Clark P. Read

STUDIES ON MEMBRANE TRANSPORT, WITH SPECIAL REFERENCE TO PARASITE-HOST INTEGRATION*

The physicochemical relationships between a host and a parasite will involve a region in space and time which may be termed the host-parasite interface. The host-parasite interface will be intimately involved in determining the nature and extent of integration, and thus the outcome of the relationship, since it represents the region of chemical juxtaposition of regulatory mechanisms of both host and parasite. In the latter regard, two distinct aspects of the interface have regulatory significance: The physicochemical characteristics of the surrounding host fluids, together with mechanisms regulating their composition; and the functional characteristics of surfaces or surface membranes of the parasites themselves. The latter includes the bounding membrane and associated organelles in acellular forms, but may possibly be considered to comprise entire organs, such as the lining of the parasite’s digestive tract, in many metazoan species. In the work to be presented, the authors have examined functional capacities of a cestode parasite, Hymenolepis diminufa, with regard to acquisition of amino acids from a surrounding fluid. The selection of a tapeworm for such a study might be regarded as unfortunate in some quarters, since it can be argued that these animals are not “typical” parasites; however, because a gut is lacking, tapeworms provide an unusually favorable material for studies of acquisition of substances by intact organisms. It cannot be decided as yet whether the interface, in the case of a cestode, should be considered to include a specific bounding membrane or the entire integument, which studies with the electron microscope have shown to be acelMar (Read, 1955; Rothman, 1963; Threadgold, 1962). Nature of absorption processes and previous studies with cestodes. Definitions of parasitism are usually couched in nutritional terms. In order for a material to serve as a nutrient it must, of course, be metabolizable, but it must also become available for metabolism. A nutrient may become available directly by entry into the parasite, or it might be altered by the parasite into a form which can then enter, or it might be utilized at the interface itself. While there have been numerous measurements of the metabolism of various substances by parasites, there have been few attempts to study the processes by which parasites acquire materials from the environment. It seems appropriate to include brief comments on the types of processes by which compounds could be directly acquired. General discussions of absorption mechanisms are quite numerous, and the subject receives periodic review in the physiological literature. Among many excellent discussions, those of Davson and Danielli ( 1952), Danielli ( 1954), Gale (1954), LeFevre ( 1954), Rosenberg (1954), Harris

The role of carbohydrates in the biology of cestodes. VIII. Some conclusions and hypotheses

Abstract 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 effect 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.

Circadian migratory behavior of a cestode symbiote in the rat host.

Under specified experimental conditions, Hymenolepis diminuta exhibits a longitudinal circadian migration in the small intestine of the rat. This was shown by changes in distribution of total worm tissue and of scolices in the gut at different times of the day in hosts on a 15-hr feeding schedule. Maximum numbers of scolices and amounts of worm tissue were in the anterior 10 inches of the small intestine during the 12 to 4 AM period, and lowest quantities at 4 to 5 PIM. There were accompanying inverse worm distribution changes in the posterior part of the small intestine. Short periods of host starvation delayed anteriad migration. In a limited number of combinations, migration occurred in infections of one to 30 worms, 10 to 21 days of age. The timing of migration was changed by altering the feeding pattern of the host. Fluoroscopic study of the rat gut during and after killing indicated that the migration was not an artifact produced by violent gut movements. In the course of other experiments on the strobilate stage of the rat tapeworm, Hymenolepis diminuta, it was noted that the worms appeared to be in different parts of the host small intestine at different times of the day. This has been examined in a more systematic fashion to determine whether there is a regular pattern of movement of the strobila and whether or not the scolex migrates during the course of a day. MATERIALS AND METHODS Young male rats of the Sprague-Dawley strain (Holtzman Rat Co.) were infected with cysticercoids of Hymenolepis diminuta; unless otherwise specified, each host received 30 worms. Ten days after infection, at specified times of the day, groups of four rats were weighed; each rat was killed by a blow on the head and the small intestine rapidly removed. With minimal stretching, the small gut was cut into three sections; the first two portions were 10 inches long, and the posteriormost third varied in length. The worm tissue in each of the three sections of the gut was flushed out with Ringers solution. The worm tissue was blotted on hard filter paper, the wet weight of the tissue determined, and the number of scolices in each of three samples was counted. In experiments in which the procedure was modified, this is described in the context of the experiment. Unless otherwise stated, the rats were given an excess of food (Purina Lab Chow) each day at 5 PM and food was removed from the cages each morning at 8 AM throughout the 10-day period of worm development. Water was available to the animals at all times. RESULTS AND DISCUSSION In the first experiments (Expt. 1, Table I), Received for publication 10 January 1969. * This work was supported in part by grants from the NIH, U. S. Public Health Service (AI 01384 and 2E-106). real differences were apparent in the distribution of worm tissue and of scolices from hosts killed at four times during the day. There appeared to be a posteriad movement of worms during the post-feeding period, this repositioning being maximal at 5 PM. Next, the worms moved anteriorly and, shortly after midnight, the distribution of worm tissue and scolices resembled that seen at 8 AM. In a second experiment (Expt. 2, Table I), the afternoon shift in worm distribution was examined more closely by killing groups of animals at 4 PM and at 6:30 PM, as well as at other times of the day. The shift in position in the gut was again obvious. At 4 PM, about half of the worms were in the posterior part of the small intestine. At 6:30 PM, 1.5 hr after host feeding began, there was an increase in the number of worms in the anterior small intestine. At 9 PM, this anteriad movement was apparently still in progress. The data of Experime ts 1 and 2 have been combined and are presented graphically in Figure 1. Special mention may be made of the changes in tissue and scolex distribution in the second 10-inch section of the small gut. The fluctuations in worm weight in this region closely paralleled those seen in the anterior section. However, in the middle region the maximum number of scolices was found about 4 hr later than in the first section and several hours before the peak was attained in the posterior zone. In Experiment 2, food was withheld from two groups of animals at the regular 5 PM feeding. At 9 PM, one group was killed and food was given to the second group which was then killed 2 hr later. In the starved 9 PM

