Comp. Biochera. Physiol., 1971, Vol. 40B, pp. 993 to 1003. Pergamon Press. Printed in Great Britain

THE EFF E CT OF BILE SALTS ON THE CARBOHYDRATE M E T A B O L I S M OF TWO SPECIES OF H Y M E N O L E P I D I D

CESTODES

RICHARD D. TKACHUCK* and AUSTIN J. MAcINNIS

Department of Zoology, University of California, Los Angeles, California

(Received 29 M a y 1971)

Abstract--1. The bile salts taurocholate and taurodeoxycholate affect the glucose metabolism of adult cestodes Hymenolepis diminuta and H. citeUi.

2. When worms were incubated in glucose in the absence of bile salts, H. citeUi maintained a higher internal level of free glucose and incorporated twice as much glucose into glycogen than did t t . diminuta. Of the glucose metabolized into succinate by I t . citelli, 90 per cent came from the exogenous glucose as compared to 80 per cent with H. dirainuta.

3. Taurocholate had no effect on glucose metabolism of 11. dirainuta, but caused a 21 per cent reduction of the internal free glucose level and a 33 per cent reduction in the amount of glucose incorporated into glycogen in H. citelli.

4. Taurodeoxycholate reduced the free pool glucose levels in 11. diminuta by 30 per cent and glucose incorporation into glycogen by 50 per cent. The amount of succinate produced was not affected, but the metabolism of exo- genously supplied glucose into succinate dropped from 80 to 60 per cent.

5. Taurodeoxycholate caused a 50 per cent decrease in glucose free pool levels in H. citelli and an 80 per cent reduction of glucose incorporation into glycogen. The amount of succinate produced was reduced slightly with the amount of exogenous glucose metabolized into succinate dropping from 90 to 60 per cent.

6. Radioactive taurocholate was not taken up by H. diminuta in vitro, nor were bile salts detected in worm tissue assayed by several procedures.

7. From these results we concluded that the action of bile salts was on the surface of the worm rather than on some internal metabolic process. These data provide additional evidence for the importance of bile salts in the host- parasite relationship.

I N T R O D U C T I O N

BmCHEMICAL and physiological properties of the host intestine may be determin- ants of host specificity of parasites (Read et al. , 1963). While it is unlikely that a single parameter determines host specificity, evidence has accumulated implicating bile salts as one of the important factors in the life cycle of parasites. Bile salts are required for the excystment or hatching of many parasites (Rothman, 1959; Smyth & Haslewood, 1963 ; Graft & Kitsman, 1965 ; Hibbert et al. , 1969; Tkachuck & MacInnis, 1971). Other work suggests that bile salts are necessary for satisfactory

* Present address: Department of Biological Sciences, Notre Dame University, Notre Dame, Indiana 46556.

993 33

994 RICHARD D. TKACHUCK AND AUSTIN J. MACINNIS

growth and development of tapeworms. Beck (1951) noted that in castrated rats infected with Hymenolepis diminuta, egg production increased if bile was added to the diet. Experiments by Goodchild (1958, 1960, 1961a, b); Vilar-Mvarez & Goodchild (1961); and Goodchild & Vilar-Alvarez (1962), have shown that if bile flow was interrupted in the rat host, changes in chemical composition, growth and reproductivity of H. diminuta occurred. However, Smyth & Haslewood (1963) suggested that the effects on the parasite observed by Goodchild might be secondary reactions to modifications in the host metabolism. Bailey (1965) reported a corre- lation between a change in bile composition and a change in the chemical compo- sition of H. microstoma. Bailey & Fairbairn (1968) and Frayha & Fairbaim (1968) have shown increased lipid absorption in the presence of bile salts.

