The in vitro fermentation of carbohydrates by two species of cestodes and one species of acanthocephala

EXPEBIMENTAL

6, 245-260 (1967)

PARASITOLOGY

The in Vitro Fermentation of Carbohydrates by Two Species of Cestodes and One Species of Acanthocephala’ John S. Laurie2 School of Hygiene and Public Health, Johns Hopkins Baltimore, Maryland (Submitted

for

publication,

11 September

University, 1956)

There is considerable evidence available indicating that several species of cestodes utilize glucose (Friedheim and Baer, 1933; Hopkins, 1952; Markov, 1939; Read, 1956; Van Grembergen, 1943-‘45; Wardle, 1937). Arabinose, galactose, glucosamine, fructose, and maltose do not appear to be converted into polysaccharide by Moniezia expansa whereas glucose is (Wardle, 1937). Read (1951) has presented evidence for the existence of a phosphorylative, glycolytic system in the tissues of Hymenolepis diminuta. Although Ward (1952) has shown that the anaerobic, in vitro utilization of endogenous glycogen by adult, female Macracanthorhynchus hirudinaceus is greater than the aerobic utilization, no data on the utilization of exogenous carbohydrates by acanthocephalans seems to be available. The present study has been carried out to obtain information concerning the ability of these helminths to ferment carbohydrates that might be present in the intestine of the host and the effect of known or potential inhibitory reagents on the fermentative metabolism of the worms. MATERIALS

AND METHODS

The substrates were obtained from commercial sources with the exception of glucosyloxime (Shriner and Fuson, 1948), cr-methylglucoside (Helferich and Schafer, 1926), gluconic acid and mucic acid (Bates, 1942), 1 These studies were carried out under the direction of Dr. Clark P. Read and were aided by a contract between the Office of Naval Research, Department of the Navy, and The Johns Hopkins University, N.R. 119-353. 2 Present address: Institute of Parasitology, MacDonald College, McGill University, Canada.

245

246

LAURIE

and dibromophlorizin (Misaki, 1925), which were prepared in the laboratory, and 3@methyl-n-glucose and 2-deoxy-n-glucose that were provided as gifts. Hymenolepis diminuta was maintained in the McCollum strain of rats, Oochoristica symmetrica in the Princeton strain of white mice, and the acanthocephalan, Monilijormis dub&s Meyer 19333, in both the McCollum strain and the Wistar strain of rats. The worms were allowed to grow at least to sexual maturity in the rodent hosts, 5 weeks for M. dubius (Moore, 1946) and 3 weeks for the cestodes, before being recovered for use. Infected rats were starved for 24 hours and infected mice for 21 hours before being killed by a blow on the head. The fasting period reduces the polysaccharide content of the tapeworms (Read, 1949, Reid, 1942), and, since such worms have a higher metabolic rate (Read, 1956), provides a clearer demonstration of the fermentation of the active substrates. The shorter fasting period for the mice reduces the incidence of cannibalism amongst the mice, and also prevents the shedding of the strobila by the worms, which occurs when the hosts are subjected to longer periods of starvation. The phenomenon of strobilar shedding as a result of starvation of the host has been reported for Raillietina cesticillus, a tapeworm in chickens (Reid, 1940). The worms were removed from the intestine of the host, cleaned of debris and placed into small Erlenmeyer flasks, one to three tapeworms or four to five acanthocephalans per flask, containing about 10 ml of Krebs-Ringer bicarbonate adjusted to pH 7.4 for atmospheric conditions. The flasks were partially immersed in a 38’C water-bath and allowed to shake slowly for 45 minutes. The salt solution was replaced at 15minute intervals. After being washed each worm was transferred to the main compartment of a Warburg flask containing Krebs-Ringer bicarbonate. The initial pH of this buffer was calculated to be 7.4 with a 5 % concentration of carbon dioxide in the gas phase. The flasks were attached to manometers, gassed with a 95 % Nz-5 % COZ gas mixture for 7 minutes, and equilibrated in the water-bath for an additional 4 minutes prior to measuring gas evolution. The worms were allowed to incubate in the buffer for 20 minutes before a substrate was added. The rate of acid production during this time was assigned a value of 100 and the total rate of acid production in the presence of the substrate in question was ser,

a Van Cleave 1811).