Carbohydrate metabolism of Hymenolepis diminuta

Abstract 1. 1. The endogenous respiratory quotient of Hymenolepis diminuta averages 0.51. In the presence of glucose the average respiratory quotient is 1.02. Aerobic respiration is stimulated by glucose. Succinate, malate, glycerophosphate, and malonate stimulated or supported respiration while citrate, α-ketoglutarate, β-hydroxybutyrate, pyruvate, and acetate were without effect on the endogenous rate. 2. 2. The rate of aerobic respiration is sharply dependent on oxygen tension between 2.1 and 5.0 volumes % in the atmosphere and less so between 5.0 and 21.0 volumes %. H. diminuta shows an oxygen debt when returned to air after periods of 1 to 3 hours of anaerobiasis. 3. 3. Polysaccharide content was found to be sharply different under two different laboratory situations. In vivo the worm uses about 5.04 g glycogen per 100 g tissue in a 20-hour period. In vitro the worm used about 4.41 g in a similar period. Differences in polysaccharide content of linear quarters of H. diminuta was demonstrated. 4. 4. Anaerobically, no metabolic gas was produced by H. diminuta and the major acid product excreted was lactic. The acid was also found to be a major product of Oochoristica symmetrica . Acid production of H. diminuta was enhanced by addition of glucose, independent of concentrations ranging from 0.005 to 0.066 M . Galactose is utilized at a somewhat lower rate than glucose. Maltose, trehalose, lactose, and fructose are not utilized to a significant extent. Sharp differences were found in rates of acid production and glucose utilization by portions of worms divided linearly into sixths. The linear differences are altered by washing the worms for various periods in Ringers solution. The worms produce 31 % more lactate under anaerobic conditions than under aerobic conditions. 5. 5. The data are compared with similar data on other cestodes. The possible significance of aerobic metabolism to cestodes is discussed in terms of energy yield vs. molecular configuration of products and in relation to the morphological and ecological peculiarities of cestodes.

Effect of behavior on development of resistance in trichinosis.

Summary The demonstration that behavioral factors cause hypertrophy of adrenals, and that injection of corticoids decreased the resistance of mice to Trichinella spiralis suggested that behavior might affect infections under relatively natural conditions. “Wild” strain house mice were infected with embryonated larvae. Twelve mice were isolated in jars and 12 were placed in 2 groups in a large can for 3 hours a day for 10 days. When killed on the fifteenth day the grouped mice had from 16–51 worms in the intestine while only 3 of the isolated mice had worms. To determine the effect on subsequent stages, 12 mice were infected. Six were isolated and 6 were grouped for 10 days. All were killed on the thirtieth day. The isolated mice averaged 1054 larvae per gram of muscle while the grouped mice averaged 1556. It is concluded that behavior may affect resistance and it is suggested that this phenomenon may be an important factor in epidemics and in development of host-parasite relations.

Observations on certain phases of nutrition and host parasite relations of Hymenolepis diminuta in white rats.