The detergent properties of bile salts may have adverse effects on excysted tape- worms. Smyth (1962, 1963) and Smyth & Haslewood (1963) examined the effect of concentration and conjugation of various bile salts on the excystment and survival of scoleces of Echinococcus granulosus, and concluded that lyric action of certain bile salts may be an important factor in host specificity. Phifer (1960) showed that 0.5% sodium taurocholate inhibited glucose uptake in H. diminuta. Rothman (1958, 1959) measured the effect of different bile salts on glucose fermentation by H. diminuta and H. citelli. He found that sodium taurocholate reduced anaerobic fermentation in H. diminuta, but had no effect on H. citelli. Bile salts also modify glucose metabolism in other tissues (Holt et al., 1963; Nunn et al., 1963; Faust, 1964; Pope et al., 1966), but the details of the effects on tapeworms are not yet known. The following experiments were undertaken to study the effects of bile salts on carbohydrate metabolism, specifically on the uptake and incorporation of glucose into glycogen, and on the production of succinic acid by two species of hymenolepidid tapeworms. These worms were chosen to represent two different host-parasite systems--H, diminuta in rats and H. citelli in hamsters. Different complements of bile salts are found in the two hosts (Prange et al., 1962; Boyd et al., 1963), thus affording an opportunity for a comparative study that might improve our understanding of host specificity.

MATERIALS AND METHODS Experiments concerned with the effects of taurocholate and taurodeoxycholate on the

metabolism of glucose were patterned after those of Fairbairn et al. (1962) and Scheibel & Saz (1966). Rats were infected with ten cysticercoids of H. diminuta and hamsters with H. citdli. Fourteen days post infection, the worms were flushed from the intestine with Krebs-Ringer-Bicarbonate (KRB), pH 7-1 (Umbreit, 1954). Three worms of H. diminuta or four of H. citelli were placed in 30-ml beakers containing 4 ml of KRB and preincubated for 30 rain at 37°C in a reciprocating water-bath. The worms were then placed in stoppered 25-ml Erlenmcyer flasks containing 9 ml of 17 mM glucose (0"01/zc/pM of uniformly labelled glucose, New England Nuclear Corp., Boston, Mass.) in KRB. Experimental solutions were gassed with Ns-CO~ (95 : 5) for 45 rain before and 10 rain after addition of the worms. After an incubation period of 2~ hr, the worms were removed from the flask, washed three times in KRB and placed in 4 ml of 70% ethyl alcohol. After several days the worms were removed from the alcohol, placed on tared planchets, dried and weighed.

B I L E SALT ~J~'~'~CTS O N CARBOHYDRATE M E T A B O L I S M OF CESTODES 995

Bile salts (obtained from Calbiochem Corp., Los Angeles, Calif.) were "A" grade and chromatographically pure as shown by T L C in solvents described by Hofmann (1962) except for taurodeoxycholate which contained a trace amount ( ~ 1%) of deoxycholate.

Glycogen isolation from dried worm tissue and subsequent quantitative analysis was according to Montgomery as described by MaeInnis & Voge (1970). Glycogen was pre- cipitated repeatedly with cold ethanol until no radioactivity above background could be detected in the supernatant. Aliquots of aqueous glycogen suspensions were dissolved in NCS solution (a quaternary ammonium base used as a tissue solubilizer, Amersham/Searle Corp., Arlington Heights, Ill.) and then added to scintillation fluid.

Succinate produced by the worms was extracted from the incubation media and assayed by the method of Busch et al. (1955). Bio-Rad ion exchange resin (AG 1-X8, 200-400 mesh, formate form, Bio-Rad Laboratories, Richmond, Calif.) was placed in chromatography columns (1 x 30 cm) to a height of 12 cm. Before application of samples, the columns were washed with 100 ml 6 N formic acid followed by 200 ml distilled water. The incubation medium contained bicarbonate ions which could attach to the ion exchange resin; subse- quent elution with acid would form CO~ gas and disrupt the packed columns. Therefore, bicarbonate was removed from the samples as follows: an aliquot of incubation medium was taken to pH 2 with 1 M H3PO~. Steam was passed through the solution until it began to bubble vigorously. The solution was allowed to cool and 1 N NaOH added until a pH of 7-9 was obtained. The sample was then applied to the column. Fifteen ml of distilled water were passed through the column; it was then re-filled with distilled water and attached to a gradient maker, as described by Busch et al. (1955). Each 2-ml aliquot of eluent was taken to dryness in vacuo, suspended in 2 ml distilled water, and titrated with 0"05 N NaOH to a phenolphthalein end-point using a microburet. COlumns were calibrated with known amounts of succinate to determine point of elution and percentage recovery. Complete recovery of the succinate added was obtained. The amount of radioactivity in the succinate was determined by evaporating the contents of tubes containing succinate, suspending the residue in 70% alcohol and determining the radioactivity in a scintillation counter.