(1953)

considers

this

to be

B synonym

of M.

moniliformis

(Brem-

HELMINTH

METABOLISM

247

then compared with this initial rate. These relative values are included in Tables I to V. The actual rates of acid production in the presence or absence of exogenous substrates are presented in Table VI. At the termination of each experiment the worms were dried to a constant weight at 90°C. The &acid (~1 acid/mg dry worm/hour) has been calculated from the dry weight of the worms and the carbon dioxide evolved from the buffer. In a reaction volume of 3.0 ml a substrate concentration of 30 PM/ml was used, except for glucuronic acid and glucosamine which were reduced to 20 PM/ml (for H. diminuta only), fructose-l, 6-diphosphoric acid to 24 PM/ml, and 3-methoxyglucose, 2-deoxyglucose and lyxose to lOpM/ml. Lactic acid was estimated by the method of Barker and Summerson (1941). Salicin and a-methylglucoside, apparently are not removed by the treatment with the alkaline copper solution and interfere in this method. The curves in Fig. 1 have been computed by the method of least squares. An average dry weight to live weight conversion factor of 5.88 has been given for H. diminuta by Beck (1951). A similar conversion factor for 0. symmetrica based on data from 78 worms is 4.14 (0.21). Conversion factors for M. dub& based on data from 320 males and 281 females are 4.23 (0.47) for the males and 4.43 (0.48) for the females. The values in parentheses for 0. symmetrica and M. dubius are standard deviations. Metabolic Gas Determination

It has been shown that H. diminuta does not produce metabolic gas under anaerobic conditions (Read, 1956). To determine whether 0. symmetrica produces metabolic gas, 21 worms were incubated, singly, in Krebs-Ringer bicarbonate for 20 minutes, glucose was then added and the incubation continued for an additional 70 minutes. At the end of the incubation period acid was added from a side-arm, in a stepwise manner, until the bicarbonate was completely decomposed. Another series of 18 Warburg flasks, containing the reaction mixture only, was carried through the above procedure except that the incubation periods were eliminated. By the use of Student’s “t” test the mean volume of gas found in these flasks was compared with the mean volume of gas found in the 21 flasks containing the worms. No statistically significant difference (P = 0.68) exists between these volumes; therefore, it is con-

248

LAURIE

eluded that 0. symmetrica did not produce metabolic gas under the conditions employed. This procedure was repeated with M. dub& using 17 males and 16 females, except that several substrates were employed in addition to glucose. There is no statistically significant difference between the mean gas volume of the flasks containing these worms and the mean gas volume of the control flasks; therefore, it is concluded that neither M. dub&s males (P = &. 08 ) or females (P = 0.77) produced metabolic gas under the conditions employed. From these results it may be considered that the carbon dioxide formed is equivalent to the number of acid groups, stronger than carbonic acid, liberated into the medium by the worms. CARBOHYDRATE

