- Email: [email protected]
The role of carbohydrates in the biology of cestodes. VIII. Some conclusions and hypotheses
EXPERIMENTAL
The
PARASITOLOGY
8,
365-382 (1959)
Role of Carbohydrates in the Biology of Cestodes. VIII. Some Conclusions and Hypotheses* Clark
Department
of Pathobiology,
(Submitted
Johns
P. Read Hopkins
for publication,
University,
Baltimore,
Maryland
9 May 1958)
In a recent address, Huff (1956) has pointed up the paucity of generalizations which can be applied in parasitology. The present author agrees with Huff’s implication that this may be due to the focusing of major attention on specific problems related to the medical and veterinary fields or on the parasite itself. It would seem that greater attention to the phenomena of parasitism or symbiosis may be expected to yield information leading to the development of principles and the present series of studies represent gropings in this direction. The high rates of carbohydrate metabolism in parasitic organisms belonging to various phyla suggest that exogenous carbohydrate may have some special roles in the microecology of these animals. An attempt has been made to analyze the effects of exogenous carbohydrate on tapeworms. The available data on Acanthocephala are also included in the present considerations. EVIDENCE FOR A QUANTITATIVE CARBOHYDRATEREQUIREMENT
There is a considerable amount of evidence that cestodes require the inclusion of carbohydrate in the host diet for normal development and reproduction. Chandler (1942) reported that, when the rat host is placed on a diet lacking carbohydrate, there is a decrease in the number of Hymenolepis diminuta which become established and that those worms which become established are quite small in size. This has been confirmed, and it was further shown that if rats harboring previously established worms are placed on a carbohydrate deficient diet for 15 days * Portions of the work discussed in this paper have been aided by a contract between the Office of Naval Research, Department1 of the Navy, and Johns Hopkins University, N.R. 119-353 and by a grant, E-1508, from the National Institutes of Health, U.S. Public Health Service.
365
36ti
READ
about one third of the worms are lost from the hosts; those worms remaining show a cessation of egg production and are reduced in wet weight to about 15% that of worms from control animals receiving carbohydrate in the diet (Read and Rothman, 1957a). Further, it was shown that the size and reproductive rate of Hymenolepis diminuta appear to be linear functions of the absolute quantity of carbohydrate ingested by the host. This relationship was valid in the range of 0.1 to 3.0 g starch ingested per day by the host. Additional starch above the 3.0 g level was without effect and caloric replacement of the starch by casein in the low carbohydrate range did not affect the worms (Read, Schiller, and Phifer, 1958). Read, Schiller, and Phifer showed that Hymenolepis citelli and H. nana also require inclusion of carbohydrate in the host diet for normal growth and reproduction. Unlike H. diminuta, H. nuna is only affected if carbohydrate is deleted during the early phase of the infection. This seems to be related to a fundamental difference in the growth patterns of these two worms. H. diminuta is an organism which does not show senescence over a considerable period of time. It grows at an essentially continuous rate for at least 18 months, shedding a number of segments daily so that a particular size is more or less maintained. On the other hand, H. nuna has an abbreviated life span. The worm probably does not grow after 14 to 16 days in the mouse. The segments already formed continue to mature and undergo reproductive activity. At about 25 days the worm dies and its life as an individual is completed. During this latter phase, deletion of carbohydrate from the host diet seems to have no discernible effect. This suggests that the sensitivity to carbohydrate lack is a function of the inherent growth capacity of a particular species. This is borne out by observations on H. citelli which seems to be intermediate between H. nana and H. diminuta in its growth characteristics. H. citelli grows rapidly during its early life in the vertebrate. Its growth then slows and it continues to grow at this lower rate for a time after reaching a definitive size. At 70 to 90 days after infection H. citelli begins to exhibit definite symptoms of senility. Growth slows and eventually halts. The segments present ripen, and the worm literally becomes smaller and smaller as segments are shed. H. citelli seems to also occupy an intermediate position with respect to its sensitivity to carbohydrate lack. After the initial phase of high growth rate it is less sensitive to deletion of carbohydrate from the host diet than H. diminuta but more sensitive than H. nana.
ROLE
OF
CARBOHYDRATES
IN
BIOLOGY
OF
CESTODES.
VIII
367
Reid (1942) showed that when the avian host is subjected to short periods of starvation Raillietina cesticillus sheds the strobila. This is correlated with a marked diminution in the glycogen content of R. cesticillus. Whiie Reid’s observations do not demonstrate with certainty what dietary component is required for maintainence of the normal physiological integrity of this species, they strongly suggest that carbohydrate is the critical material. Read (1949) made similar observations on Hymenolepis diminuta. Read (1957) showed that starvation of the dogfish for 7 days resulted in a marked loss of worms and reduction in size of surviving worms belonging to the trypanorhynchid species, Lacistorhynchus tenuis. If dogfish were given carbohydrate by mouth during the same period, the number and size of L. tenuis obtained at autopsy remained very near the normal level. Oochoristica symmetrica is also sensitive to lack of carbohydrate in the diet of the mouse host, cessation of reproduction and marked reduction in the size of the worms occurring very rapidly when the hosts are given a carbohydrate-deficient diet (Read and Rothman, unpublished). The lack of effect of host dietary carbohydrate quantity is probably greatest in such worms as Schistocephalus, studied by Hopkins (1950, 1951). In this case, the worm reaches its maximum size in the intermediate host, rapidly undergoes differentiation without strobilar growth in the definitive host, and dies in a very few days. Hopkins’ data support the conclusion that this species relies on food materials stored during larval life as the major source of energy for its life in the definitive host. There is evidence that the acanthocephalan, Moniliformis dubius, is dependent on host dietary carbohydrate for normal growth and reproduction. Burlingame and Chandler (1941) reported that starvation of the rat host resulted in the loss of a considerable percentage of previously established AI. d&us. More recently it was demonstrated that growth of M. dubius is arrested if the host is placed on a carbohydrate deficient diet, that the polysaccharide content of the worm is sharply affected by the carbohydrate ingested by the host, and that a dramatic loss of body weight of the worm occurs when carbohydrate intake is curtailed (Read and Rothman, 1958b). Thus, there is experimental evidence that five cestode species (four cyclophyllidean and one trypanorhynchid) and one acanthocephalan species require the inclusion of carbohydrate in the host diet for normal growth and reproduction. Among the cestodes, the quantitative degree to which carbohydrate deficiency is manifested seems to be correlated
368
READ
with the rate and pattern of growth of the strobila. The higher the rate of growth, the greater is the sensitivity to a lack of carbohydrate in the host diet. THE
QUALITY
OF CARBOHYDRATES
UTILIZED
BY CESTODES
in Vitro
Since there is evidence that the cestodes and acanthocephalans have a quantitative biological requirement for carbohydrate, it is of great importance to ascertain what kinds of carbohydrates these animals may be capable of utilizing. Recently, systematic studies have been carried out to determine the spectrum of carbohydrates utilized by several cestode and one acanthocephalan species. Read (1956) found that Hymenolepis diminuta metabolized glucose and galactose but did not metabolize maltose, trehalose, lactose, or fructose. Laurie (1957) demonstrated that the cyclophyllideans, Hymenolepis diminuta and Oochoristica symmetrica, were capable of metabolizing glucose and galactose to acidic end products. Mannose and xylose were metabolized to a dubious extent by H. diminuta, and the former sugar to a slight extent by 0. symmetrica. These cestodes did not ferment fructose, arabinose, maltose, sucrose, lactose, trehalose, inositol, sorbitol, dulcitol, mannitol, 3-methoxyglucose, Z-deoxyglucose, a-methylglueoside, glucosamine, and several other substrates. Wardle (1937) showed that, when incubated for 6 hours in the presence of glucose, the glycogen content of Moniezia expansa increases. Arabinose, galactose, fructose, maltose, glucosamine, and some amino acids were not glycogenic. However, Wardle divided the worms into thirds, using one third for determining initial glycogen content and the other portions for determining glycogen after incubation in the presence and absence of substrate. Since it has been shown that there is a gradient in the glycogen content of at least one cyclophyllidean cestode (Read, 1956; Daugherty and Taylor, 1956), there may be some question concerning the reliability of the conclusions derived from Wardle’s experiments. It has recently been shown that Mesocestoideslatus, Hymenolepis cite& and H. nana utilize glucose and galactose, but none of a variety of other carbohydrates tested; on the other hand, Cittotaenia was found to metabolize glucose, galactose, and the disaccharides, maltose and sucrose (Read and Rothman, 1958a). Oddly, though it ferments sucrose, Cittotaenia apparently does not utilize fructose. Since Cittotaenia seems to be exceptional, among those species studied, in its capacity to metabolize disaccharides, studies on other anoplocephalids would be highly desirable.
ROLE
OF
Carbohydrates
CARBOHYDRATES
s 8 8
Cestodes
%I
,o s -2 17
++ f?
H. nana
t+++
t+t
-
H. citelli
t++t
Oochoristica symmetrica Mesocestoides latuc
Moniezia
ex-
pansa Cittotaenia
OF
Carbohydrate
$ g i-
t+++
Hymenolepis diminuta
BIOLOGY
TABLE I in vitro by Tapeworms
Metabolized
Species
IN
-
-
and
VIII
Acanthocephalans
$ 2 s m
P 0 ‘1 :4
-
-
-
-
-
-
++ -
-
-
-
t++t
t+-t f?
-
-
-
+-t+t
4-f -
-
-
-
-
7
2
I-?
rium verticillatum Lacistorhynthus tenuis Acanthocephala Moniliformis dubius
Author
Read, 1956 ; Laurie, 1957 Read and Rothman, 1958a Read and Rothman, 1958a Laurie, 1957
-t-+
-t
? -
I+++-
+t
-
-
-
-
-
Read and Rothman, 1958a Wardle, 1937 Read and Rothman, 1958a Read, 1957
I t k ++-
-
-
-
-
Read,
+++-t
-
-
-
?
t++
-+i
SP-
Callioboth-
369
-
-
-
s ,o “1 Ft”
CESTODES.