Investigation of taurocholate uptake by H. diminuta was by the general methods used for studying transport standardized by Read et al. (1963). Ten-day-old worms were incu- bated in Krebs-Ringer-Tris maleate buffer with 5 mM sodium taurocholate-24-14C (Tracerlab, Waltham, Mass., sp. act. 0-01/~c/pM). Worms were placed in 70% ethanol and radioactivity in aliquots of the ethanol was determined by scintillation counting.

Techniques for the extraction and detection of bile salts from worm tissue are described in context.

R E S U L T S

T o determine wha t effects taurochola te and taurodeoxychola te have on glucose metabol i sm into g lycogen and succinate, H. diminuta and H. citelli were incubated in 17 m M glucose in the presence and absence o f the bile salts. Care was taken to ensure tha t control and experimental g roups were ma tched as closely as possible by wet weight. Af te r a 2½-hr incubat ion, the alcohol extractable free pool, g lycogen and succinate were assayed as descr ibed in the Materials and M e t h o d s section. T h e results (Table 1) descr ibed below show tha t the two species o f w o r m differ in their response to the experimental condit ions.

Glucose metabolism in absence of bile salt

After incuba t ion in the absence o f ei ther bile salt, H. diminuta had 30 per cent less radioactivi ty in the alcohol extractable free pool t han did H. citelli. Since mos t

996 RICHARD D. TKACHUCK AND AUSTIN J. MACINNIS

of the activity is probably in the form of glucose, these results suggest that H. citelli maintains a higher internal level of free glucose.

H. citelliincorporated nearly twice as much glucose into glycogen per g dry worm weight than did H. dirninuta. After a 2½-hr incubation, glycogen extracted from H. diminuta had 1.7 per cent of its weight represented by exogenously incorporated glucose, whereas H. dtelli had 3.2 per cent of its glycogen arising from the external glucose.

TABLE 1--SUMMARY OF GLUCOSE METABOLISM BY H. diminuta AND H. citelli AFTER 2½ h r INCUBATION IN THE PRESENCE OR ABSENCE OF BILE SALTS

Alcohol Specific extractable free Glucose Succinate activity of

pool incorporated into produced succinate (/zM/g dry glycogen (/zM/g dry (counts/min worm wt.) (/zM/g glycogen) worm wt.) per/zM)

H. dirainuta C 120.5 4- 19.2 95 _ 13 199 4- 0 7953 _ 360 T2o 126"7 4- 4.9 113 _+ 10 210 4- 13 8120 4- 220 Tx5 107"24-29"4 104+ 4 1924- 8 79204-120 T10 121"9 4- 6"9 103 4- 4 187 4- 7 8320 4- 100 D2o 85"44- 8"6* 474- 4* 2314- 7 61204-520* D15 88"44- 2"3* 704- 8* 241 4-43 67604-360* Dlo 98"8 __ 15.9 52 4- 8* 203 4- 20 6080 + 660*

H. citelli C 171.3 + 10.0 179 4- 28 137 4- 12 9060 4- 160 T20 131"8+ 3"8* 1034- 6* 1204- 4 9220+540 T15 141"5 + 5"4* 139 4- 18 140 4- 10 9700 4- 120 Tlo 131"6+ 0"3* 1184- 1 1264- 4 96804-120 D2o 84"9+ 0"9* 334- 2* 1004- 7* 6040+140" Dis 86"1+ 3"2* 31 4- 1" 924- 6* 6680+840* Dlo 79"0" 26" 84" 6460"

T, taurocholate; D, taurodeoxycholate; C, control. Number following T or D is concentration in raM.

The amount of metabolism in the free pool is expressed as glucose, although other compounds were probably present. Asterisk indicates groups considered to be different from controls at P > 0'95 as determined by Student's t-test.

Each determination is the mean + S.E. of three samples except two samples for I-I. diminuta Tx~, Dis, Dr0 and Tz0 and Tx0 of H. citelli, and one sample in the D10 of H. citelli. Samples consisted of three worms with H. diminuta, and four worms for H. citelli.

