Studies on the Absorption and Metabolism of D-(u-^14C) Glucose by Polymorphus minutus (Acanthocephala) in vitro

The large amount of research on the carbohydrate metabolism of parasitic helminths, reviewed recently by von Brand (1966), has established that several cestodes and the acanthocephalan, Moniliformis dubius, have an absolute requirement for carbohydrate which can be supplied only through their hosts’ diet. It is also evident from the literature that, since these helminths do not possess an alimentary tract, most of their dietary carbohydrate must be absorbed through their body surfaces in the form of glucose present in the lumen of their hosts’ intestine.

The acanthocephalan, Polymorphus minutus, inhabits a region of the small intestine of ducks where a considerable range of glucose concentrations has been measured (Crompton, 1966). It is most probable that P. minutus will be found to have a requirement for carbohydrate similar to that of M. dubius, and the primary aim of the work reported in this paper was to investigate the absorption of glucose by P. minutus in vitro, under conditions designed to simulate those in the worm’s environment. The other aim was to expand the work of Crompton & Ward (1967a), who found that lactic and succinic acids were the main excretory products from the carbohydrate metabolism of P. minutus, by analyzing extracts of the worms for other metabolites originating from glucose. It was intended to compare these results with those obtained from studies of the carbohydrate metabolism of M. dubius by Graff (1964) and Bryant & Nicholas (1965). Fairbairn (1958) identified trehalose in extracts of M. dubius, a result confirmed by Laurie (1959) and Fisher (1964), who also detected trehalose in Macracanthorhynchus hirudinaceus. Thus, it was decided to search carefully for trehalose in P. minutus, a parasite having crustacean and avian hosts in contrast to Moniliformis dubius and Macracanthorhynchus hirudinaceus, both of which have insect and mammalian hosts.

Throughout the work care was taken to maintain P. minutus in a healthy physiological condition and so ensure that the results would have some application to worms living in their final hosts.

P. minutus of known age, sex and wet weight were recovered from freshly killed Khaki Campbell ducks and maintained in vitro as described by Crompton & Ward (1967a). The tests used by Crompton & Ward for bacterial contamination and the physiological condition of the worms at the end of the experiments were also employed during this work.

The principle of the experimental procedure was to incubate the worms at 41·7 ± 0·5° C., at pH 7·7, under commercial nitrogen in a known volume of physiological saline (Hanks, 1948) containing D-(u-¹⁴C) glucose, of specific activity from 2 to 4 mc/mM, obtained from the Radiochemical Centre, Amersham, Bucks, U.K. The glucose concentrations used in these experiments were at the lower end of the range measured in the worm’s environment by Crompton (1966). The absorption of glucose was determined by assuming that the difference between the glucose concentration in the medium before and after an incubation experiment represented the amount of glucose absorbed by the worms. Radioactivity was determined with an Ecko Scanner after samples of the incubation medium had been examined by paper chromatography. This instrument is a 4π system incorporating two 2BM2 thin window GM tubes and, while the efficiency of the counting system is not more than 10%, the use of paper chromatography facilitated the complete separation of glucose from the other radioactive metabolites present in the small volumes of medium used.

The metabolism of glucose was studied by analyzing extracts of worms for radio-active metabolites. A 2π counting system, involving manual feeding of paper strips through the top tray of a lead castle containing a GM tube, was used during this phase of the work as well as the 4π system. Background radioactivity was determined by scanning strips of paper free from radioactive compounds and frequent checks were made for self absorption.

Several 7-day-old worms were maintained for 40 min. in saline containing 1 mg. non-radioactive glucose/ml. followed by 20 min. in glucose-free saline before being transferred to the experimental medium. The radioactive glucose medium was contained in the apparatus shown in Fig. 1. These tubes enabled either the absorption of glucose by worms from one host to be studied simultaneously at five different glucose concentrations, or worms to be transferred quickly between different glucose solutions by means of the nylon baskets. The incubation periods lasted for 1 hr., and the wet and dry weights of the worms were determined at the end of each incubation. A typical experiment involved 7 mg. wet weight of worm and 150 μl. of incubation medium.