FERMENTATIONS

The rate of acid evolution from H. diminuta is essentially equivalent to the rate of glucose disappearance from the medium (Read, 1956); therefore, an increased rate of carbon dioxide evolution, subsequent to the addition of the substrate in question, is considered to indicate that the substance is being fermented to acidic end products. Glucose and galactose are fermented, at least in part, to acidic catabolites by H. diminuta (Table I). The response of these animals to exogenous mannose and xylose is somewhat equivocal in that the &acid in the presence of these substances is not increased very much beyond that of the controls. The sugar alcohols sorbitol, dulcitol, and mannitol, and the disaccharides lactose, maltose, sucrose, and trehalose are not fermented, nor is arabinose, glucosamine, glucosyloxime, fructose, glucantime, 3methoxyglucose, 2-deoxyglucose, cY-methylglucoside, salicin, i-inositol, mucic acid, glucuronic acid, gluconic acid, glycerophosphoric acid, or fructose-l ,6-diphosphoric acid. At concentrations of 0.005 M and 0.01 M, 2-deoxyglucose does not inhibit the fermentation of glucose (0.01 M and 0.06 M) by H. diminuta. In contrast, this derivative of glucose rapidly enters the extrahepatic tissues of eviscerated rabbits and inhibits the penetration of glucose into these tissues (Wick, Drury, and Morita, 1955). Also, 3-methoxyglucose (0.005 M and 0.03 M) does not inhibit the fermentation of glucose (0.01 M and 0.06 M) by this tapeworm. Lactic acid has been found to account for 37 % to 98% of the total acid excreted by H. diminuta in the presence of exogenous glucose. 0. symmetrica ferments glucose and galactose (Table I) but not sorbitol, dulcitol, mannitol, arabinose, xylose, fructose, glucosamine, glu-

HELMINTH

249

METABOLISM

TABLE I The Relative Rate of Acid Production by Helminths f an Exogenous Substrate Worms incubated in Krebs-Ringer bicarbonate (pH 7.4). Substrate added from the side-arm to yield a final concentration of 0.03 M. Gas phase 95% Nt-5% CO* . 38°C. Time

1 Additions

1 Glucose

1 Galactose

1 Maanose

( Xylose

1 Fructose

1 Maltose

/Eodogenaus

H. diminuta O-20 20-50 50-80

none sugar sugar nb

100~ 422 540 7

100 167 162 6

100 119 106 8

100 139 128 5

-

-

106 75 59 14

-

-

-

100 55 46 10

0. symmetrica O-20 20-50 60-90

none sugar sugar n

100 311 327 49

loo 197 172 8

100 118 70

8 M. dubius

O-20 20-50 60-90

none sugar sugar n

a The Qscid during b n = the number

$8 100 198 196 11

99 100 479 510 9

the preliminary of observations.

dd 109 177 157 12

PO 100 179 143 9

incubation

0-d 100 249 218 12

99 ddOO 100 - 406 - 403 - 8--

period

is assigned

88 100 383 397 9 a relative

99 100 546 586 9

dd 106 137 163 8

value

99 dd 99 100 100 100 383 47 44 442 45 44 9 15 9

of 100.

cosyloxime, 3-methoxyglucose, 2-deoxyglucose, cu-methylglucoside, mucic acid, glucuronic acid, gluconic acid, or the disaccharides lactose, maltose, sucrose and trehalose. The fermentability of mannose is questionable (Table I). Unless otherwise specifically designated, all results to be given for M. dubius apply to both males and female. These worms have been found to ferment glucose, galactose, mannose, fructose, and the disaccharide maltose (Table I). There is no indication that the disaccharides cellobiose, lactose, melibiose, sucrose, trehalose and turanose are fermented, nor is sorbitol, dulcitol, mannitol, glycerol, i-inositol, raffinose, arabinose, xylose, lyxose, rhamnose, glucosamine, glucosyloxime, (Y-

250

LAURIE

methylglucoside, 3-methoxyglucose, 2-deoxyglucose, sorbose, mucic acid, glucuronic acid, gluconic acid, glycerophosphoric acid, or fructose-l, 6diphosphoric acid. Inasmuch as chitin is considered to be present in the inner egg membranes of Macracanthorhynchus hirudinaceus (von Brand, 1940), it is possible that glucosamine may be taken up by M. dubius females and incorporated into the egg membranes. The possibility that the various substrates may be metabolized by any of the above species of worms, completely or partially, to non-acidic catabolites that are not accounted for by the measuring technique, cannot be ruled out. It should be kept in mind that the above results apply only with a gas phase of nitrogen and carbon dioxide. An alteration in the gas phase may change the results obtained with a given substrate, e.g., it was shown that with air as the gas phase, glycerophosphoric acid increases the rate of oxygen consumption of the tapeworms, Moniexia benedeni (Van Grembergen, 1943-‘45) and H. diminuta (Read, 1956). EFFECTS OF POTENTIAL INHIBITORY