+t+-
+++
+t
-k+,f-
tt+.
t
-
t++-
?
Laurie, 1957
1957
370
READ
The tetraphyllidean, Calliobothrium verticillatum, and the trypanorhynchid, Lacistorhynchus tenuis, metabolize glucose and galactose but do not metabolize xylose, mannose, fructose, maltose, trehalose, sucrose, lactose, or raffinose (Read, 1956). In these latter experiments, analyses were made of the carbohydrate removed from the suspending media, as well as observation of changes in rate of acid production after the addition of each substrate. Laurie (1957) has shown that the acanthocephalan, Moniliformis &&US, can ferment a somewhat broader spectrum of carbohydrates than cestodes. This worm metabolized the monosaccharides, glucose, galactose, mannose, and fructose, and the disaccharide, maltose. A number of other monosaccharides, disaccharides, and sugar alcohols are not fermented by Moniliformis. The data available on the kinds of carbohydrates metabolized by cestodes and acanthocephalans in vitro are summarized in Table 1. The information available supports the conclusion that most cestodes are capable of metabolizing a very limited number of monosaccharide carbohydrates and are not capable of metabolizing disaccharides which may be available in the environment. The single species of acanthocephalan which has been studied is less limited than most cestodes in the number of monosaccharides metabolized and is capable of utilizing at least one disaccharide, maltose. It differs from all cestodes studied in its ability to metabolize fructose. These considerations might have some bearing on the fact that in light infections Moniliformis frequently occurs in the most anterior portion of the small gut; in being less selective, this acanthocephalan may utilize the incompletely hydrolyzed products of carbohydrate digestion which would be present in the anterior portion of the small intestine. EFFECTS
OF THE QUALITY
OF HOST DIETARY
CARBOHYDRATE
Several years ago it was noted that the quality of carbohydrate in the host diet had an effect on the size of Hymenolepis diminuta (Chandler, Read, and Nicholas, 1950). This phenomenon has been studied in greater detail by Read and Rothman (1957a, b, and c) and by Read, Schiller, and Phiier (1958). In these latter studies hosts infected with single worms were used as experimental material. It was shown with H. diminuta that the worm’s size (wet weight, dry weight, length, and number of segments) is affected by the quality of carbohydrate in the host diet. Studies of the initial growth of the worms in hosts receiving the diets,
ROLE
OF
CARBOHYDRATES
IN
BIOLOGY
OF
CESTODES.
VIII
371
differing only in containing starch or sucrose, showed that the growth rate of the worm was dependent on the kind of carbohydrate ingested by the host. Studies of the morphology of worms from hosts on diets containing different carbohydrates revealed that there were significant differences in mean gravid segment volume and, by inference, reproductive capacity as functions of the quality of dietary carbohydrate. Differences in actual reproductive rate, as measured by the number of eggs in the feces, corresponded to the morphological differences induced by specific dietary carbohydrates. From the standpoint of the growth rate, size maintained, and reproductive rate, starch was the most beneficial carbohydrate tested. Glucose, dextrins-maltose, or sucrose as sole carbohydrate components of the diet were less effective in maintaining high growth rate, definitive size, and reproductive rate. When fructose, which is not utilized by the worm in vitro, was supplied as the sole carbohydrate component of the diet, the reproductive rate of the worms was reduced to a very low level, approximating that observed in animals receiving no dietary carbohydrate. It was shown that eggs from worms in hosts receiving fructose were abnormal in size and shape and did not contain the material staining red with iodine (polysaccharide?) which is present in normal eggs. When fructose was fed to animals receiving weighed amounts of food such that the carbohydrate component (starch) limited the size and reproductive rate of the worm, the worms were larger, and had a higher reproductive rate than those from hosts not receiving fructose. This was tentatively interpreted as evidence that fructose may interfere with the absorption by the host of the products of starch hydrolysis, although in animals given free access to diets containing fructose and glucose the worms were not affected by the addition of fructose to the diet. When animals were fed on a diet containing lactose as the sole carbohydrate constitutent, the worms were greatly reduced in size. Further, when lactose was added to diets containing adequate amounts of starch it produced a significant deleterious effect on the worms. This was tentatively interpreted in terms of the known effects of lactose on the physical and chemical characteristics of the gut. It is known that the feeding of lactose produces changes in the pH, oxidation-reduction potential emptying time, and other features of the intestine (see Read, 1950, for review). Since Rothman (1958) has shown that the metabolism of H. diminuta is inhibited by low concentrations of bile salts, normally present in the gut, and that the degree of inhibition is a function of pH in the range of 7.0 to 7.4, it seems probable that the
372
READ
deleterious effects of lactose are indirect ones. Laurie’s (1957) findings that H. diminuta does not metabolize lactose in vitro and that the metabolism of exogenous glucose by the worm is not inhibited by lactose lend further support to this view. In studies on Hymenolepis citelli and H. nana (Read, Schiller, and Phifer 1958), it was shown that the quality of carbohydrate in the host diet also has an effect on the size of these worms. The furnishing of sucrose as the sole carbohydrate component resulted in worms which were smaller than those from starch-fed animals. In the case of H. nana, the effect was produced in young growing worms but older senescent worms were not affected by the quality of carbohydrate. The rate of senescence of this species was apparently not affected by dietary carbohydrate quality (or quantity). This lends further support to the concept that the quantity of carbohydrate required by these animals is a function of the quantity of growth which is characteristic of a particular stage in the life of each species. In this connection, the differences in the growth characteristics of H. diminuta, H. citelli, and H. nana may again be emphasized. In working with the first-named species during the past ten years, no evidence has ever been obtained to indicate that the worm may become senescent. H. diminuta seems to live as long as the rat host and remains young, in the sense that a high growth rate is maintained for an indefinite period. As a matter of fact, data on the size of worms in animals of different ages suggests that the worms may grow at a higher rate as the host grows older (Read, Schiller, and Phifer, 1958). On the other hand, H. cite& and H. nana show phenomena of ageing and senescence to differing degrees. The cited studies on the effects of host dietary carbohydrate quality strongly support the concept that there is a relationship between the degree of response to alteration of the host dietary carbohydrate and the growth characteristics of a particular species. It seems plain that some of the effects of dietary carbohydrate quality on the tapeworms, Hymenolepis diminuta, H. citelli, and H. nana may be interpreted in terms of quantity of utilizable carbohydrate available to the worms in the host. The effects of glucose, sucrose, and starch may be compared in this manner. The worms seem to have less utilizable carbohydrate available to them when glucose or sucrose are fed to the host than when starch is fed. It is known that these species can utilize glucose but cannot metabolize sucrose, fructose, or maltose, a product of starch digestion (Read, 1956; Laurie, 1957; Read and Rothman,
ROLE
OF
CARBOHYDRATES
IN
BIOLOGY
OF
CESTODES.
VIII
373
1958a). Thus, after digestion they can use half of the sucrose molecule and only the final degradation product of starch hydrolysis. There is evidence that the enzyme sucrase may be, for the most part, present in the cells lining certain parts of the digestive tract and in sloughed epithelial cells (Florey et al., 1941). There is also evidence that sucrase activity is primarily located in the anterior portion of the small intestine (Wilson and Vincent, 1955). If the fructose and glucose are absorbed by the host through an active process, a fact which seems to be reasonably well established (Jervis et al., 1956), the amount available to the worm would represent a vectorial sum of hydrolysis rate, absorption rate, and the rate of propulsion down the digestive tract. In the case of glucose there is excellent direct evidence that when glucose is given to the rat per orum, most of the carbohydrate is absorbed in the upper part of the small intestine (Reynell and Spray, 1956). It was shown by Reynell and Spray that retention of glucose in the stomach occurs so that the amount entering the small intestine does not seem to be a direct function of the quantity taken in by mouth. It can be concluded that the deleterious effects of glucose as the dietary carbohydrate are a manifestation of the fact that only a small amount of ingested glucose is directly available to a parasite in the lower two thirds of the small intestine. Direct time relationships should also be of great significance since it has been shown that the rate of glucose absorption by the mammal is independent of the concentration over a wide range (Reynell and Spray, 1956) and that the rate of utilization of exogenous substrate by Hymenolepis diminuta is also independent of concentration over a considerable range (Read, 1956). A somewhat more complicated series of hydrolytic events in the degradation of starch to glucose is undoubtedly involved in the beneficial effects of that carbohydrate on the tapeworms studied. Again, a direct time relationship would seem to be indicated with glucose available at rather low absolute concentrations for an extended time period. This concept is supported by the observation that, when starved hosts are given glucose or starch, glycogenesis in the worms is greatest in the hosts receiving starch (Read, 1955). It seems apparent that the effects on tapeworms of the quality of carbohydrate in the host diet may be of at least two general types: First, there are those effects which are actually manifestations of the quantity of utilizable carbohydrate which is available to the worm. These are mediated through the physiology of the normal digestion and ab-
374
READ