Glucose activity, 20,508 counts/min per ftM. Mean dry weight of samples: H. citelll, 94"1 +1 .4rag ; H. dlminuta, 138.0+3.4mg.

Scheibel & Saz (1966) showed that glucose is metabolized directly to two three- carbon fragments and these to succinate by COs fixation. Therefore, if 1 mole glucose is metabolized to 2 mole of succinate, the amount of activity in 2 mole succinate will be related to the amount of non-radioactive glucose added to the

BILE SALT EFFECTS ON CARBOHYDRATE METABOLISM OF CESTODES 9 9 7

succinate pool from non-radioactive glycogen reserves. H. dtelli produced 30 per cent less succinate than did H. diminuta (Table 1). The specific activity of the succinate shows that 90 per cent of the succinate excreted by H. citelli came from exogenously supplied glucose as compared to 80 per cent with H. diminuta. These results suggest that H. diminuta uses more of its endogenous glucose reserve than H. citelli under control conditions. The greater amount of succinate produced by H. diminuta shows that it has a higher overall glucose utilization rate than H. citelli.

Glucose metabolism in the presence of bile salts

The presence of taurocholic acid had no effect on any parameter measured for H. diminuta, but with H. citelli it reduced the amount of alcohol extractable radio- activity by about 21 per cent, and the amount of glucose incorporated into glycogen by 33 per cent (Table 1). The amount of succinate excreted by H. citelli and the specific activity of the succinate did not differ from controls. From these results it was concluded that endogenous glucose levels in H. citelli were not lowered to such an extent as to require any greater mobilization of the glycogen reserves during the experiments, but were lowered enough to reduce incorporation into glycogen.

Taurodeoxycholate affected the metabolism of glucose in both species. In the presence of tanrodeoxycholate the alcohol extractable free pools were reduced by about 30 per cent in H. diminuta and 50 per cent in H. citelli(Table 1). The amount of glucose incorporated into glycogen decreased by 50 per cent in H. diminuta and about 80 per cent in H. citelli. Suecinate production did not change in H. diminuta but was reduced slightly in H. citelli. The specific activity of the succinate was reduced in both species, indicating decreased utilization of external glucose from 80 to 60 per cent in H. diminuta and a change from 90 to 60 per cent in H. citelli. There was no difference in the amount of glycogen per g dry worm weight, nor was there any difference in the dry worm weights between controls or experimental groups within species so that the differences observed can not be attributed to differences in size.

Attempts to demonstrate bile salts in worm tissues

The above experiments indicate that bile salts modify carbohydrate metabolism, but they do not indicate whether the effect is at the worm's surface, or internally. If the effect is internal it should be possible to show the presence of bile salt in worm tissue. Cramer (1967) stated that she was able to extract bile salts from H. diminuta tissue. Her procedure involved a soxhlet extract of 190 mg of dry worm tissue with chloroform-methanol, saponification with an ether extraction, acidification and ether extraction, yielding a material which gave a positive Gregory-Pasco test, indicating the presence of bile salt. However, when this procedure was followed starting with more than ten times the tissue used by Cramer, no bile salt could be detected. In addition, when the procedures of Levin et al. (1960), Sjovall (1964) and Nakayama (1967) were used to attempt isolation of bile salt from worm tissue, no bile salt could be detected.

998 RICHARD D. TKACHUCK AND AUSTIN J. MACINNIS

Attempts to demonstrate uptake of radioactive bile salt

To further confirm the above observations, experiments were performed to determine if H. diminuta is able to take up taurocholate from the incubation medium. The worms were incubated in 5 mM sodium taurocholate-24-t4C as described in the Materials and Methods section. The results (Table 2) showed no significant uptake of taurocholate. The low amount of radioactivity recovered probably represents bile salt bound to the surface of the worm. From the facts that bile salt could not be extracted from worm tissue, nor were significant amounts of radioactive bile salt taken up by the worm, it was concluded that the effects of bile salts are primarily at the surface and affect the movement of glucose across the surface.

