid (Daniel and Kline, 1947)], are not required from yeast extract. On the other hand, in the absence of amino acids (8a) development was retarded, and it would seem probable that the flukes require a source of at least one of the acid labile amino acids; serine, threonine, and tryptophan, all of which occur in yeast extract, though serine only in trace quantities (Difco, 1958; Pyke, 1958). The results obtained using alkaline hydrolyzed yeast were similar to those obtained using acid hydrolyzed material. With amino acids added (9b) the level of development was not significantly different from that in untreated extract. This substantiates the conclusions reached using acid hydrolyzed material; viz., that the B vitamins, B13 (Hartley et al., 1950) and pantothenic acid are not required from yeast. It further precludes the possibility that thiamine (Rosenberg, 1945), riboflavine (Wagner-Jauregg, 1954), and biotin (Merck, 1960) are essential contributions from yeast extract, as these are destroyed by boiling alkali. Without amino acids development is greatly reduced (8b). Alkaline hydrolysis destroys cysteine, cystine and arginine, in addition to the acid labile amino acids, serine and threonine. The reduction in development in the absence of an amino acid supplement thus suggests the conclusion that some of the amino acids in yeast are essential for the flukes. The results obtained with dialyzed yeast were of particular interest, as dialysis removes the small molecular substances which include nicotinic acid, DPN, and TPN. Nicotinic acid occurs at a high concentration in yeast extract (279 pg p.g.-Difco, 1958), and because of its importance in energy release, it seemed likely that, during the active phase of maturation in the gut of a bird, the flukes might have substantial requirements for the vitamin and coenzymes. However, the results indicated that in the presence of an amino acid supplement the important part of yeast extract was in the large molecular fraction. In the absence of amino acids (8~) dialyzed yeast was of little value. It appeared, therefore, that yeast was contributing (i) a dialyzable fraction, the active part of which

STRIGEID

TREMATODES.

III.

125

consisted of amino acids, and (ii) a large molecular, nondialyzable fraction. No attempt was made to determine which amino acids in yeast the worms were utilizing as this aspect of nutrition will be more easily studied when a successful medium containing no serum or albumen is developed. The amino acid supplement used in these experiments, while no doubt containing several redundant acids, suffices as a substitute for the amino acids in yeast. However, if yeast extract is to be replaced by a chemically defined additive, the nature of the active part in the large molecular fraction must be determined. To test the possibility that the active part of the nondialyzable fraction was a small molecule attached to a protein or peptone, and not a large molecule, yeast extract was first hydrolyzed and then dialyzed (9d, 8d). Whereas separately neither of these procedures caused loss of activity, when combined they resulted in a loss of half of the activity of the yeast extract. This suggests that at least part of the activity in the nondialyzable fraction is due to a small molecular substance, which is freed by acid hydrolysis without damage, but thereafter is lost on dialysis. Lastly, to obtain a guide to the probable nature of the substances being contributed by yeast, extracts were passed through ion exchange columns. An acid hydrolyzed extract of yeast was passed through a cation exchange column. ZeoKarb 215, the resin used, absorbs in the H+ form at pH 4, all the amino acids, even aspartic and glutamic (Samuelson, 1953, p. 212). It also absorbs proteins, peptones and the bases. Not surprisingly therefore, the effluent showed almost complete loss of activity (medium 8f). The results obtained using the effluent from anion exchange are more difficult to interpret. All that can be concluded is that, since vitelline production remained normal (se), the nondialyzable factor passed through the column and hence is basically charged. The poor development obtained when the amino acid supplement was omitted (se) is probably due to the total loss of the acidic amino acids and partial loss of the neutral amino acids. (The effluent used did not contain a washing from the column.)

126

WILLIAMS,

HOPKINS,

In summary of this first part of the work we concluded that the active nondialyzable component of yeast extract was a small molecular substance, resistant to mild acid and alkaline hydrolysis, probably basically charged and usually occurring in a large nondialyzable complex. Of the substances reported in yeast, pyridoxine or some other form of vitamin Ba fitted this description. This substance occurs as a nondialyzable protein complex in yeast (Kuhn and Wendt, 1938) is basically charged and resistant to 5 N acid and alkali at 100” C (Robinson, 1951). It should be added, however, that the proportion of BF that remains in a nondialyzable complex after the autolysis which occurs in the preparation of yeast extract is not known. Certainly proteins are not completely hydrolyzed as is shown by the form01 titration relative to total nitrogen (Sykes, 1956). It should also be noted that nicotinic acid is resistant to acid and alkaline hydrolysis, and occurs in fresh tissue in protein complexes (Hundley, 1954). Earlier experiments using the B vitamin supplement of medium 703 (Healy et al., 1954) had given poor results, though rather better results had been obtained using Waymouth’s B vitamin supplement (Medium MB 752/l, quoted by Paul, 1959). At the time the difference was not associated with the pyridoxine level, but in retrospect it was realized that Waymouth’s medium contained 20X the BG concentration in medium 703. The experiment using Waymouth’s B vitamin solution was repeated (medium 10, Table II), and cultures were also set up using Bo (medium 10a). The results could not be directly compared with earlier figures as the method of assessment differed: in the earlier experiments assessment was made by selecting, under x2.5 magnification, 10 flukes which appeared the best developed, and then examining these under X200 magnification for sperm and vitellaria. However, as in the earlier work some flukes formed sperm and vitellaria but the results were again much poorer than those obtained with yeast extract. The interesting point was that Be gave as good results as whole B vitamin supplement. The higher percentage of flukes with yolk reservoirs in medium 10a is prob-