sorption of carbohydrate by the host intestine. It may be appropriate to reiterate a point emphasized elsewhere (Read, 1950) : Carbohydrates, as a class of compounds, seem to be unique in that they are absorbed from the vertebrate gut and do not undergo exocrino-enteric circulation. Further, it may be noted that the cestodes seem to be capable of utilizing those specific carbohydrates which the host is capable of absorbing against a concentration gradient. Second, there are effects which do not directly involve the quantity of utilizable carbohydrate available but are ecological in nature. These are mediated through a change in the intestinal environment, a change wrought by the particular carbohydrate involved. It is also obvious that, with the information available, a more specific recognition of effects on tapeworms of dieta.ry carbohydrate quality is not feasible. HOST STARVATION AND CARBOHYDRATE UTILIZATION It has been shown that starvation of the host for short periods results in the depletion of the polysaccharide of tapeworms (Reid, 1942; Read, 1949; Read, 1956). In the presence of glucose in o&o, worms from such hosts have a higher metabolic rate (Read, 1956; Read and Rothman, 1957b) and a higher rate of glycogenesis than worms from unstarved hosts (Daugherty, 1956; Read and Rothman, 195710). 1n GO, such starved worms show a rapid reconstitution of glycogen if the host is fed carbohydrate (Read, 1955). Since the metabolic rate of starved worms is higher than that of unstarved worms, it might be hypothesized that in a starved host, the worms would compete with the host to a greater extent when food became available than would be the case in a previously unstarved host. This possibility was tested by Read and Rothman (1957b), using Hymenolepis diminuta in the rat. It was found that as starvation proceeds the wet weight of worm tissue in the host declines rapidly. This loss of weight essentially compensates for the increase in metabolic rate, calculated on the basis of tissue weight. Thus, the absolute quantity of carbohydrate utilized by a worm from a previously starved host was slightly less than that utilized by a worm from an unstarved host. That the increase in the metabolic rate was real rather than only apparent was demonstrated by the use of unstarved control worms, reduced in size by cutting the strobila. Of some interest was the observation that the proportion of glucose converted to glycogen in vitro was greater in worms from starved hosts than in those from unstarved hosts. This indicates that a rapid alteration of assimilative
ROLE OF CARBOHYDRATES
IN BIOLOGY OF CESTODES. VIII
375
patterns may occur during starvation and is further evidence of the sensitivity of the tapeworm to alterations of the host’s intake of carbodrate. INTERACTIONS
OF WORMS
It has been recognized for some time that as the number of tapeworms in an individual host increases, the average size of each worm decreases. This has been termed the crowding effect (reviewed by Read, 1951). In considering this stunting of tapeworms observed in heavy infections, Reid (1942) dismissed carbohydrate as a probable limiting factor. Read (1951) concurred in this view. However, subsequent observations demonstrating effects of host dietary carbohydrate quantity and quality on tapeworm size and reproduction suggest that carbohydrate ingested by the host cannot be ruled out as a limiting factor in tapeworm ecology. It was shown by Read, Schiller, and Phifer (1958) that the body weight and reproductive rate of Hymenolepis are, within a certain range, essentially linear functions of the quantity of carbohydrate ingested by the host and that alteration of the quality of carbohydrate ingested by the host induces parallel changes in size and reproductive rate of this worm. Subsequently, a study was made of the effects of crowding under conditions in which the hosts ingested diets containing a worm-limiting quantity of starch or of sucrose, a carbohydrate of suboptimal quality. The slopes of the plots of the weight of worms against worm numbers from hosts on experimental diets were essentially identical with those from hosts on the control diet, but were displaced to considerably lower values in infections with the same number of worms (Read and Phifer, 1958). It has been indicated above that Hymenolepis cannot directly utilize the carbohydrates involved in these experiments (starch or sucrose) and that the effects of quality, in this case, are probably effects of quantity of utilizable carbohydrate available to the worm. In view of these considerations, it is suggested that the quantity of carbohydrate available per unit of time per worm may be involved in the crowding effect. It seemed feasible to evaluate this hypothesis by comparison with a model. The data obtained have been compared with what might be expected if a definite quantity of utilizable carbohydrate is required to support a given quantity of tapeworm tissue, and a hypothetical curve has been constructed. In constructing this curve it has been assumed that (1) a given quantity of carbohydrate ingested by the host is equally shared
376
READ
by the worms in the host, (2) the presence of varying numbers of worms does not affect the physiology of digestion and absorption of carbohydrate by the host, and (3) the body weight maintained by an individual worm is a function of the proportion of the available carbohydrate obtained by the worm. Thus, it is hypothesized that in an infection of 30 worms each worm would have available one-sixth of the quantity of carbohydrate available to the individuals in a five worm infection. Such a calculated curve is a rectangular hyperbola and is shown in Fig. 1 along with the data obtained experimentally. In constructing the curve it has been assumed that, in a five worm infection, each worm weighs 300 milligrams and that worm size decreases as the worm number increases. All other points on the hypothetical curve are calculated on the basis of the foregoing assumptions. The data of Read and Phifer (1958) have been plotted for comparison with this hypothetical curve. The similarities in the predicted and observed values are obvious and support the concept that competition for utilizable carbohydrate is the limiting factor involved in determining the size of individual tapeworms in infections of varying intensity. Incidentally, the data obtained experimentally support the view that infections of 30 worms do not affect digestion and absorption of carbohydrate by the host to any greater extent than do infections of 5 worms. Read and Phifer (l.c.) also showed that the two species, Hymenolepis
D
A fii:q B
C
IO 5
10 Number
of worms per
2030
host
FIG. 1. The average weight of individual worms in infections with various numbers of worms. Hosts were maintained on diets differing in carbohydrate quantity or quality. A = high starch diet, B = low starch diet, C = Sucrose diet, D = hypothetical curve.
See text
for
discussion.
ROLE OF CARBOHYDRATES
IN BIOLOGY OF CESTODES. VIII
377
diminuta and H. cite%, show some reduction in body size when the hamster is infected with a single worm of each species.When the hosts were placed on low carbohydrate diets (0.5 g starch per day) H. citelli was reduced in size to the same extent in the presence or absence of H. diminuta. On the other hand, H. diminuta from hosts on such restricted diets, was reduced in size to a greater extent in the presence of H. cite& than in its absence. This has shown that the effects on these worms of quantity of carbohydrate available may be modified to differing extents by the presence of another species.It would be of interest to determine the effects of dietary modification on the interactions observed between Hymenolepis and Monilijormis in the rat (Holmes, 1957). GENERAL
SIGNIFICANCE
OF A CARBOHYDRATE
REQUIREMENT
There is great interest in the search for the chemical needs which bind obligate parasites to their hosts. That is, bind them in the sense that the organism is incapable of reproducing itself outside the environment furnished by a host (excepting, of course, the special cultural conditions concocted by man for the welfare of these organisms). From our knowledge of the nutrition of free-living animal organisms it is plain that the requirements may vary tremendously. Among the protozoa many flagellates require only a single vitamin, ammonia, carbon dioxide, and minute quantities of certain minerals (Hutner and I’rovasoli, 1951) while ciliates may have highly complex requirements (Kidder and Dewey, 1951). It would seem that among the free-living animals there is little evidence to indicate that an increase in number of requirements for micronutrients has interfered with the evolution of the animal organism. Indeed, it may readily be seen that such requirements are limiting ecological parameters and, as such, serve as mechanisms for isolation. We know that some obligately parasitic microorganisms require micronutrients which are relatively unstable and are characteristically found only in living organisms. Such is Hemophilus, which requires diphosphopyridine nucleotide, or the gonococcus, which requires thiamine pyrophosphate (Snell, 1951). However, there is no particular reason for assuming that a parasite which lives outside cells should be different from predatory organisms with respect to its requirements for micronutrients. A tapeworm living in the intestinal tract of a vertebrate is probably not in contact with compounds of special micronutritional significance occurring only in that habitat. In speculating as to the probable chemical requirements of extracellular parasites it has fre-
378
READ
quently been assumed that these organisms would have highly complex and peculiar nutritional requirements. However, we may have neglected the biological significance of nutrient materials required in large quantity. It seems to be widely accepted that animals require protein, or at least amino acids, for the synthesis of protoplasm. In the usual definition of a “balanced diet”, protein or amino acid content is considerable; interestingly, carbohydrate is also usually included as a sizeable component, although there are few free-living organisms which have a requirement for carbohydrate. The honey bee, and perhaps a few other organisms showing a high degree of specialization, are exceptional in the realm of free-living animals. Carbohydrate is a highly convenient source of calories representing, in the final sense, the most efficient energy storage material arrived at in the evolution of the diverse organisms present on the earth. Other materials may act as energy depots but none in so universal a manner. Animal organisms take advantage of this ubiquitous distribution of carbohydrates and seem to be almost universally capable of utilizing them. However, in the case of most free-living animals protein or fat and protein in sufficient quantity can meet the caloric requirements for maintenance and growth. On the other hand, this may not hold true for many extracellular, and proba.bly some intracellular, parasites. The meager data available strongly suggest that many parasites have a requirement for carbohydrate. In some cases this may be an absolute requirement and in others what may be termed a biological requirement. The latter term is intended to mean that carbohydrate is required for reproduction and hence for species survival. Such seems to be the case with tapeworms. There is suggestive evidence that the cyclic nature of ingestion of carbohydrate by hosts may affect tapeworms under what may be termed natural conditions. Oliger (1950), studying the seasonal changes in tapeworms of tetraonid birds in European Russia, reported that when the birds change to a winter diet (high in fat) in the late autumn, there is a loss of the strobilar portions of their tapeworm parasites. Segments are not produced during the winter feeding. In late February or early March the birds change to a summer diet (high carbohydrate content). At this time the tapeworms produce segments and again become patent. According to Oliger the hosts remain quite well-nourished on both winter and summer diets. It has been observed that bears shed large portions of the strobila of Dibothriocephalus when the feeding habits are altered before hibernation (E. L. Schiller, personal communication). Levine
ROLE
OF
CARBOHYDRATES
IN
BIOLOGY
OF
CESTODES.