Five incubations, designated A, B, C, D and E in the text, were undertaken and details of each one are given in Table 1. After 4 hr., the worms were removed from the medium and washed for three periods of 1 min. each in saline followed by two periods of 1 min. each in distilled water. The worms were then homogenized in 1 ml. absolute ethanol and the homogenate, after being allowed to evaporate to dryness, was taken up in a known volume of 60% ethanol (Table 1). This ethanolic extract was analyzed by paper chromatography and high-voltage paper electrophoresis.

Samples of the extracts and incubation media were examined by one-dimensional, descending chromatography on Whatman no. 1 paper at room temperature in a Shandon Panglas 300 Chromatank, or by one-dimensional, ascending chromatography in a 10 × 10 in. Universal tank. There was no equilibration period for any of the solvent systems used. Standard solutions of authentic substances were made up in water, 60% ethanol and Hanks’s saline. Details of the solvent systems used in this work are listed below, and their compositions are given by Smith (1960). For sugars:

EtAcPy, 8 parts ethyl acetate: 2 parts pyridine: 1 part water.

For organic acids:

Etam, 16 parts ethanol: 3 parts water: 1 part 0.880 ammonia,

PrF, 5 parts n-propanol: 5 parts cineole: 2 parts formic acid. This mixture was saturated with water just before use.

For sugar nucleotides:

AmAcEt, 3 parts M ammonium acetate: 7·5 parts 96% ethanol (Paladini & Leloir, 1952).

(HVPE) Whatman no. 1 paper, onto which samples of extract and standard solutions had been loaded, was hung in a vertical position in an electrophoresis tank for 2 hr. at 2 kV. and pH 6·5, or for varying times at either 2 kV and pH 2·0.

For sugars:

silver nitrate reagent (Smith, 1960),

aniline phthalate reagent (Wilson, 1959),

0·5% sodium metaperiodate (Evans & Dethier, 1957) followed by silver nitrate.

For amino acids and amino sugars:

0·1% ninhydrin and 5% collidine in acetone. The identification procedure of Stoffyn & Jeanloz (1954) for amino sugars was also used.

For sugar phosphates:

examination of papers under UV light (2537 Å) after spraying with the reagent of Haynes & Isherwood (Bandurski & Axelrod, 1951).

For sugar nucleotides:

examination of papers under UV light (2370 Å).

For organic adds:

bromcresol green reagent (Smith, 1960),

0·1% acridine in absolute ethanol followed by examination of papers under UV light (2537 Å) (Smith, 1960).

About 10 mg. wet weight of worms were sealed in a glass ampoule with about 0·125 of 30% KOH, and heated at 110° C. for 1/2 hr. The polysaccharide was precipitated with 1·2 vol. of absolute ethanol, and the sample was centrifuged at 5000 rev./min. for 5 min. The supernatant was decanted and the precipitate was dissolved in 1 ml. 0·9% NaCl. The polysaccharide was re-precipitated by the addition of 12 ml. ethanol and, after centrifugation, it was dissolved in 0·3 ml. of 0·6 N-HCI and hydrolyzed under an air condenser for 2 hr. at 100° C. The hydrolysate was examined by paper chromatography for radioactive glucose.

Figure 2 is a graph showing the results of plotting the uptake of glucose by P. minutus expressed as μg. glucose/mg. wet weight of worm/hr., against the glucose concentration in the medium. The glucose concentrations used for the graph were those calculated to exist after half the incubation period had passed.

Curve a was obtained after five preliminary experiments, during which worms were incubated with radioactive glucose for 4 hr. The medium was sampled hourly and, for all the concentrations studied, it was found that the rate of glucose uptake was not affected by the decreasing glucose concentration in the medium during the first $112$ hr. of the incubation period. This information was needed before the uptake of glucose over periods of an hour could be investigated in more detail, and curve a was constructed from data applying to the first hour only of the incubation.