AGENTS

Glucosides Abderhalden and Effkemann (1934) have reported that salicin inhibits glucose absorption by the rat intestine, and that arbutin inhibits phosphorylation in frog muscle, glucose reabsorption by frog kidneys, and glucose absorption by the rat intestine. Phlorizin has been reported to inhibit the absorption of several sugars from the vertebrate intestine (Bogdanove and Barker, 1950; Bruckner, 1951). This inhibition can be reversed by rinsing the intestine with Ringer’s solution (Ohnell and HBber, 1939). Phlorizin also inhibits reabsorption of sugars by the vertebrate kidney, with the site of inhibition being localized in the proximal tubules (Ellinger and Lambrechts, 1937; Reinecke, Rudolph, and Bryson, 1947; Richards, 1934; Walker and Hudson, 1937). It does not block the diffusion of glucose out of the kidney tubule (Richards, 1934), or the diffusion of glucose and other monosaccharides out of the peritoneal cavity (Wertheimer, 1934). Phlorizin has been reported to interfere in the phosphate metabolism of various animal tissues by blocking such reactions as the phosphorolysis of glycogen but it does not inhibit the formation of hexose diphosphate from hexose monophosphate, or the dissimilation of hexose diphosphate to lactic acid (Kaplan and Greenberg, 1944; Parnas, Mejbaum and Sobczuk, 1936; Shapiro, 1947). This glucoside decreases acid production by sea-urchin eggs (Rothschild, 1939) and partially inhibits glucose fermentation by yeast (Wertheimer,

HELMINTH

251

METABOLISM

TABLE

II

The E#ect of Glucosides (2.18 X IO-’ M) on the Relative Rate of Acid Production by Tapeworms in the Presence of Ezogenous Sugars Conditions as in Table I

-

H. diminuto

Additions

Time

Glu- Galaccase tose --

-O-20 20-50 50-80 60-69

-

Phlorizin

none sugar glucoside glucoside n

100 636 354 4

100 212 163 7

0. symmetrica

Dibromophlorizin Gluaxe --

106 348 308 256 8

Galacme

100 251 213 148 8

Pblorizin

Dibromophlorizin

Glu- G&cGlu- GalacCOSC tose case tase ----

100 302 _ 318 12

100 179 143 9

100 360 380 8

100 189 131 8

1934). Dibromophlorizin produces a glycosuria in the mammalian kidney (Lambrechts, 1937). Salicin (0.015 M), lactose (0.01 M and 0.02 M), glucantime (0.015 M), esculin and amygdalin (2.12 X 10m4M) do not inhibit the fermentation of glucose by H. diminuta, nor does lactose, at the concentrations stated, inhibit the fermentation of galactose. Arbutin (2.12 X 10e4 M) does not inhibit the fermentation of glucose and galactose by either H. diminuta or 0. symmetrica. At a concentration of 12.69 X 10e4 M, arbutin still has no effect on the fermentation of glucose by H. diminuta. Phlorizin inhibits the fermentation of glucose and galactose by H. diminuta and galactose fermentation by 0. symmetrica but has no influence in the fermentation of glucose by 0. symmetrica (Table II), or on the fermentation of glucose, mannose, fructose, and maltose by M. d&us. Analysis of the data from experiments with a glucose-H. diminuta-phlorizin system by the method of Lineweaver and Burk (1934) demonstrates that the inhibition possesses the characteristics of the noncompetitive type (Fig. 1). Dibromophlorizin inhibits the fermentation of glucose and galactose by H. diminuta and galactose fermentation but not glucose fermentation by 0. symmetrica (Table II). The fermentation of glucose, mannose, fructose, and maltose by M. dubius is unaffected by this glucoside.