VIII
379
(1938) showed that the normal shedding of segments by Davainea is correlated with the cyclic nature of feeding of the avian host. Levine also demonstrated that reduction of the nutritive value of the host diet by increasing the bran content resulted in a marked lowering of worm reproduction; starvation for a day caused cessation of proglottid production, as indicated by examination of the feces of the chicken. It has been shown that there are diurnal fluctuations in the polysaccharide content of Raillietina, Hymenolepis, and Moniliformis (Reid, 1942; Read, 1949; Read and Rothman, 195813). These fluctuations are correlated with the feeding behavior of the host. The data available indicate that the effects on tapeworms of the quality of host dietary carbohydrate are more subtle than might be anticipated by demonstration that most of these worms can only use certain monosaccharide sugars. This is most striking in experiments comparing the effects of starch and partially predigested starch as dietary carbohydrate components. The observed effects on the worms of the latter carbohydrate serve to illustrate the point that the imposition of the dynamics of gut physiology on the specific carbohydrate requirement of cestodes may be of some importance in intra- and inter-host species distribution, apparent age or sex-linked resistance, and, of course, speciation when the interaction serves as a mechanism for isolation. Just as feeding habits of the host are frequently involved in the completion of the cestode life cycle, so they seem to be delicately involved in determining whether the cestode may prosper in a particular host. The imposition of the physiology of carbohydrate degradation in the vertebrate gut plus food preference or habit on the specialized carbohydrate requirements of cestodes seems to constitute a phenomenon which fits Lewis’ balance hypothesis of parasitism (Lewis, 1953) and the basically similar nutrition-inhibition hypothesis of Garber (1956). Obviously, it cannot be claimed that this proves the validity of the hypotheses, and it is plain that many other nutritional and physical factors may be involved in determining whether a particular cestode will live in a particular host. An important consideration in parasitism is the question of the extent to which a given parasite competes with the host for materials of nutritional significance. In the interaction of hosts and tapeworms, with particular reference to carbohydrate, it would seem that competition may be negligible. The location of the worms below the region of maximal intestinal absorption, the lack of significant carbohydrate in the intes-
380
READ
tinal secretion, and dependence of the worms on the quantity in the host ingesta favor the hypothesis that the carbohydrate requirements of the worms are met by amounts which represent an excess for the host. Competition between the worms and the intestinal flora for carbohydrate seems more likely than competition between worms and host. It is obvious, however, that tapeworms may compete with the host for other kinds of nutritional materials, such as amino acids and vitamins.
SUMMARY It is concluded that: (1) Tapeworms require a source of carbohydrate for growth and reproduction; this requirement is satisfied by the utilization of carbohydrate in the host ingesta. The higher the rate of growth, the greater is the apparent sensitivity to carbohydrate deficiency. (2) The kinds of carbohydrates which can be utilized in vitro by tapeworms are quite limited. Most species studied are capable of using only certain monosaccharides. Anoplocephalids may be exceptional in being able to ferment certain disaccharides. (3) The quality of carbohydrate ingested by the host has marked effects on growth rate, size attained, and reproductive rates. The effects are explained in terms of interactions of host physiology and physiological capacities of the worms. Certain carbohydrates, such as lactose, probably affect the worms by altering the physicochemical characteristics of the habitat. (4) The crowding e$ect in cestode infections may be interpreted in terms of competition for utilizable carbohydrate by the individual worms in the populations. (5) The competitive effects of one tapeworm species on another are modified by variations in the quantity of carbohydrate ingested by the host, different species not being affected proportionally. (6) The significance of a carbohydrate requirement is discussed in terms of (a) its uniqueness in the animal kingdom, (b) its probable role in cyclic changes in tapeworms, (c) possible special effects in distribution of tapeworms.
REFERENCES BURLINOAME,
P. L., AND CHANDLER, A. C. 1941. Host-parasite relations of Monilijormis dubius (Acanthocephala) in albino rats, and the environmental nature of resistance to single and superimposed infections with this parasite. Am. J. Hyg. 33(D), 1-21. CHANDLER, A. C. 1942. Studies on the nutrition of tapeworms. Am. J. Hyg. 37, 121-130. CHANDLER, READ, C.P., AND NICHOLAS, H. 0.1950. Observations on certain phases of nutrition and host-parasite relations of Hymenolepis diminuta in white rats. J. Parasitol. 36, 523-535.
A. C.,
ROLE
OF
CARBOHYDRATES
IN
BIOLOGY
OF
CESTODES.
VIII
381
DAUOHERTY, J. W. 1956. The effect of host castration and fasting on the rate of glycogenesis in Hymenolepis diminuta. J. Parasitol. 42, 17-20. DAUGHERTY, J. W., AND TAYLOR, D. 1956. Regional distribution of glycogen in the rat cestode, Hymenolepis diminuta. Exptl. Parasitol. 6, 376-390. DICKENS, F. 1951. Anaerobic glycolysis, respiration, and the Pasteur effect. In: The Enzymes. Vol. II. Edited by J. B. Sumner and K. Myrbilck, Academic Press, New York, pp. 624683. FLOREY, H. W., WRIGHT, R. D., AND BROWN, R. A. 1941. The secretions of the intestine. Physiol. Rev. 21, 36-69. GARBER, E. D. 1956. A nutrition-inhibition hypothesis of pathogenicity. Am. Naturalist 40, 183-194. HOLMES, J. C. 1957. The effects of concurrent infections with the spiny-headed worm, Monilijormis d&&s, on the rat tapeworm, Hymenolepis diminuta. J. Parositol. 43 (Suppl.), 24. HOPKINS, C. A. 1950. Studies on cestode metabolism. I. Glycogen metabolism in Schistocephalus
solidus
in vivo.
J. Parasitol.
36, 384-390.
HOPKINS, C. A. 1951. Studies on cestode metabolism. II. The utilization of glycogen by Schistocephalus solidus in vitro. Exptl. Parasitol. 1, 196-213. HUFF, C. G. 1956. Parasitism and parasitology. J. Parasitol. 42, l-10. HUTNER, S. H., AND PROVASOLI, L. 1951. The phytoflagellates In: Biochemistry and Physiology of the Protozoa. Vol. I. Edited by A. Lwoff, Academic Press, New York, pp. 27-128. JERVIS, E. L., JOHNSON, F. R., SHEFF, M. F., AND SMYTH, D. H. 1956. The effect of phlorizin on intestinal absorption and intestinal phosphatase. J. Physiol. 134, 675488. KIDDER, G. W., AND DEWEY, V. C. 1951. The biochemistry of ciliates in pure culture. In: Biochemistry and Physiology of the Protozoa. Vol. I. Edited by A. Lwoff, Academic Press, New York, pp. 324400. LAURIE, J. 1957. The in vitro fermentation of carbohydrates by two species of cestodes and one species of Acanthocephala. Exptl. Parasitol. 6, 245-260. Levine, P. P. 1938. Observations on the biology of the poultry cestode, Daoainea proglottina (Dav.). J. Parasitol. 24, 423-431. LEWIS, R. W. 1953. The balance hypothesis of parasitism. Am. Naturalist 37, 273-281. OLIGER, I. M. 1950. Causes of destrobilization in cestodes of Tetraonidae. Doklady Akad. Nauk SSSR 74, 869-872 (in Russian). READ, C. P. 1949. Fluctuation in the glycogen content of the cestode Hymenolepis diminuta. J. Parasitol. 36 (Suppl.), 26. READ, C. P. 1950. The vertebrate small intestine as an environment for parasitic helminths. Rice Inst. Pamphl. 37(2), 94 pp. READ, C. P. 1951. The “crowding effect” in tapeworm infections. J. Parasitol. 37, 174-178. READ, C. P. 1955. Intestinal physiology and the host-parasite relationship. In: Some Physiological Aspects and Consequences of Parsasitism. Edited by W. H. Cole, Rutgers Univ. Press, pp. 2743. READ, C. P. 1956. Carbohydrate metabolism of Hymenolepis diminuta. Exptl. Parasitol.
6. 325-344.
382
READ
C. P. 1957. The role of carbohydrates in the biology of cestodes. III. Studies on two species from dogfish. Exptl. Parasitol. 6.288-293. READ, C. P., AND PHIFER, K. 0. 1959. The role of carbohydrates in the biology of cestodes. VII. Interactions between individual tapeworms of the same and different species. Exptl. Parasitol. 6, 4650. READ, C. P., AND ROTHMAN, A. H. 1957a. The role of carbohydrates in the biology of cestodes. I. The effect of dietary carbohydrate quality on the size of Hymenolepis diminuta. Exptl. Parasitol. 6, 1-7. READ, C. P., AND ROTHMAN, A. H. 195713.The role of carbohydrates in the biology of cestodes. II. The effect of starvation on glycogenesis and glucose consumption in Hymenolepis. Exptl. Parasitol. 6, 280-287. READ, C. P., AND ROTHYAN, A. H. 1957c. The role of carbohydrates in the biology of cestodes. IV. Some effects of host dietary carbohydrate on growth and reproduction of Hymenolepis. Exptl. Parasitol. 6, 294-305. READ, C. P., AND ROTHMAN, A. H. 1958a. The role of carbohydrates in the biology of cestodes. VI. The carbohydrates metabolized in vitro by some cyclophyllidean species. Exptl. Paraaitol. 7, 217-223. READ, C. P., AND ROTHMAN, A. H. 1958b. The carbohydrate requirement of Monilijormis (Acanthocephala). Exptl. Parasitol. 7, 191-197. READ, C. P., SCHILLER, E. L., AND PHIFER, K. 0.1958. The role of carbohydrates in the biology of cestodes. V. Comparative studies on the effects of host dietary carbohydrate on Hymenolepis spp. Exptl. Parasitol. 7, 198-216. REID, W. M. 1942. Certain nutritional requirements of the fowl cestode Raillietina cesticillus (Molin) as demonstrated by short periods of starvation of the host. J. Parasitol. 26, 319340. REYNELL, P. C., AND SPRAY, G. H. 1956. The absorption of glucose by the intact rat. J. Physiol. 164, 531-537. ROTHMAN, A. H. 1958. The role of bile salts in the biology of tapeworms. I. Effects of bile salts on the metabolism of Hymenolepis diminuta and Oochoristica symmetrica. Exptl. Parasitol. 7, 328-337. SNELL, E. E. 1951. Bacterial nutrition-growth factors. In: Bacterial Physiology. Edited by C. H. Werkman and P. W. Wilson, Academic Press, New York, pp. 215-255. WARDLE, R. A. 1937. The physiology of the sheep tapeworm, Moniezia expansa Blanchard. Con. J. Research 16D, 117-126. WILSON, T. H., AND VINCENT, T. N. 1955. Absorption of sugars in vitro by the intestine of the golden hamster. J. Biol. Chem. 216,851-866. READ,
The
PARASITOLOGY
8,
365-382 (1959)
Role of Carbohydrates in the Biology of Cestodes. VIII. Some Conclusions and Hypotheses* Clark
Department
of Pathobiology,
(Submitted
Johns
P. Read Hopkins
for publication,
University,
Baltimore,
Maryland
9 May 1958)
In a recent address, Huff (1956) has pointed up the paucity of generalizations which can be applied in parasitology. The present author agrees with Huff’s implication that this may be due to the focusing of major attention on specific problems related to the medical and veterinary fields or on the parasite itself. It would seem that greater attention to the phenomena of parasitism or symbiosis may be expected to yield information leading to the development of principles and the present series of studies represent gropings in this direction. The high rates of carbohydrate metabolism in parasitic organisms belonging to various phyla suggest that exogenous carbohydrate may have some special roles in the microecology of these animals. An attempt has been made to analyze the effects of exogenous carbohydrate on tapeworms. The available data on Acanthocephala are also included in the present considerations. EVIDENCE FOR A QUANTITATIVE CARBOHYDRATEREQUIREMENT
There is a considerable amount of evidence that cestodes require the inclusion of carbohydrate in the host diet for normal development and reproduction. Chandler (1942) reported that, when the rat host is placed on a diet lacking carbohydrate, there is a decrease in the number of Hymenolepis diminuta which become established and that those worms which become established are quite small in size. This has been confirmed, and it was further shown that if rats harboring previously established worms are placed on a carbohydrate deficient diet for 15 days * Portions of the work discussed in this paper have been aided by a contract between the Office of Naval Research, Department1 of the Navy, and Johns Hopkins University, N.R. 119-353 and by a grant, E-1508, from the National Institutes of Health, U.S. Public Health Service.