Curve a is not unlike a hyperbola in shape, a result suggesting that a carrier system of the type discussed by Davson (1964) could be operative in P. minutus. Consequently, a hypothetical curve (b) was constructed by substituting values in the equation, ⁠, where u represents uptake rate, x the glucose concentration in the medium, the maximum uptake rate (assumed to be 15 μg./mg./hr.), and the glucose concentration in the medium necessary to promote half the maximum uptake rate (assumed to be 0·3 mg./ml.). Sixteen more absorption experiments were then carried out and the results were plotted as solid circles on Fig. 2. Curve c was drawn through seven of the points and was found to be in close agreement with the hypothetical curve b.

These data are compatible with the assumption that a carrier system is involved in the absorption of glucose. If this assumption be accepted, it can be seen that the carrier is half-saturated at an external glucose concentration of about 0·25 mg./ml. and is tending towards full saturation at an external glucose concentration of about 2·0 mg./ml. when glucose concentration no longer affects the rate of uptake. Figure 3 is a Lineweaver-Burk plot for the points through which curve c was drawn. The resulting straight line, having an intercept on the Y axis, demonstrates that curve c is a rectangular hyperbola, like curve b, and that the assumption that a carrier system is involved in glucose uptake may be applied to P. minutus. The data plotted in Fig. 3 may also be used to determine the Michaelis-Menten constant for the carrier if this is further assumed to be an enzyme.

The results shown in Fig. 2 suggest that P. minutus will be able to absorb glucose against a concentration gradient, and an experiment was designed to test this inference. Fifteen 7-day-old worms were incubated in a radioactive glucose solution containing 1·55 mg./ml. After 1 hr., 8 of the worms were rinsed and transferred to another radioactive glucose solution containing 0·125 mg./ml., while the other worms were used for a determination of their internal glucose concentration. It was found that, on transfer to the lower glucose concentration, the internal glucose concentration of the worms was about 0·166 mg./ml. The volume of solvent within the worms, in which the glucose was dissolved, was taken to be the difference between their wet and dry weights, the greater part of this difference being due to their pseudocoelomic fluid. The concentration of non-radioactive glucose in the worms was not measured, but it follows that the actual concentration of glucose within the worms would have been greater than 0·166 mg./ml. After incubation of the worms for $12$ hr. at the lower glucose concentration, it was found that the concentration of the medium had fallen from 0·125 to 0·086 mg./ml., and the internal glucose concentration of the worms had risen to about 0·4 mg./ml. Thus evidence was obtained to show that P. minutus can absorb glucose against a concentration gradient when the external glucose concentration is low.

The uptake of glucose against a concentration gradient must depend upon either active transport or a combination of this process and facilitated diffusion. Simple diffusion or facilitated diffusion, however, might be involved in glucose uptake when the glucose concentration of the medium exceeds that in the worms. There was no evidence of this happening (Fig. 2), but if it were the case glucose might be expected to diffuse out of the worms into the medium when the concentration gradient was reversed. This possibility was tested by incubating ten worms for 2 hr. in a solution of radioactive glucose containing 0·55 mg./ml., and then transferring the worms to 0·15 ml. glucose-free saline and examining it for radioactive glucose over a period of an hour. The results are shown in Figs. 4 and 5, from which it is apparent that no significant amount of radioactive glucose was detected in the saline after an hour (Fig. 4), and that the internal glucose concentration of the worms was about 0·02 mg./ ml. at the end of the experiment (Fig. 5). These results indicate that glucose does not pass through the surface of P. minutus by simple diffusion. It is unlikely that the surface membrane acts as a chemical rectifier permitting the passive diffusion of glucose in one direction only.