LAURIE

control

I

I

I

2( ) 00 l/S FIG. 1. The effect of phlorisin on the production of acid by H. diminuta in the presence of exogenous glucose. Worms incubated in Krebs-Ringer bicarbonate (pH 7.4) + glucose f phloriain for one hour. Gas phase 95% N,-5% CO2 . 38°C. 1. l/v = 0.048 + 0.000014(1/5 - 78), 0 M phloriain, s = 0.005, n = 10. 2. l/v = 0.078 + 0.000228(1/S - 78), 2.12 X RF M phlorizin, s = 0.019, n = 15. 3.1/v -= 0.108 +0.000583(1/S - 78), 3.17 X 10d4M phlorizin, s = 0.043, n = 10. 4. i/v = 0.130 + 0.000644(1/5 - 65), 4.23 X lo-4 N phlorizin, s E 0.046, n = 18. 5. l/v = 0.142 $0.090676(1/S - 72), 6.35 X 10m4M phlorizin, s = 0.054, n = 19. v = &acid , S = molar substrate cone., s = standard deviation, n = number of observations. 1725

50

Reversal of Phlorizin

Inhibition

To test for the reversibility of phlorizin inhibition the following procedures were carried out: Individual worms, H. diminuta, were incubated anaerobically in the buffer for 20 minutes prior to adding glucose and phlorizin. After 30 minutes incubation the worms were

HELMINTH

253

METABOLISM

TABLE III The Reversal of Phlorizin (2.12 X 1W M) Inhibition Glucose Fermentation by H. diminuta

of

Conditions as in Table I. See text for procedures. The O-20 20-50 030

Additions

none glucose + phlorizin glucose n

Part A

Part B

100 328 682 8

100 491 815 12

transferred, singly, to flasks containing 10 ml of Krebs-Ringer bicarbonate (pH 7.4 in air) and washed with slow shaking in the 38” water bath for 15 minutes (part A), or for 3 minutes (part B). During the washing period the buffer was replaced at 5-minutes intervals (part A), or at l-minute intervals (part B). At the end of the washing procedure the worms were transferred to a second group of Warburg flasks and the latter were attached to manometers. The system was gassed and equilibrated, glucose was tipped into the main compartment, and the worms were allowed to incubate for 30 minutes. The time elapsing between the end of the first 30-minute incubation and the beginning of the second was about 35 minutes for part A and about 12 minutes for part B. The results demonstrate that the phlorizin inhibition of glucose fermentation by this worm may be reversed readily by brief washing (Table III). Sulfhyclryl Inhibitors

Iodoacetic acid and p-chloromecuribenzoic acid ( 10m3 M) completely block glucose fermentation by H. diminuta (Read, unpublished); however, only the latter exerts an inhibitory affect of the fermentation of hexoses by M. d&us. The p-chloromecuribenzoic acid solution absorbed carbon dioxide during the first 20 minutes. This was indicated by the appearance of a precipitate in the side-arm and was of sufficient magnitude to obscure any evolution of carbon dioxide from the buffer. The net changes in the gas volumes of the system were nil or occasionally slightly negative. This has made it necessary to assign the &acid for the 20-50-minute time interval the arbitrary value of 100 (Table IV). Heterocyclic Bases

Culbertson (1940) reported on the expulsion of tapeworms from mice after the administration of quinacrine and commented on the color imparted to the worms by this chemical. Mustakallio and Saikkonen

254

LAURIE

TABLE The

Eflect

of Iobdoacetic Relative

Concentration

of the halogen substituted Iodoacetic

Time

Additions

* The pCMBA

none sugar acid n precipitated

acids, 10-a M. Conditions acid

Glucose

100 497 525 5

as in Table I

Glucose

QQ

cFd

QQ

100 397 472 3

100 302 372 5

100 531 556 3

from solution

on the

pCMBA Fructose

dd

O-20 20-50 60-90

IV

acid and p-Chloromecuribenzoic Acid (pCMBA) Rate of Acid Production by M. dubius in the Presence of Ezogenoua Sugars

in the sidearm

during

thii

Fructose

$6’

QQ

cl-8

-* 100 58 7

100 65 3

100 52 4

time.