TABLE 2----UPTAKE OF TAUROCHOLATE BY 10-DAY-OLD Hymenolepis diminuta INCUBATED IN 5 m M TAUROCHOLATE-24-14C SPECIFIC ACTIVITY, 0"01 f tc/f tM

I n c u b a t i o n t ime (min)

Incubation medium n-moles uptake per (ml) mg dry worm wt.

2 4 0"24 + 0"04 (4) 30 8 0"82 + 0"07 (2) 60 12 1-04 + 0"04 (4)

Five worms per sample. Figures in parentheses indicates number of samples used.

DISCUSSION

Smyth & Haslewood (1963) suggested that bile salts may act as agents of host specificity in three different manners: (1) as a stimulant to hatching or excystment of resting stages; (2) by acting lyrically and destroying the parasite; and (3) by stimulating metabolism of the parasitic organism. Our results support and amplify this hypothesis. The present in vitro experiments show that bile salts affect the metabolism of the two species in different ways. H. diminuta is exposed to tauro- cholate and taurochenodeoxycholate in the rat intestine. H. citelli encounters both the glycine and taurine conjugates of cholic, chenodeoxycholic and deoxycholic acids in the hamster intestine. Voge (1956) stated that the rat was an unsuitable host for H. citelli and that hamsters could be laboratory hosts as a substitute for ground squirrels from which this worm was originally isolated. H. diminuta was seemingly affected only in the presence of a non-host bile salt, whereas H. citelli was affected by salts normally found in hamster bile. The inhibitory action of taurodeoxycholate on H. diminuta could be explained by the fact that this bile salt is not normally encountered by this worm. This interpretation must be viewed with caution, since H. citelli was affected by bile salts it normally encounters. Read & Phifer (1959) showed that although H. diminuta can establish in hamsters, they are smaller than worms from rats, implying that H. diminuta is not limited merely by the presence or absence of a single bile salt. H. citelli, which develops

B I L E SALT ~ ~ C T S O N CARBOHYDRATE M E T A B O L I S M OF CESTODES 999

poorly in the rat, is not exposed to any bile salt different than that encountered in the hamster, therefore factors which modify the effects of bile salts must be sus- pected. It is probable that within the intestine, interactions with certain compo- nents of the intestinal contents with bile salts could reduce and modify the activity of the bile salt on the worm, and this interaction may determine what role bile salts play in host specificity. Bile salts in vertebrates solubilize lipids by forming lipid- bile salt complexes or micells. Since the activity of a bile salt is proportional to the number of individual molecules, reduction of the number of bile salt molecules by lipid micell formation would reduce the overall detergent action of the bile salt (Dietschy, 1967). It would be imeresting to determine if the action of bile salts observed in present work could be modified by the addition of lipids to the incu- bation medium.

Our results indicating that H. citelli maintained a higher internal pool of alcohol extractable radioactive material than did H. diminuta are consistent with Read's (1967) observation that H. citelli takes up more glucose from the medium than H. diminuta. However, it should be noted that the average weight per worm for H. citelli was 23 mg and 47 mg for H. dirninuta. This difference in size may have an affect on the rate of glucose uptake by these worms, and may account for the difference observed. Our data show that H. citelli produced less succinate than did H. diminuta. Since succinate production is a measure of glucose utilization, our observations further confirm the works of Rothman (1958, 1959) and Read & Rothman (1958), which show that H. diminuta has a higher rate of glucose utiliza- tion than H. citelli.

The specific activity of the succinate shows that most of the glucose metabolized to succinate by both species comes from exogenous glucose in the medium and that only a small percentage comes from endogenous glucose or glycogen. This suggests that the worms continuously metabolize glycogen and thus add non-radioactive glucose to the metabolic pool. It is doubtful that the incubation medium did not supply enough glucose to meet the worms' requirements since only about one- tenth of the glucose (sum of glucose metabolized into glycogen and succinate per worm sample) in the incubation medium was utilized over the entire incubation period. H. citelli incorporated twice as much glucose into glycogen than did H. diminuta. Although this observation is consistent with the results of Read (1967), the difference he reported is not as great.