Experiments were undertaken to obtain information about the absorptive process over a range of glucose concentrations to which the worms had previously been acclimatized. The results were expected to be more relevant to P. minutus in vivo, where they may be exposed to constant glucose concentrations for periods longer than an hour. Forty 7-day-oId worms were separated into five groups and each group was placed in a Nylon basket and suspended in 50 ml. of a known non-radioactive glucose solution. After 1 hr., when it was assumed that any change in the glucose concentration of such a large volume as 50 ml. would be immeasurable, the worms were transferred quickly in their baskets to 0·5 ml. of a radioactive glucose solution of the same concentration as that in which they had just spent an hour. This second incubation was stopped after 2 hr. and the rate of glucose absorption for the first hour, expressed as µg. glucose/mg. dry weight of worm/hr., was determined. The results are shown in Fig. 6 and the curve, which is of the same general shape and has similar characteristics to curve c (Fig. 2), provides further evidence in support of the hypothesis for glucose uptake by a carrier system. The curve obtained from acclimatized worms, however, flattened out after the glucose concentration in the medium exceeded 1·5 mg./ml., a feature which may have resulted from the accumulation of excretory products in the medium. If the results had been expressed for mg. wet weight of worms, the maximum uptake of glucose by acclimatized worms would have been between 4 and 5 μg./mg. worm/hr., once full saturation of the carrier system had been achieved. This value could represent the usual rate of glucose uptake by P. minutus in vivo, and its reliability was tested by sealing, in a glass vessel, fourteen worms of known weight in saline containing sufficient glucose to promote 150 hr. spontaneous movement. The amount of glucose added was calculated from the figure of 4 μg./mg. worm/hr., and it had already been observed that spontaneous movement by P. minutus stops after about 40 ± 10 hr. in glucose-free saline (Crompton—unpublished observations). The worms under observation stopped spontaneous movement after 168 ± 6 hr., a result which does not contradict the possibility that the normal uptake rate of glucose by P. minutus in vivo is 4–5 μg./mg. worm/hr.

The results of analyzing extracts of P. minutus for radioactive metabolites are reported in this section. The results shown in Fig. 7 were obtained every time a sample of worm extract from incubations A to E was examined by paper chromatography using the EtAcPy solvent system. The radioactive material at position (i), which gave a positive reaction with both silver nitrate and aniline phthalate, was identified as glucose, since it reached the same position on the chromatogram as authentic glucose run under the same conditions. The material at position (ii) was found to be negative to ninhydrin, silver nitrate and aniline phthalate, but positive to reagents for organic acids. It was tentatively identified as organic acid material at this stage of the work because it reached the same position on the chromatogram as fumaric and malic acids. The material at position (iii) was positive to almost any location reagent applied. It can also be seen from Fig. 7 that neither age nor sex of the worms affected the result of this analysis, and it was calculated that young worms contain more radioactivity, per unit weight, than old male or female worms.

More information about the radioactive metabolites within the worms was obtained after subjecting three samples of 200 μ. each of worm extract from incubation C to HVPE at pH 6·5 and 2 kV. for 2 hr. Most of the radioactivity was found to be associated with material which remained near the origin and gave positive results to tests for carbohydrates, amino groups and organic acids. Some radioactive material, however, had moved towards the anode and it gave a positive reaction to the test for sugar phosphates and had reached the same position as authentic G-6-P and G-l-P. Similar results were obtained with samples of extracts from incubations D and E.

The material which remained near the origin after HVPE at pH 6·5 was eluted and subjected to HVPE at pH 2·0 and 1 kV. for 2 hr. Most radioactivity was again detected near the origin, except for a considerable amount of activity which was associated with material containing amino groups and at the same position as that reached by authentic glucosamine. Similar results were obtained when samples of extracts from incubation D and E were subjected to HVPE at pH 2·0 and 5 kV. For 25 min. This radioactive substance did not correspond in position with those reached by any of the sixteen amino acids run under the same conditions. Only a very small amount of radioactivity was found to be associated with amino acids in the extracts of P. minutus, and these have not yet been identified. An attempt was made to confirm the identity of glucosamine by eluting the radioactive material from that position on the electrophoresis strip and testing it by the procedure of Stoffyn & Jeanloz (1954). The results were negative for both the radioactive eluate and the control eluate from filter paper, but positive when applied to a sample of worm extract before electrophoresis. Both radioactive glucosamine and galactosamine appeared to be present in the worm extract and it is possible that inefficient elution was responsible for the negative result.