(1954) have established that this chemical penetrates into the tissues of tapeworms and that it exhibits an affinity for muscle, reproductive tissue and nuclei. Quinacrine inhibits glycolysis in Schistosoma mansoni (Bueding, 1950) and in frog muscle (Derouaux and Lecomte, 1950). Quinacrine inhibits the fermentation of glucose and galactose by 0. symmetrica and glucose fermentation by H. diminuta; whereas acriflavine only inhibits the fermentation of glucose (Table V). Chloroquine diphosphate blocks the fermentation of glucose by both tapeworm species and the fermentation of galactose by 0. symmetrica. Thorazine is a moderately effective inhibitor of glucose and galactose fermentation by these TABLE The Effect of Heterocyclic Bases Acid Production by Tapeworms

V

(i.26 X W3 M) on the Relative in the Presence of Exogenoue

Rate Sugars

of

The concentration of acriflavine is based on the assumption that the commercial preparation used was $5 contaminated with proflavine (Albert, A., 1949, p. 320). Conditions as in Table I. 0. symmetrica Quinacrine Tim.2

O-20

20-50 60-90

Additions

none sugar base n

Glucose

100 286 70 9

Il. dininrtfa Acriflavine

Galactose

100 218 80 7

Quinacrine

Acriflavine

Glucose

Galactose

Glucose

Glucose

Galactose

100 191 103 9

100 165 161 10

100 595 177 6

100 495 342 7

100 282 282 8

HELMINTH

255

METABOLISM

TABLE VI The Effect of Exogenous Carbohydrates on Acid Production by Helminths Time 20-80 (H. diminuta only) or 20-90. Conditions aa in Table I. I

mean &id

Animal GlUCOSt!

H. 0. M. hf. l

diminuta symmetrica at&us CM dubiu.3 0 0 The figures

22.96 18.37 14.84 16.86 in parentheses

(7)’ (49) (11) (9)

Galactose

Mannose

3.48 11.72 6.01 5.22

5.28 6.66 14.07 13.14

represent

(6) (8) (12) (9)

the number

Xylose

(8) (8) (12) (8)

5.22

Fructose (5)

-

of observations

17.97 17.12

Endogenous

Maltose

(9) (9)

6.79 11.32

used to compute

(8) (9)

3.20 3.10 1.66 1.09

the mean

(14) (10) (16) (9) Qacid

worms. All four of these bases reduce the rate at which glucose, mannose, fructose, and maltose is fermented by M. dubius. Exposure to any of these bases evokes a contraction of the worms; however, this phenomenon is most easily observed with the tapeworms that have been exposed to either quinacrine or chloroquine. The degree of contraction induced in the worms by thorazine or acriflavine is not as noticeable. The bases were used at a fairly high concentration, 1.25 X 1O-3 M. DISCUSSION

The capacity of these animals to ferment a quite limited number of monosacharides, and a disaccharide in one instance, may partially account for the specificity of the environment inhabited by the adults. Their dependence on another organism for splitting a polysaccharide into its monosaccharide components (Read, 1955), or into maltose in the case of M. dubius, would probably prevent these worms from surviving as free-living organisms. The cestodes and acanthocephalans lack a mouth, alimentary canal and anus; consequently, materials entering or leaving these animals must pass through the external surface either by diffusion, by active transport, or by both methods. The failure of the tapeworms to ferment disaccharides implies that hydrolytic enzymes for acting on these substrates are neither liberated into the environment, nor are present in the surface of these worms. It is not known if maltose passes directly into M. dubius, or is split at the surface into its glucose constituents which then enter as distinct entities. Whether these worms possess a multiplicity of kinases such as have been shown in the trematode, Schistosoma mansoni (Bueding and MacKinnon, 1955), is unknown; however, a