Some of the results reported by Rothman (1958, 1959) do not agree with our findings. He reported that taurocholate affected glucose fermentation in H. diminuta but not in H. citelli, while we found the opposite, that H. citelli is affected by taurocholate. Rothman's assay depended upon the detection of excretion of acidic compounds into the medium. Succinate makes up about 70 per cent of the acids excreted by H. diminuta with the remainder made up of lactic, acetic and other keto acids (Fairbairn et al., 1962). The amount of succinate produced did not change in the presence of taurocholate with respect to controls in the present study. Therefore the reduced acid excretion noted by Rothman for H. diminuta could be a result of a reduction in lactic, acetic or keto acid production. The

1000 RICHARD D. TKACHUCK AND AUSTIN J. MACINNIS

possibility of a contaminant in the bile salts used by Rothman would probably not explain the difference observed, since he showed that bile salts had no effect on H. citelli, and our studies show that H. citelli is more sensitive to bile salts than H. diminuta. Further work is required to determine the cause of the difference.

Bile salts have been shown to affect glucose metabolism in two ways (Dietschy, 1967): by inhibiting glucose transport through the cell membrane, and/or by affecting intracellular metabolic activities. Inhibition of glucose transport and metabolism by bile salts has been reported on several occasions (see review, Pope et al., 1966; Dietschy, 1967). However, the reports reviewed are conflicting with respect to which bile salts cause the effect and at what concentrations. Pope et al. (1966) showed that the glycine and taurine conjugates of cholic acid had no effect on glucose metabolism in intestinal mucosal tissue and that the effects observed by previous workers were due to impure bile salt preparations. Pope et al. (1966) found that a concentration of 0.2 mM deoxycholate would give the effects described to higher concentrations of taurocholate. No studies conducted by them concerned the effect of taurodeoxycholate on glucose uptake so it is not known if the taurine conjugation modifies the effect noted. Work discussed by Dietschy (1967) has shown that unconjugated bile salts appear in the small intestine of the rat due to bacterial action upon conjugated bile salts and that these bile salts may attain a concentration of about 2 raM. He has also shown that the effect of unconjugated bile salts is lessened by the presence of conjugated forms due to micellar formation with the conjugated forms. Since the taurodeoxycholate used in the present study contained a trace of deoxycholate, it is not possible to say definitely that the con- jugated forms are responsible for the results observed. However, we feel that the effects observed are not due to the presence of the contaminant for the following reasons. Effects were observed at a concentration of 10 mM taurodeoxycholate. The deoxycholate contaminant at this level of taurodeoxycholate was at a concen- tration of about 0-1 mM. The concentration of deoxycholate within the gut of the rat is about ten times this value. H. citelli was affected by taurocholate which had no deoxycholate contaminant, indicating that a worm can be affected by a con- jugated bile salt and not only by unconjugated forms. This view is also supported by Smyth & Haslewood (1963) who showed that taurodeoxycholate caused lysis of Echinococcus larvae. Since unconjugated bile salts appear within the gut of the host, further studies are necessary to determine precisely the effects of these two forms of bile salt on tapeworm metabolism of glucose.

The majority of evidence presented in this paper supports the proposal that bile salts modify glucose metabolism in tapeworms by a surface action resulting in reduced permeability of glucose. However, if bile salts do enter the tissues (possi- bly at concentrations not detectable by the procedures employed) one may speculate that the internal presence of the bile salt might cause reduced phosphorylation, thus lower ATP levels leading to less glycogen synthesis and less ATP for active trans- port, leading to lower free pool levels of glucose. Since no radioactive taurocholate was taken up by H. diminuta, nor were bile salts detected in the worms, the results

BILE SALT EFFECTS ON CARBOHYDRATE METABOLISM OF CESTODES 3001

of the present work lend suong suppor t for action of the bile salts on outer m e m - branes.

T h e complete action of bile salts upon hymenolepidid cestodes remains unclear. T h e data show that bile salts have the potential of restricting the metabol ism of glucose markedly. I t is obvious that the interaction between the intestine, its contents and the parasitic worm is complex, and that much remains to be learned before a complete understanding is acquired.

Acknowledgements--This research was supported by grants from the National Institutes of Health (AI-00070) and the National Science Foundation (GB8753).