365
36ti
READ
about one third of the worms are lost from the hosts; those worms remaining show a cessation of egg production and are reduced in wet weight to about 15% that of worms from control animals receiving carbohydrate in the diet (Read and Rothman, 1957a). Further, it was shown that the size and reproductive rate of Hymenolepis diminuta appear to be linear functions of the absolute quantity of carbohydrate ingested by the host. This relationship was valid in the range of 0.1 to 3.0 g starch ingested per day by the host. Additional starch above the 3.0 g level was without effect and caloric replacement of the starch by casein in the low carbohydrate range did not affect the worms (Read, Schiller, and Phifer, 1958). Read, Schiller, and Phifer showed that Hymenolepis citelli and H. nana also require inclusion of carbohydrate in the host diet for normal growth and reproduction. Unlike H. diminuta, H. nuna is only affected if carbohydrate is deleted during the early phase of the infection. This seems to be related to a fundamental difference in the growth patterns of these two worms. H. diminuta is an organism which does not show senescence over a considerable period of time. It grows at an essentially continuous rate for at least 18 months, shedding a number of segments daily so that a particular size is more or less maintained. On the other hand, H. nuna has an abbreviated life span. The worm probably does not grow after 14 to 16 days in the mouse. The segments already formed continue to mature and undergo reproductive activity. At about 25 days the worm dies and its life as an individual is completed. During this latter phase, deletion of carbohydrate from the host diet seems to have no discernible effect. This suggests that the sensitivity to carbohydrate lack is a function of the inherent growth capacity of a particular species. This is borne out by observations on H. citelli which seems to be intermediate between H. nana and H. diminuta in its growth characteristics. H. citelli grows rapidly during its early life in the vertebrate. Its growth then slows and it continues to grow at this lower rate for a time after reaching a definitive size. At 70 to 90 days after infection H. citelli begins to exhibit definite symptoms of senility. Growth slows and eventually halts. The segments present ripen, and the worm literally becomes smaller and smaller as segments are shed. H. citelli seems to also occupy an intermediate position with respect to its sensitivity to carbohydrate lack. After the initial phase of high growth rate it is less sensitive to deletion of carbohydrate from the host diet than H. diminuta but more sensitive than H. nana.
ROLE
OF
CARBOHYDRATES
IN
BIOLOGY
OF
CESTODES.
VIII
367
Reid (1942) showed that when the avian host is subjected to short periods of starvation Raillietina cesticillus sheds the strobila. This is correlated with a marked diminution in the glycogen content of R. cesticillus. Whiie Reid’s observations do not demonstrate with certainty what dietary component is required for maintainence of the normal physiological integrity of this species, they strongly suggest that carbohydrate is the critical material. Read (1949) made similar observations on Hymenolepis diminuta. Read (1957) showed that starvation of the dogfish for 7 days resulted in a marked loss of worms and reduction in size of surviving worms belonging to the trypanorhynchid species, Lacistorhynchus tenuis. If dogfish were given carbohydrate by mouth during the same period, the number and size of L. tenuis obtained at autopsy remained very near the normal level. Oochoristica symmetrica is also sensitive to lack of carbohydrate in the diet of the mouse host, cessation of reproduction and marked reduction in the size of the worms occurring very rapidly when the hosts are given a carbohydrate-deficient diet (Read and Rothman, unpublished). The lack of effect of host dietary carbohydrate quantity is probably greatest in such worms as Schistocephalus, studied by Hopkins (1950, 1951). In this case, the worm reaches its maximum size in the intermediate host, rapidly undergoes differentiation without strobilar growth in the definitive host, and dies in a very few days. Hopkins’ data support the conclusion that this species relies on food materials stored during larval life as the major source of energy for its life in the definitive host. There is evidence that the acanthocephalan, Moniliformis dubius, is dependent on host dietary carbohydrate for normal growth and reproduction. Burlingame and Chandler (1941) reported that starvation of the rat host resulted in the loss of a considerable percentage of previously established AI. d&us. More recently it was demonstrated that growth of M. dubius is arrested if the host is placed on a carbohydrate deficient diet, that the polysaccharide content of the worm is sharply affected by the carbohydrate ingested by the host, and that a dramatic loss of body weight of the worm occurs when carbohydrate intake is curtailed (Read and Rothman, 1958b). Thus, there is experimental evidence that five cestode species (four cyclophyllidean and one trypanorhynchid) and one acanthocephalan species require the inclusion of carbohydrate in the host diet for normal growth and reproduction. Among the cestodes, the quantitative degree to which carbohydrate deficiency is manifested seems to be correlated
368
READ
with the rate and pattern of growth of the strobila. The higher the rate of growth, the greater is the sensitivity to a lack of carbohydrate in the host diet. THE
QUALITY
OF CARBOHYDRATES
UTILIZED
BY CESTODES
in Vitro
Since there is evidence that the cestodes and acanthocephalans have a quantitative biological requirement for carbohydrate, it is of great importance to ascertain what kinds of carbohydrates these animals may be capable of utilizing. Recently, systematic studies have been carried out to determine the spectrum of carbohydrates utilized by several cestode and one acanthocephalan species. Read (1956) found that Hymenolepis diminuta metabolized glucose and galactose but did not metabolize maltose, trehalose, lactose, or fructose. Laurie (1957) demonstrated that the cyclophyllideans, Hymenolepis diminuta and Oochoristica symmetrica, were capable of metabolizing glucose and galactose to acidic end products. Mannose and xylose were metabolized to a dubious extent by H. diminuta, and the former sugar to a slight extent by 0. symmetrica. These cestodes did not ferment fructose, arabinose, maltose, sucrose, lactose, trehalose, inositol, sorbitol, dulcitol, mannitol, 3-methoxyglucose, Z-deoxyglucose, a-methylglueoside, glucosamine, and several other substrates. Wardle (1937) showed that, when incubated for 6 hours in the presence of glucose, the glycogen content of Moniezia expansa increases. Arabinose, galactose, fructose, maltose, glucosamine, and some amino acids were not glycogenic. However, Wardle divided the worms into thirds, using one third for determining initial glycogen content and the other portions for determining glycogen after incubation in the presence and absence of substrate. Since it has been shown that there is a gradient in the glycogen content of at least one cyclophyllidean cestode (Read, 1956; Daugherty and Taylor, 1956), there may be some question concerning the reliability of the conclusions derived from Wardle’s experiments. It has recently been shown that Mesocestoideslatus, Hymenolepis cite& and H. nana utilize glucose and galactose, but none of a variety of other carbohydrates tested; on the other hand, Cittotaenia was found to metabolize glucose, galactose, and the disaccharides, maltose and sucrose (Read and Rothman, 1958a). Oddly, though it ferments sucrose, Cittotaenia apparently does not utilize fructose. Since Cittotaenia seems to be exceptional, among those species studied, in its capacity to metabolize disaccharides, studies on other anoplocephalids would be highly desirable.
ROLE
OF
Carbohydrates
CARBOHYDRATES
s 8 8
Cestodes
%I
,o s -2 17
++ f?
H. nana
t+++
t+t
-
H. citelli
t++t
Oochoristica symmetrica Mesocestoides latuc
Moniezia
ex-
pansa Cittotaenia
OF
Carbohydrate
$ g i-
t+++
Hymenolepis diminuta
BIOLOGY
TABLE I in vitro by Tapeworms
Metabolized
Species
IN
-
-
and
VIII
Acanthocephalans
$ 2 s m
P 0 ‘1 :4
-
-
-
-
-
-
++ -
-
-
-
t++t
t+-t f?
-
-
-
+-t+t
4-f -
-
-
-
-
7
2
I-?
rium verticillatum Lacistorhynthus tenuis Acanthocephala Moniliformis dubius
Author
Read, 1956 ; Laurie, 1957 Read and Rothman, 1958a Read and Rothman, 1958a Laurie, 1957
-t-+
-t
? -
I+++-
+t
-
-
-
-
-
Read and Rothman, 1958a Wardle, 1937 Read and Rothman, 1958a Read, 1957
I t k ++-
-
-
-
-
Read,
+++-t
-
-
-
?
t++
-+i
SP-
Callioboth-
369
-
-
-
s ,o “1 Ft”
CESTODES.