256

LAURIE

fructokinase appears to be lacking in the tapeworms. As a result of the response of M. dubius to the substrates which it is capable of fermenting, one might postulate the existence of more than one kinase in its tissues. Because of the relatively small structural changes between the fermentable and non-fermentable monosaccharides it is probable that the differences found in the ability of the worms to ferment these substrates cannot be explained solely on the basis of permeability and are due, at least in part, to the presence or absence of enzymatically catalyzed reactions. The inhibitory activity of phlorizin (or dibromophlorizin) may arise from blocking the phosphorylation of the sugar by a kinase, or perhaps by interfering in the function of a prior “sugar transfer system” such as has been postulated to exist in some mammalian tissues (Levine and Goldstein, 1955). The ready reversibility of the inhibition, and the observations that methylation of the phenolic hydroxyl groups (Lambrechts, 1937) or exposure to a pH of about 8.3 (Lundsgaard, 1933) inactivates phlorizin, suggests that hydrogen bonding between the non-ionized, phenolic hydroxyl groups of phlorizin and a locus on the surface of H. diminuta may be essential for producing the inhibition. The inhibitory activity of phlorizin and dibromophlorizin is not dependent on the presence of oxygen as has been suggested for the former by Shapiro (1947). The response of the worms to the halogen substituted acids may arise from a selective permeability to these substances. Quinacrine has been shown to inhibit a number of enzymes associated with glycolysis (Marshall, 1948; Speck and Evans, 1945); hence, this compound, chloroquine and thorazine might be expected to function in a somewhat similar manner in the helminths. In- the tapeworms the inhibitory activity of acriflavine suggests that the fermentation of glucose involves at least one enzyme which does not take part in the dissimilation of galactose. The interaction of chloroquine with proteins (Parker and Irvin, 1952a, b) and the affinity quinacrine has for tapeworm muscle, suggests that the contraction of the worms evoked by the four bases is at least partially brought about by a direct action of the bases on the protein molecules of the muscle tissue. Within the physiological pH range, the positively charged bases could function as intra- and inter-molecular linkages by forming hydrogen bonds with suitable groups on the muscle protein molecules and thereby disrupt the normal structure and activity of these molecules. The bases may also affect the functioning of the musculature by interfering in the energy metabolism of the worms.

HELMINTH

METABOLISM

257

It is apparent from the experiments with phlorizin, dibromophlorizin and acriflavine that the methods of penetration of fermentable substrates into the worms and/or the subsequent dissimilation of the substrates are not identical either in the individual animal or in members of different species. Such information indicates that generalizations concerning the metabolic characteristics of the large taxonomic groups of helminths should be made with some caution. SUMMARY

Under anaerobic conditions, the tapeworms, Hymenolepis diminuta and Oochoristica symmetrica, ferment glucose and galactose, at least partially, to acidic end-products. The fermentation of mannose and xylose by the former and of mannose by the latter is somewhat equivocal. The acanthocephalan, Monilijormis dubius, ferments glucose, galactose, mannose, fructose and maltose to acidic catabolites. No evidence of the acidic fermentation of a variety of other carbohydrate substrates by these helminths has been found. Phlorisin, non-competitively, inhibits the fermentation of glucose by H. diminuta but has no effect on the fermentation of glucose, mannose, fructose, and maltose by M. dubius. Dibromophlorizin inhibits glucose and galactose fermentation by H. diminuta but has no influence on sugar fermentation by M. &&us. These glucosides inhibit galactose fermentation but not glucose fermentation by 0. symmetrica. The fermentation of glucose and fructose by M. dubs is inhibited by p-chloromecuribenzoic acid but not by iodoacetic acid. Quinacrine inhibits the fermentation of glucose and galactose by 0. symmetrica and glucose fermentation by H. diminuta; whereas, acriflavine inhibits glucose fermentation but not galactose fermentation by these cestodes. ACKNOWLEDGMENTS I wish to express my appreciation to Dr. B. E. Fisher, Agricultural Research Service, United States Department of Agriculture, Peoria, Illinois, and to Dr. R. D. DeMoss, McCollum-Pratt Institute, The Johns Hopkins University, Baltimore for providing gifts of 3-O-methyl-n(+)glucose and 2-deoxy-n-glucose, respectively, and to Dr. A. C. Chandler, Rice Institute, Houston for providing the initial supply of Moniliformis dubius.

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258

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