REFERENCES BAILEY H. H. (1965) The effect of in vivo alteration of bile acid composition on Hymenolepis

microstoma. Doctoral dissertation, University of Oklahoma. BAILEY H. H. & FAI~AIRN D. (1968) Lipid metabolism in helminth parasites--V. Absorp-

tion of fatty acids and monoglycerides from micellar solution by Hymenolepis diminuta (Cestoda). Comp. Biochem. Physiol. 26, 819-836.

BECK J. W. (1951) Effect of diet upon singly-established Hymenolepis diminuta in rats. Expl Parasit. 1, 46-59.

BOYD G. S., EASTWOOD M. A. & K~MACK B. (1963) Bile salt metabolism in the rat. In Biochemical Problems of Lipids (Edited by FRAZER A. C.), pp. 24-36. Elsevier, Amster- dam.

BuscH H., HtmLBERT R. B. & Pom~n V. R. (1952) Anion exchange chromatography of acids of the citric acid cycle. J. biol. Chem. 196, 717-727.

CRAM~R A. L. (1967) Effects of dietary lipid alteration and infection with Hymenolepis diminuta (Rudolphi, 1819) in the rat. Doctoral dissertation, Emory University, Georgia.

DI~rscHY J. M. (1967) Effects of bile salts on intermediate metabolism of the intestinal mucosa. Fedn Proc. Fedn Am. Socs exp. Biol. 26, 1589-1598.

FAmRAIRN D., WEnTHEIM G., I-IABPUR R. P. & SCHILLER E. L. (1961) Biochemistry of normal and irradiated strains of Hymenolepis diminuta, Expl Parasit. 11, 248-263.

FAUST R. G. (1964) Effects of bile salts, sodium deoxycholate, strophanthin-G and metabolic inhibitors on the absorption of D-glucose by the rat jejunum, in vitro..~, cell. comp. Physiol. 63, 55-64.

FRAYHA G. J. & FAIm3AIRN D. (1968) Lipid metabolism in helminth parasites--VII. Absorp- tion of cholesterol by Hymenolepis diminuta (Cestoda). ~. Parasitol. 54, 1144-1146.

GOODCHILD C. G. (1958) Growth and maturation of the cestode Hymenole~'s diminuta in bileless hosts. ~. Parasitol. 44, 352-362.

GOODCHILD C. G. (1960) Effects of starvation and lack of bile upon growth, egg production and egg viability in established rat tapeworms, Hymenolepis diminuta..7. Parasitol. 46, 615-623.

GOODCHILD C. G. (1961a) Carbohydrate contents of the tapeworm Hymenolepis diminuta from normal, bileless, and starved rats. ~. Parasitol. 47, 401--405.

GOODCHILD C. G. (1961b) Protein contents of the tapeworm Hymenolepis diminuta from normal, bileless, and starved rats. ~. Parasitol. 47, 830-832.

GOODCHZLD C. G. & VILAR-ALVA~Z C. M. (1962) Hymenolepis diminuta in surgically altered hosts--II . Physical and chemical changes in tapeworms grown in shortened small intestines. ~7. Parasitol. 48, 379-383.

GRAVF D. J. & KXTZMAN W. B. (1965) Factors influencing the activation of acanthocephalin cystacanths. ~. Parasitol. 51, 424-429.

1002 RICHARD D. TKACHUCK AND AUSTIN J. MACINNIS

HIBBERT L. E., HAMMOND D. M. & SIMMONS J. R. (1969) The effects of pH, buffers, bile and bile acids on excystation of sporozoites of various Eimeria species, ft. Protozool. 16, 441 A. I A..

HOFMANN A. F. (1962) Thin-layer adsorption chromatography of free and conjugated bile acids on silicic acid. a~. Lipid Res. 3, 127-128.

HOLT P. R., Ha~SSLER H. A. & ISSELBACHER K. J. (1963) Effects of bile salts on glucose metabolism by slices of hamster small intestine, ft. clin. Invest. 42, 777-786.

LEVIN~ S. L., JOHNSTON C. G. & BOYLE A. J. (1961) Spectrophotometric determination of several bile acids as conjugates, extraction with ethyl acetate. ~lnalyt. Chem. 33, 1407- 1411.

MAclNNIS A. J. & VOGE M. (1970) Experiments and Techniques in Parasitology. Freeman, San Francisco.

NAKAYAMA F. (1967) Quantitative microanalysis of bile. ft. Lab. clin. Med. 69, 594-- 609.