+t+-
+++
+t
-k+,f-
tt+.
t
-
t++-
?
Laurie, 1957
1957
370
READ
The tetraphyllidean, Calliobothrium verticillatum, and the trypanorhynchid, Lacistorhynchus tenuis, metabolize glucose and galactose but do not metabolize xylose, mannose, fructose, maltose, trehalose, sucrose, lactose, or raffinose (Read, 1956). In these latter experiments, analyses were made of the carbohydrate removed from the suspending media, as well as observation of changes in rate of acid production after the addition of each substrate. Laurie (1957) has shown that the acanthocephalan, Moniliformis &&US, can ferment a somewhat broader spectrum of carbohydrates than cestodes. This worm metabolized the monosaccharides, glucose, galactose, mannose, and fructose, and the disaccharide, maltose. A number of other monosaccharides, disaccharides, and sugar alcohols are not fermented by Moniliformis. The data available on the kinds of carbohydrates metabolized by cestodes and acanthocephalans in vitro are summarized in Table 1. The information available supports the conclusion that most cestodes are capable of metabolizing a very limited number of monosaccharide carbohydrates and are not capable of metabolizing disaccharides which may be available in the environment. The single species of acanthocephalan which has been studied is less limited than most cestodes in the number of monosaccharides metabolized and is capable of utilizing at least one disaccharide, maltose. It differs from all cestodes studied in its ability to metabolize fructose. These considerations might have some bearing on the fact that in light infections Moniliformis frequently occurs in the most anterior portion of the small gut; in being less selective, this acanthocephalan may utilize the incompletely hydrolyzed products of carbohydrate digestion which would be present in the anterior portion of the small intestine. EFFECTS
OF THE QUALITY
OF HOST DIETARY
CARBOHYDRATE
Several years ago it was noted that the quality of carbohydrate in the host diet had an effect on the size of Hymenolepis diminuta (Chandler, Read, and Nicholas, 1950). This phenomenon has been studied in greater detail by Read and Rothman (1957a, b, and c) and by Read, Schiller, and Phiier (1958). In these latter studies hosts infected with single worms were used as experimental material. It was shown with H. diminuta that the worm’s size (wet weight, dry weight, length, and number of segments) is affected by the quality of carbohydrate in the host diet. Studies of the initial growth of the worms in hosts receiving the diets,
ROLE
OF
CARBOHYDRATES
IN
BIOLOGY
OF
CESTODES.
VIII
371
differing only in containing starch or sucrose, showed that the growth rate of the worm was dependent on the kind of carbohydrate ingested by the host. Studies of the morphology of worms from hosts on diets containing different carbohydrates revealed that there were significant differences in mean gravid segment volume and, by inference, reproductive capacity as functions of the quality of dietary carbohydrate. Differences in actual reproductive rate, as measured by the number of eggs in the feces, corresponded to the morphological differences induced by specific dietary carbohydrates. From the standpoint of the growth rate, size maintained, and reproductive rate, starch was the most beneficial carbohydrate tested. Glucose, dextrins-maltose, or sucrose as sole carbohydrate components of the diet were less effective in maintaining high growth rate, definitive size, and reproductive rate. When fructose, which is not utilized by the worm in vitro, was supplied as the sole carbohydrate component of the diet, the reproductive rate of the worms was reduced to a very low level, approximating that observed in animals receiving no dietary carbohydrate. It was shown that eggs from worms in hosts receiving fructose were abnormal in size and shape and did not contain the material staining red with iodine (polysaccharide?) which is present in normal eggs. When fructose was fed to animals receiving weighed amounts of food such that the carbohydrate component (starch) limited the size and reproductive rate of the worm, the worms were larger, and had a higher reproductive rate than those from hosts not receiving fructose. This was tentatively interpreted as evidence that fructose may interfere with the absorption by the host of the products of starch hydrolysis, although in animals given free access to diets containing fructose and glucose the worms were not affected by the addition of fructose to the diet. When animals were fed on a diet containing lactose as the sole carbohydrate constitutent, the worms were greatly reduced in size. Further, when lactose was added to diets containing adequate amounts of starch it produced a significant deleterious effect on the worms. This was tentatively interpreted in terms of the known effects of lactose on the physical and chemical characteristics of the gut. It is known that the feeding of lactose produces changes in the pH, oxidation-reduction potential emptying time, and other features of the intestine (see Read, 1950, for review). Since Rothman (1958) has shown that the metabolism of H. diminuta is inhibited by low concentrations of bile salts, normally present in the gut, and that the degree of inhibition is a function of pH in the range of 7.0 to 7.4, it seems probable that the
372
READ
deleterious effects of lactose are indirect ones. Laurie’s (1957) findings that H. diminuta does not metabolize lactose in vitro and that the metabolism of exogenous glucose by the worm is not inhibited by lactose lend further support to this view. In studies on Hymenolepis citelli and H. nana (Read, Schiller, and Phifer 1958), it was shown that the quality of carbohydrate in the host diet also has an effect on the size of these worms. The furnishing of sucrose as the sole carbohydrate component resulted in worms which were smaller than those from starch-fed animals. In the case of H. nana, the effect was produced in young growing worms but older senescent worms were not affected by the quality of carbohydrate. The rate of senescence of this species was apparently not affected by dietary carbohydrate quality (or quantity). This lends further support to the concept that the quantity of carbohydrate required by these animals is a function of the quantity of growth which is characteristic of a particular stage in the life of each species. In this connection, the differences in the growth characteristics of H. diminuta, H. citelli, and H. nana may again be emphasized. In working with the first-named species during the past ten years, no evidence has ever been obtained to indicate that the worm may become senescent. H. diminuta seems to live as long as the rat host and remains young, in the sense that a high growth rate is maintained for an indefinite period. As a matter of fact, data on the size of worms in animals of different ages suggests that the worms may grow at a higher rate as the host grows older (Read, Schiller, and Phifer, 1958). On the other hand, H. cite& and H. nana show phenomena of ageing and senescence to differing degrees. The cited studies on the effects of host dietary carbohydrate quality strongly support the concept that there is a relationship between the degree of response to alteration of the host dietary carbohydrate and the growth characteristics of a particular species. It seems plain that some of the effects of dietary carbohydrate quality on the tapeworms, Hymenolepis diminuta, H. citelli, and H. nana may be interpreted in terms of quantity of utilizable carbohydrate available to the worms in the host. The effects of glucose, sucrose, and starch may be compared in this manner. The worms seem to have less utilizable carbohydrate available to them when glucose or sucrose are fed to the host than when starch is fed. It is known that these species can utilize glucose but cannot metabolize sucrose, fructose, or maltose, a product of starch digestion (Read, 1956; Laurie, 1957; Read and Rothman,
ROLE
OF
CARBOHYDRATES
IN
BIOLOGY
OF
CESTODES.
VIII
373
1958a). Thus, after digestion they can use half of the sucrose molecule and only the final degradation product of starch hydrolysis. There is evidence that the enzyme sucrase may be, for the most part, present in the cells lining certain parts of the digestive tract and in sloughed epithelial cells (Florey et al., 1941). There is also evidence that sucrase activity is primarily located in the anterior portion of the small intestine (Wilson and Vincent, 1955). If the fructose and glucose are absorbed by the host through an active process, a fact which seems to be reasonably well established (Jervis et al., 1956), the amount available to the worm would represent a vectorial sum of hydrolysis rate, absorption rate, and the rate of propulsion down the digestive tract. In the case of glucose there is excellent direct evidence that when glucose is given to the rat per orum, most of the carbohydrate is absorbed in the upper part of the small intestine (Reynell and Spray, 1956). It was shown by Reynell and Spray that retention of glucose in the stomach occurs so that the amount entering the small intestine does not seem to be a direct function of the quantity taken in by mouth. It can be concluded that the deleterious effects of glucose as the dietary carbohydrate are a manifestation of the fact that only a small amount of ingested glucose is directly available to a parasite in the lower two thirds of the small intestine. Direct time relationships should also be of great significance since it has been shown that the rate of glucose absorption by the mammal is independent of the concentration over a wide range (Reynell and Spray, 1956) and that the rate of utilization of exogenous substrate by Hymenolepis diminuta is also independent of concentration over a considerable range (Read, 1956). A somewhat more complicated series of hydrolytic events in the degradation of starch to glucose is undoubtedly involved in the beneficial effects of that carbohydrate on the tapeworms studied. Again, a direct time relationship would seem to be indicated with glucose available at rather low absolute concentrations for an extended time period. This concept is supported by the observation that, when starved hosts are given glucose or starch, glycogenesis in the worms is greatest in the hosts receiving starch (Read, 1955). It seems apparent that the effects on tapeworms of the quality of carbohydrate in the host diet may be of at least two general types: First, there are those effects which are actually manifestations of the quantity of utilizable carbohydrate which is available to the worm. These are mediated through the physiology of the normal digestion and ab-
374
READ