They were also found to be independent of alimentary vitamins with the exception of some unidentified substance associated with the B-complex in brewers yeast, the need for which is influenced by the sex hormones. In the present paper some of these experiments have been repeated and extended, using more adequately controlled experimental diets, to determine whether H. diminuta obtains its vitamins from the host, from bacterial synthesis in the intestine, or by its own synthesis. For this purpose thiamin was selected for experimentation, since (1) it is a typical vitamin; (2) as far as known, it is needed in the metabolism of all kinds of animals; (3) good quantitative methods for its determination are available; and (4) its preparation with radioactive sulfur (S35) was known to be feasible, permitting tracer experiments. We incidentally obtained information concerning the effect of various dietary factors on the growth of tapeworms, including sulfasuxidine, various carbohydrates, and thiamin. Information on the source of thiamin for the worms was obtained by fluorimetric analysis of thiamin in worms, intestinal mucosa, and feces under various dietary regimes, and by determination of radioactivity in thiamin extracted from worms and intestinal mucosa, respectively, after parenteral administration of radioactive thiamin. At the conclusion of the paper we have discussed the mechanism whereby tapeworms are able to obtain substances from their hosts, independent of the diet.

Studies on membrane transport—VI. Absorption of amino acids by fascioliid trematodes

Abstract 1. 1. Ligation of Fasciola or Fascioloides did not affect the 2-min absorption of 14C-cycloleucine. 2. 2. Absorption of cycloleucine, arginine, proline or methionine was a linear function of concentration and competitions were not demonstrable. Amino acids entered the tegument by diffusion. 3. 3. Efflux experiments indicated that cycloleucine may be accumulated in an inner “compartment” in Fasciola. 4. 4. Four hours after administration of 14C-cycloleucine to infected hosts, the amino acid was equally distributed in tissue water of host blood and Fasciola.

Sugar Transport and Metabolism in Schistosoma mansoni

The absorption kinetics of some 14-C-labeled simple sugards in adults of Schistosoma mansoni are described. The influx of fructose and 3-0-methylglucose was by diffusion alone, while glucose, 2-deoxyglucose (2DOG), galactose, glucosamine, and mannose were absorbed by mediated transport as well as by diffusion. Although absorbed glucose was rapidly metabolized, uptake rates of radio-glucose in 2-min incubations corresponded with the amount of glucose (determined chemically) removed from the incubation medium. In 30-min incubations 2DOG was slowly metabolized and accumulated against an apparent concentration difference. The mediated transport of glucose and 2DOG was inhibited in Na+-free media, and by the presence of ouabain, phlorizin, phloretin, and other sugars. Accordingly, influxes of glucose of 2DOG and 22-Na+ were coupled. On a per mg protein basis, female worms transported more 2DOG and glucose, but less glycine, than did males. However, the rate of glucose metabolism by male and female worms incubated together was greater than that of either males or females incubated separately. The nature of sugar transport in schistosomes and other flatworms is similar to that in vertebrates.

The role of carbohydrates in the biology of cestodes. I: The effect of dietary carbohydrate quality on the size of Hymenolepis diminuta☆

Abstract Comparisons have been made in the size attained by Hymenolepis diminuta when the hosts are given diets containing single carbohydrate components. The worms from hosts receiving dextrose or sucrose as the carbohydrate are markedly smaller than worms from hosts receiving starch as the sole carbohydrate. When dextrins-maltose, an enzymatically degraded product of starch, is substituted as the carbohydrate component, the worms are much smaller than when glucose or sucrose is substituted. Possible explanations for these effects are briefly discussed.

STUDIES ON MEMBRANE TRANSPORT. I. A COMMON TRANSPORT SYSTEM FOR SUGARS AND AMINO ACIDS

1. The uptake of cycloleucine (l-aminocyclopentane-1-carboxylic acid) by dogfish intestinal tissue is inhibited by galactose in 10-minute incubations but not in 2-minute incubations.2. Preincubation of the tissue in galactose inhibits subsequent uptake of cycloleucine in 2-minute incubations without sugar. Addition of phlorizin to the preincubation with galactose abolishes inhibition. The inhibition by galactose, and other actively transported sugars, is effected during or after absorption of the sugar and is not a competition for a common site at the external interface. Leucine on the other hand, competitively inhibits cycloleucine uptake in 2-minute incubations.3. The inhibitory effects of galactose are dependent on time of preincubation and concentration of the sugar.4. In 2,4-dinitrophenol-inhibited tissues, the previous absorption of galactose inhibits the subsequent uptake of cycloleucine.5. Both galactose uptake and cycloleucine uptake are sodium-dependent. However while potassium antagonizes sodiu...