NUNN A. S., BAKER R. D. & SmRLS G. W. (1963) Inhibition of intestinal glucose absorption by bile salt. Life Sci. 2, 646-650.

PHIFER K. (1960) Permeation and membrane transport in animal parasites: the absorption of glucose by Hymenolepis diminuta, y. Parasitol. 46, 137-144.

POPE J. L., PARKINSON T. M. & OLSON J. A. (1966) Action of bile salts on the metabolism and transport of water-soluble nutrients by perfused rat jejunum in vitro. Biochim. biophys. Acta 130, 218-232.

PRANGE I., CHRISTENSEN F. & DAM H. (1962) Alimentary production of gallstones in hamsters - - I I . Relation between diet and composition of the bladder bile. Z. Ernahr- Wiss. 3, 59-78.

READ C. P. (1967) Carbohydrate metabolism in Hymenolepis (Cestoda). ft. Parasitol. 53, 1023-1029.

READ C. P. & PHIFER K. (1959) The role of carbohydrates in the biology of ces todes--VII . Interactions between individual tapeworms of the same and different species. Expl Parasit. 8, 46-50.

READ C. P. & ROTHMAN A. H. (1958) The role of carbohydrates in the biology of cestodes-- VI. The carbohydrates metabolized in vitro by some cyclophyllidean species. Expl Parasit. 7, 217-223.

READ C. P., ROTHMAN A. H. & SIMMONS J. E. (1963) Studies on membrane transport, with special reference to parasite-host integration. Ann. N .Y . Aead. Sci. 113, 153-205.

ROTHMAN A. H. (1958) Role of bile salts in the biology of tapeworms-- I . Effects on the metabolism of Hymenolepis diminuta and Oochoristica symmetries. Expl Parasit. 7, 328- 337.

ROTHM~'~ A. H. (1959a) Studies on the exeystment of tapeworms. Expl Parasit. 8, 336- 364.

ROTHMAN A. H. (1959b) The role of bile salts in the biology of t apeworms-- I I . Fur ther observations on the effects of bile salts on metabolism, a q. Parasitol. 45, 379-383.

SCX-mIBEL L. W. & Saz H. J. (1966) The pathway for anaerobic carbohydrate dissimilation in Hymenolepis diminuta. Comp. Biochem. Physiol. 18, 151-162.

SJOVALL J. (1964) Separation and determination of bile acids. Meth. Biochem. Anal. 12, 97-141.

SMYTH J. D. (1962) Lysis of Echinococcus granulosus by surface active agents in bile and the role of this phenomenon in the determination of host specificity in helminths. Proc. R. Soc. B 156, 553-572.

SMYTH J. D. (1963) The biology of eestode life-cycles. Technical Communication No. 34, Commonwealth Bureau of Helminthology.

SMYTH J. D. & HASL~VOOD G. A. D. (1963) The biochemistry of bile as a factor in deter- mining host specificity in intestinal parasites, with particular reference to Echinococcus granulosus. Ann. N .Y . .4cad. Sci. 113, 234-260.

BILE SALT EFFECTS ON CARBOHYDRATE METABOLISM OF CESTODES 1 0 0 3

TKACHUCK R. D. & MACINNIS A. J. (1971) The role of bile salts in the excystment of cysti- cercoids. (In preparation.)

UMnR~IT W. W., Btnmxs R. H. & STAVTnm J. F. Ed. (1964) Manometric Techniques, 4th edn. Burgess, Minneapolis.

VILAR-ALvAREZ C. M. & GOODCHILD C. G. (1961) Hymenolepis diminuta in surgically altered hosts--I. Attempts to cultivate cestodes in isolated sections of the small intestine: observations on changes in the isolated and rejoined intestines, j~. Parasitol. 47, 53-60.

VooE M. (1956) Studies on the life history of Hymenolepis citelli (Cestoda: Cyclophyllidea) (McLeod, 1933). ~. Parasitol. 42, 485-490.

Key Word Index---Bile salts; taurocholic acid; taurodeoxycholic acid; glucose meta- bolism; succinate production; tapeworm physiology; host specificity; Hymenolepis diminuta; Hymenolepis citelli.