sorption of carbohydrate by the host intestine. It may be appropriate to reiterate a point emphasized elsewhere (Read, 1950) : Carbohydrates, as a class of compounds, seem to be unique in that they are absorbed from the vertebrate gut and do not undergo exocrino-enteric circulation. Further, it may be noted that the cestodes seem to be capable of utilizing those specific carbohydrates which the host is capable of absorbing against a concentration gradient. Second, there are effects which do not directly involve the quantity of utilizable carbohydrate available but are ecological in nature. These are mediated through a change in the intestinal environment, a change wrought by the particular carbohydrate involved. It is also obvious that, with the information available, a more specific recognition of effects on tapeworms of dieta.ry carbohydrate quality is not feasible. HOST STARVATION AND CARBOHYDRATE UTILIZATION It has been shown that starvation of the host for short periods results in the depletion of the polysaccharide of tapeworms (Reid, 1942; Read, 1949; Read, 1956). In the presence of glucose in o&o, worms from such hosts have a higher metabolic rate (Read, 1956; Read and Rothman, 1957b) and a higher rate of glycogenesis than worms from unstarved hosts (Daugherty, 1956; Read and Rothman, 195710). 1n GO, such starved worms show a rapid reconstitution of glycogen if the host is fed carbohydrate (Read, 1955). Since the metabolic rate of starved worms is higher than that of unstarved worms, it might be hypothesized that in a starved host, the worms would compete with the host to a greater extent when food became available than would be the case in a previously unstarved host. This possibility was tested by Read and Rothman (1957b), using Hymenolepis diminuta in the rat. It was found that as starvation proceeds the wet weight of worm tissue in the host declines rapidly. This loss of weight essentially compensates for the increase in metabolic rate, calculated on the basis of tissue weight. Thus, the absolute quantity of carbohydrate utilized by a worm from a previously starved host was slightly less than that utilized by a worm from an unstarved host. That the increase in the metabolic rate was real rather than only apparent was demonstrated by the use of unstarved control worms, reduced in size by cutting the strobila. Of some interest was the observation that the proportion of glucose converted to glycogen in vitro was greater in worms from starved hosts than in those from unstarved hosts. This indicates that a rapid alteration of assimilative
ROLE OF CARBOHYDRATES
IN BIOLOGY OF CESTODES. VIII
375
patterns may occur during starvation and is further evidence of the sensitivity of the tapeworm to alterations of the host’s intake of carbodrate. INTERACTIONS
OF WORMS
It has been recognized for some time that as the number of tapeworms in an individual host increases, the average size of each worm decreases. This has been termed the crowding effect (reviewed by Read, 1951). In considering this stunting of tapeworms observed in heavy infections, Reid (1942) dismissed carbohydrate as a probable limiting factor. Read (1951) concurred in this view. However, subsequent observations demonstrating effects of host dietary carbohydrate quantity and quality on tapeworm size and reproduction suggest that carbohydrate ingested by the host cannot be ruled out as a limiting factor in tapeworm ecology. It was shown by Read, Schiller, and Phifer (1958) that the body weight and reproductive rate of Hymenolepis are, within a certain range, essentially linear functions of the quantity of carbohydrate ingested by the host and that alteration of the quality of carbohydrate ingested by the host induces parallel changes in size and reproductive rate of this worm. Subsequently, a study was made of the effects of crowding under conditions in which the hosts ingested diets containing a worm-limiting quantity of starch or of sucrose, a carbohydrate of suboptimal quality. The slopes of the plots of the weight of worms against worm numbers from hosts on experimental diets were essentially identical with those from hosts on the control diet, but were displaced to considerably lower values in infections with the same number of worms (Read and Phifer, 1958). It has been indicated above that Hymenolepis cannot directly utilize the carbohydrates involved in these experiments (starch or sucrose) and that the effects of quality, in this case, are probably effects of quantity of utilizable carbohydrate available to the worm. In view of these considerations, it is suggested that the quantity of carbohydrate available per unit of time per worm may be involved in the crowding effect. It seemed feasible to evaluate this hypothesis by comparison with a model. The data obtained have been compared with what might be expected if a definite quantity of utilizable carbohydrate is required to support a given quantity of tapeworm tissue, and a hypothetical curve has been constructed. In constructing this curve it has been assumed that (1) a given quantity of carbohydrate ingested by the host is equally shared
376
READ
by the worms in the host, (2) the presence of varying numbers of worms does not affect the physiology of digestion and absorption of carbohydrate by the host, and (3) the body weight maintained by an individual worm is a function of the proportion of the available carbohydrate obtained by the worm. Thus, it is hypothesized that in an infection of 30 worms each worm would have available one-sixth of the quantity of carbohydrate available to the individuals in a five worm infection. Such a calculated curve is a rectangular hyperbola and is shown in Fig. 1 along with the data obtained experimentally. In constructing the curve it has been assumed that, in a five worm infection, each worm weighs 300 milligrams and that worm size decreases as the worm number increases. All other points on the hypothetical curve are calculated on the basis of the foregoing assumptions. The data of Read and Phifer (1958) have been plotted for comparison with this hypothetical curve. The similarities in the predicted and observed values are obvious and support the concept that competition for utilizable carbohydrate is the limiting factor involved in determining the size of individual tapeworms in infections of varying intensity. Incidentally, the data obtained experimentally support the view that infections of 30 worms do not affect digestion and absorption of carbohydrate by the host to any greater extent than do infections of 5 worms. Read and Phifer (l.c.) also showed that the two species, Hymenolepis
D
A fii:q B
C
IO 5
10 Number
of worms per
2030
host
FIG. 1. The average weight of individual worms in infections with various numbers of worms. Hosts were maintained on diets differing in carbohydrate quantity or quality. A = high starch diet, B = low starch diet, C = Sucrose diet, D = hypothetical curve.
See text
for
discussion.
ROLE OF CARBOHYDRATES
IN BIOLOGY OF CESTODES. VIII
377
diminuta and H. cite%, show some reduction in body size when the hamster is infected with a single worm of each species.When the hosts were placed on low carbohydrate diets (0.5 g starch per day) H. citelli was reduced in size to the same extent in the presence or absence of H. diminuta. On the other hand, H. diminuta from hosts on such restricted diets, was reduced in size to a greater extent in the presence of H. cite& than in its absence. This has shown that the effects on these worms of quantity of carbohydrate available may be modified to differing extents by the presence of another species.It would be of interest to determine the effects of dietary modification on the interactions observed between Hymenolepis and Monilijormis in the rat (Holmes, 1957). GENERAL
SIGNIFICANCE
OF A CARBOHYDRATE
REQUIREMENT
There is great interest in the search for the chemical needs which bind obligate parasites to their hosts. That is, bind them in the sense that the organism is incapable of reproducing itself outside the environment furnished by a host (excepting, of course, the special cultural conditions concocted by man for the welfare of these organisms). From our knowledge of the nutrition of free-living animal organisms it is plain that the requirements may vary tremendously. Among the protozoa many flagellates require only a single vitamin, ammonia, carbon dioxide, and minute quantities of certain minerals (Hutner and I’rovasoli, 1951) while ciliates may have highly complex requirements (Kidder and Dewey, 1951). It would seem that among the free-living animals there is little evidence to indicate that an increase in number of requirements for micronutrients has interfered with the evolution of the animal organism. Indeed, it may readily be seen that such requirements are limiting ecological parameters and, as such, serve as mechanisms for isolation. We know that some obligately parasitic microorganisms require micronutrients which are relatively unstable and are characteristically found only in living organisms. Such is Hemophilus, which requires diphosphopyridine nucleotide, or the gonococcus, which requires thiamine pyrophosphate (Snell, 1951). However, there is no particular reason for assuming that a parasite which lives outside cells should be different from predatory organisms with respect to its requirements for micronutrients. A tapeworm living in the intestinal tract of a vertebrate is probably not in contact with compounds of special micronutritional significance occurring only in that habitat. In speculating as to the probable chemical requirements of extracellular parasites it has fre-
378
READ
quently been assumed that these organisms would have highly complex and peculiar nutritional requirements. However, we may have neglected the biological significance of nutrient materials required in large quantity. It seems to be widely accepted that animals require protein, or at least amino acids, for the synthesis of protoplasm. In the usual definition of a “balanced diet”, protein or amino acid content is considerable; interestingly, carbohydrate is also usually included as a sizeable component, although there are few free-living organisms which have a requirement for carbohydrate. The honey bee, and perhaps a few other organisms showing a high degree of specialization, are exceptional in the realm of free-living animals. Carbohydrate is a highly convenient source of calories representing, in the final sense, the most efficient energy storage material arrived at in the evolution of the diverse organisms present on the earth. Other materials may act as energy depots but none in so universal a manner. Animal organisms take advantage of this ubiquitous distribution of carbohydrates and seem to be almost universally capable of utilizing them. However, in the case of most free-living animals protein or fat and protein in sufficient quantity can meet the caloric requirements for maintenance and growth. On the other hand, this may not hold true for many extracellular, and proba.bly some intracellular, parasites. The meager data available strongly suggest that many parasites have a requirement for carbohydrate. In some cases this may be an absolute requirement and in others what may be termed a biological requirement. The latter term is intended to mean that carbohydrate is required for reproduction and hence for species survival. Such seems to be the case with tapeworms. There is suggestive evidence that the cyclic nature of ingestion of carbohydrate by hosts may affect tapeworms under what may be termed natural conditions. Oliger (1950), studying the seasonal changes in tapeworms of tetraonid birds in European Russia, reported that when the birds change to a winter diet (high in fat) in the late autumn, there is a loss of the strobilar portions of their tapeworm parasites. Segments are not produced during the winter feeding. In late February or early March the birds change to a summer diet (high carbohydrate content). At this time the tapeworms produce segments and again become patent. According to Oliger the hosts remain quite well-nourished on both winter and summer diets. It has been observed that bears shed large portions of the strobila of Dibothriocephalus when the feeding habits are altered before hibernation (E. L. Schiller, personal communication). Levine
ROLE
OF
CARBOHYDRATES
IN
BIOLOGY
OF
CESTODES.