AND

WYLLIE

ably not significant. Yolk reservoir counts are a useful index, when vitellaria are recorded as present or absent, in that flukes with large quantities of vitellaria normally have yolk reservoirs, whereas flukes with only a few vitelline cells do not, but at intermediate levels of vitelline production considerable variation in the percentage forming yolk reservoirs occurs. The following questions also arose from these experiments. Are pyridoxine and pyridoxal equally effective forms of BG, and would a higher concentration of Bc improve development? In media lob, c and d, the concentration of BG was increased to 50 yg per ml of medium. Development was better in the case of pyridoxine and pyridoxamine but the flukes died in this concentration of pyridoxal. This was unexpected as it is usually accepted that in higher animals the three forms of Bs are interchangeable (Fruton and Simmons, 1953). Further experiments with other concentrations of Bg were not attempted, as development in amino acid and Bs supplemented media was very variable, and results would have been difficult to interpret. It has not yet been possible in any medium to eliminate variation in the level of development of flukes, and even in supposedly identical control cultures containing yeast extract, the flukes develop to different extents at different times, causing difficulty in making valid comparisons between media. During the last three years, media 8 and 9 have been used extensively, and well over 2,000 flukes have been examined from these cultures. Under “good conditions” excellent results are obtained, masses of sperm in the vas deferens and seminal vesicle, extensive vitellaria and large yolk reservoirs being present. The sperm and vitelline masses are easily visible and in medium 9 may occur in up to 90% of the flukes. Yet at another time examination, may reveal sperm in only SO%,, and vitellaria in only 30% of the flukes. Several possible explanations exist but we think that differences in the serum and albumen lead to much of this variability in the level to which the flukes develop (cf. Wyllie et al., 1960). To eliminate this variability as much as possible, the only results included in Tables I and II are those ob-

THE

in

ZdP’0

CULTIVATION

tained from experiments in which at least 60% of the flukes in control cultures of medium 9 have produced active sperm and SO%, vitellaria. Once these variable factors are eliminated it will be possible to look more thoroughly at the B6 requirement of D. phoxini. While there is no doubt that B6 permits more development to take place, it does so only at a concentration far exceeding that in yeast. The conclusion is, therefore, that there is some substance(s) in yeast extract other than those considered in this work which is used by the flukes, the effect of which can be partly replaced by a high concentration of pyridoxine. REFERENCES

E. J., AND HOPKINS, C. A. 1956. The development of Diplostomum phoxini (Strigeida, Trematoda). Annals of Tropical Medicine and Parasitology 50, 275-282. BELL, E. J., AND SMYTH, J. D. 1958. Cytological and histochemical criteria for evaluating development of trematodes and pseudophyllidean cestodes in vivo and in vitro. Parasitology 48, 131148. DANIEL, E. P., AND KLINE, 0. L. 1947. Factors affecting folic acid determination. Journal of Biological Chemistry 170, 739-746. DIFCO LABORATORIES. 1958. Private communication. FRUTON, J. S., AND SIMMONDS, S. 1953. General Biochemistry. John Wiley & Sons, New York. HARTLEY, F., &ROSS, P., AND STUCKEY, R. S. 1950. Some pharmaceutical aspects of vitamin B,,. Journal of Pharmacy and Pharmacology 2, 648659. BELL,

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TREMATODES.

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G. M., FISHER, D. C., AND PARKER, R. C. 1954. Nutrition of animal cells in tissue culture. IX. Synthetic medium No. 703. Canadian Journal of Biochemistry 32, 327-337. HUNDIZY, J. M. 1954. In The Vitamins. (Sebrell, W. H., and Harris, R. S., eds.). Academic Press, New York, vol. 2. KUHN, R., AND WENDT, G. 1938. Uber das antidermatitische Vitamin der Hefte. Berichte der Chemischen Gesellschaft 71, 780-782. LEPKOVSKY, S. 1954. In The Vitamins. (Sebrell, W. H., and Harris, R. S., eds.). Academic Press, New York, vol. 2. LESTER-SMITH, E. 1956. Vitamin B13. British Medical Bulletin 12, No. 1, 52-56. MERCK INDEX OF CHEMICALS AND DRUGS. 7th ed. 1960. Merck & Co., Rahway, N.J., U.S.A. PAUL, J. 1959. Cell and Tissue Culture. Livingstone, Edinburgh and London. PYKE, M. 1958. (The Distillers Co. Ltd.) Private communication. ROBINSON, F. A. 1951. The Vitamin B Complex. Chapman and Hall, London. ROSENBERG, H. R. 1945. Chemistry and Physiology of the Vitamins. Interscience Publishers, New York. SAMUELSON, 0. 1953. Ion Exchanges in Analytical Chemistry. John Wiley and Sons, New York. SYKES, G. 1956. Constituents of Bacteriological Media. Cambridge University Press, England. WAGNER- JAUREGG, T. 1954. In The Vitamins. (Sebrell, W. H., and Harris, R. S., eds.) Academic Press, New York, vol. 3. WYLLIE, M. R., WILLIAMS, M. O., AND HOPKINS, C. A. 1960. The in vitro cultivation of strigeid trematodes. 11. Replacement of a yolk medium. Experimental Parasitology 10, 51-57. HEALY,