VIII
379
(1938) showed that the normal shedding of segments by Davainea is correlated with the cyclic nature of feeding of the avian host. Levine also demonstrated that reduction of the nutritive value of the host diet by increasing the bran content resulted in a marked lowering of worm reproduction; starvation for a day caused cessation of proglottid production, as indicated by examination of the feces of the chicken. It has been shown that there are diurnal fluctuations in the polysaccharide content of Raillietina, Hymenolepis, and Moniliformis (Reid, 1942; Read, 1949; Read and Rothman, 195813). These fluctuations are correlated with the feeding behavior of the host. The data available indicate that the effects on tapeworms of the quality of host dietary carbohydrate are more subtle than might be anticipated by demonstration that most of these worms can only use certain monosaccharide sugars. This is most striking in experiments comparing the effects of starch and partially predigested starch as dietary carbohydrate components. The observed effects on the worms of the latter carbohydrate serve to illustrate the point that the imposition of the dynamics of gut physiology on the specific carbohydrate requirement of cestodes may be of some importance in intra- and inter-host species distribution, apparent age or sex-linked resistance, and, of course, speciation when the interaction serves as a mechanism for isolation. Just as feeding habits of the host are frequently involved in the completion of the cestode life cycle, so they seem to be delicately involved in determining whether the cestode may prosper in a particular host. The imposition of the physiology of carbohydrate degradation in the vertebrate gut plus food preference or habit on the specialized carbohydrate requirements of cestodes seems to constitute a phenomenon which fits Lewis’ balance hypothesis of parasitism (Lewis, 1953) and the basically similar nutrition-inhibition hypothesis of Garber (1956). Obviously, it cannot be claimed that this proves the validity of the hypotheses, and it is plain that many other nutritional and physical factors may be involved in determining whether a particular cestode will live in a particular host. An important consideration in parasitism is the question of the extent to which a given parasite competes with the host for materials of nutritional significance. In the interaction of hosts and tapeworms, with particular reference to carbohydrate, it would seem that competition may be negligible. The location of the worms below the region of maximal intestinal absorption, the lack of significant carbohydrate in the intes-
380
READ
tinal secretion, and dependence of the worms on the quantity in the host ingesta favor the hypothesis that the carbohydrate requirements of the worms are met by amounts which represent an excess for the host. Competition between the worms and the intestinal flora for carbohydrate seems more likely than competition between worms and host. It is obvious, however, that tapeworms may compete with the host for other kinds of nutritional materials, such as amino acids and vitamins.
SUMMARY It is concluded that: (1) Tapeworms require a source of carbohydrate for growth and reproduction; this requirement is satisfied by the utilization of carbohydrate in the host ingesta. The higher the rate of growth, the greater is the apparent sensitivity to carbohydrate deficiency. (2) The kinds of carbohydrates which can be utilized in vitro by tapeworms are quite limited. Most species studied are capable of using only certain monosaccharides. Anoplocephalids may be exceptional in being able to ferment certain disaccharides. (3) The quality of carbohydrate ingested by the host has marked effects on growth rate, size attained, and reproductive rates. The effects are explained in terms of interactions of host physiology and physiological capacities of the worms. Certain carbohydrates, such as lactose, probably affect the worms by altering the physicochemical characteristics of the habitat. (4) The crowding e$ect in cestode infections may be interpreted in terms of competition for utilizable carbohydrate by the individual worms in the populations. (5) The competitive effects of one tapeworm species on another are modified by variations in the quantity of carbohydrate ingested by the host, different species not being affected proportionally. (6) The significance of a carbohydrate requirement is discussed in terms of (a) its uniqueness in the animal kingdom, (b) its probable role in cyclic changes in tapeworms, (c) possible special effects in distribution of tapeworms.
REFERENCES BURLINOAME,
P. L., AND CHANDLER, A. C. 1941. Host-parasite relations of Monilijormis dubius (Acanthocephala) in albino rats, and the environmental nature of resistance to single and superimposed infections with this parasite. Am. J. Hyg. 33(D), 1-21. CHANDLER, A. C. 1942. Studies on the nutrition of tapeworms. Am. J. Hyg. 37, 121-130. CHANDLER, READ, C.P., AND NICHOLAS, H. 0.1950. Observations on certain phases of nutrition and host-parasite relations of Hymenolepis diminuta in white rats. J. Parasitol. 36, 523-535.
A. C.,
ROLE
OF
CARBOHYDRATES
IN
BIOLOGY
OF
CESTODES.
VIII
381
DAUOHERTY, J. W. 1956. The effect of host castration and fasting on the rate of glycogenesis in Hymenolepis diminuta. J. Parasitol. 42, 17-20. DAUGHERTY, J. W., AND TAYLOR, D. 1956. Regional distribution of glycogen in the rat cestode, Hymenolepis diminuta. Exptl. Parasitol. 6, 376-390. DICKENS, F. 1951. Anaerobic glycolysis, respiration, and the Pasteur effect. In: The Enzymes. Vol. II. Edited by J. B. Sumner and K. Myrbilck, Academic Press, New York, pp. 624683. FLOREY, H. W., WRIGHT, R. D., AND BROWN, R. A. 1941. The secretions of the intestine. Physiol. Rev. 21, 36-69. GARBER, E. D. 1956. A nutrition-inhibition hypothesis of pathogenicity. Am. Naturalist 40, 183-194. HOLMES, J. C. 1957. The effects of concurrent infections with the spiny-headed worm, Monilijormis d&&s, on the rat tapeworm, Hymenolepis diminuta. J. Parositol. 43 (Suppl.), 24. HOPKINS, C. A. 1950. Studies on cestode metabolism. I. Glycogen metabolism in Schistocephalus
solidus
in vivo.
J. Parasitol.
36, 384-390.
HOPKINS, C. A. 1951. Studies on cestode metabolism. II. The utilization of glycogen by Schistocephalus solidus in vitro. Exptl. Parasitol. 1, 196-213. HUFF, C. G. 1956. Parasitism and parasitology. J. Parasitol. 42, l-10. HUTNER, S. H., AND PROVASOLI, L. 1951. The phytoflagellates In: Biochemistry and Physiology of the Protozoa. Vol. I. Edited by A. Lwoff, Academic Press, New York, pp. 27-128. JERVIS, E. L., JOHNSON, F. R., SHEFF, M. F., AND SMYTH, D. H. 1956. The effect of phlorizin on intestinal absorption and intestinal phosphatase. J. Physiol. 134, 675488. KIDDER, G. W., AND DEWEY, V. C. 1951. The biochemistry of ciliates in pure culture. In: Biochemistry and Physiology of the Protozoa. Vol. I. Edited by A. Lwoff, Academic Press, New York, pp. 324400. LAURIE, J. 1957. The in vitro fermentation of carbohydrates by two species of cestodes and one species of Acanthocephala. Exptl. Parasitol. 6, 245-260. Levine, P. P. 1938. Observations on the biology of the poultry cestode, Daoainea proglottina (Dav.). J. Parasitol. 24, 423-431. LEWIS, R. W. 1953. The balance hypothesis of parasitism. Am. Naturalist 37, 273-281. OLIGER, I. M. 1950. Causes of destrobilization in cestodes of Tetraonidae. Doklady Akad. Nauk SSSR 74, 869-872 (in Russian). READ, C. P. 1949. Fluctuation in the glycogen content of the cestode Hymenolepis diminuta. J. Parasitol. 36 (Suppl.), 26. READ, C. P. 1950. The vertebrate small intestine as an environment for parasitic helminths. Rice Inst. Pamphl. 37(2), 94 pp. READ, C. P. 1951. The “crowding effect” in tapeworm infections. J. Parasitol. 37, 174-178. READ, C. P. 1955. Intestinal physiology and the host-parasite relationship. In: Some Physiological Aspects and Consequences of Parsasitism. Edited by W. H. Cole, Rutgers Univ. Press, pp. 2743. READ, C. P. 1956. Carbohydrate metabolism of Hymenolepis diminuta. Exptl. Parasitol.
6. 325-344.
382
READ
C. P. 1957. The role of carbohydrates in the biology of cestodes. III. Studies on two species from dogfish. Exptl. Parasitol. 6.288-293. READ, C. P., AND PHIFER, K. 0. 1959. The role of carbohydrates in the biology of cestodes. VII. Interactions between individual tapeworms of the same and different species. Exptl. Parasitol. 6, 4650. READ, C. P., AND ROTHMAN, A. H. 1957a. The role of carbohydrates in the biology of cestodes. I. The effect of dietary carbohydrate quality on the size of Hymenolepis diminuta. Exptl. Parasitol. 6, 1-7. READ, C. P., AND ROTHMAN, A. H. 195713.The role of carbohydrates in the biology of cestodes. II. The effect of starvation on glycogenesis and glucose consumption in Hymenolepis. Exptl. Parasitol. 6, 280-287. READ, C. P., AND ROTHYAN, A. H. 1957c. The role of carbohydrates in the biology of cestodes. IV. Some effects of host dietary carbohydrate on growth and reproduction of Hymenolepis. Exptl. Parasitol. 6, 294-305. READ, C. P., AND ROTHMAN, A. H. 1958a. The role of carbohydrates in the biology of cestodes. VI. The carbohydrates metabolized in vitro by some cyclophyllidean species. Exptl. Paraaitol. 7, 217-223. READ, C. P., AND ROTHMAN, A. H. 1958b. The carbohydrate requirement of Monilijormis (Acanthocephala). Exptl. Parasitol. 7, 191-197. READ, C. P., SCHILLER, E. L., AND PHIFER, K. 0.1958. The role of carbohydrates in the biology of cestodes. V. Comparative studies on the effects of host dietary carbohydrate on Hymenolepis spp. Exptl. Parasitol. 7, 198-216. REID, W. M. 1942. Certain nutritional requirements of the fowl cestode Raillietina cesticillus (Molin) as demonstrated by short periods of starvation of the host. J. Parasitol. 26, 319340. REYNELL, P. C., AND SPRAY, G. H. 1956. The absorption of glucose by the intact rat. J. Physiol. 164, 531-537. ROTHMAN, A. H. 1958. The role of bile salts in the biology of tapeworms. I. Effects of bile salts on the metabolism of Hymenolepis diminuta and Oochoristica symmetrica. Exptl. Parasitol. 7, 328-337. SNELL, E. E. 1951. Bacterial nutrition-growth factors. In: Bacterial Physiology. Edited by C. H. Werkman and P. W. Wilson, Academic Press, New York, pp. 215-255. WARDLE, R. A. 1937. The physiology of the sheep tapeworm, Moniezia expansa Blanchard. Con. J. Research 16D, 117-126. WILSON, T. H., AND VINCENT, T. N. 1955. Absorption of sugars in vitro by the intestine of the golden hamster. J. Biol. Chem. 216,851-866. READ,