The material which still remained near the origin after HVPE at pH 2·0 was eluted and examined by paper chromatography with the EtAcPy solvent system and the results are shown in Fig. 8. Most radioactivity was found to be associated with glucose, but some activity was connected with a reducing carbohydrate which had moved only a short distance from the origin to the same position as authentic maltotriose. A third radioactive substance was also present on the chromatogram in a position in front of glucose. This compound was not identified.

It was expected that trehalose would have been detected at this stage of the analysis had it been present in the worm extracts. A careful search was made for trehalose by preparing an aqueous extract from 50 mg. wet weight of worms which had been stored at − 20 °C. after the end of experiments on the absorption of glucose. Amino acids and organic acids were removed from this extract by passing it down columns of ion-exchange resins, and the resulting fraction was concentrated and examined by paper chromatography using the EtAcPy solvent system. No evidence was found for the presence of trehalose in P. minutus.

Two large female worms from incubation C were analyzed for glycogen, which was extracted and hydrolyzed with HCl. Subsequent examination of the hydrolysate revealed that glucose containing above-background radioactivity was the sole component of the hydrolysate (Fig. 9). The presence of HCl was responsible for the fact that the glucose did not form a very precise spot on the chromatogram. Thus it was demonstrated that glycogen was synthesized from absorbed glucose during the course of the incubation period.

Glucose, G-6-P, G-l-P and maltotriose are known to be intermediate metabolites of glycogenesis in other animals, and, in order to complete the reaction sequence, an attempt was made to detect radioactive UDPG in the worm extract. A batch of worms, which had been incubated for an hour in radioactive glucose, was homogenized in ice-cold perchloric acid, and the resulting extract was examined by paper chromate-graphy using the solvent system of Paladini & Leloir (1952). Counting with the 4π system indicated that above-background radioactivity was present at the same position as that reached by authentic UDPG. This position was cut out of the chromatogram and counted in a Packard Tri-carb Liquid Scintillation Spectrophotometer and a significant amount of radioactivity was detected at the position which UDPG would have reached.

Information about radioactive organic acids within the worms (Fig. 7 (ii)) was obtained by eluting the material from this position on similar chromatograms and examining it with the EtAm and PrF solvent systems. Fig. 10 shows the results obtained using the EtAm solvent system. It is possible that five different organic acids were present within the complex which reached a position between 3 and 6 in. from the start line, and a sixth radioactive organic acid was also separated from the eluate and found to have reached the same position as authentic lactic acid. Succinic and lactic acids can be identified with certainty because they have been found to be excretory products of P. minutus (Crompton & Ward, 1967a), and the identification of malic and fumaric acids is also reliable because they have been detected in extracts of M. dubius by Graff (1964) and Bryant & Nicholas (1965).

An attempt was made to determine the percentage of radioactivity associated with each of the radioactive metabolites detected in the worms in incubation C. The results were as follows: glucose 2%, sugar phosphates 0·2%, maltotriose 0·06%, glycogen 20%, organic acids 3%, amino acids 1 %, and amino sugars 1%. Thus, 27·26% of the radioactivity absorbed by the worms during this incubation period was present within them, and a further 58 % had been excreted in the form of lactic and succinic acids. Nearly 15% of the radioactivity absorbed by the worms could not be traced, but it was most likely that much of this would be associated with the resistant eggs of the parasite which would be unaffected by the extraction procedures. Most of the worms involved in incubation C were mature female worms in which egg production was occurring.

The results obtained during these investigations on the absorption and metabolism of glucose may be assumed to apply to P. minutus living in vivo, because the worms have been shown to be in a healthy physiological condition at the end of the experiments and, therefore, capable of becoming re-established in their natural environment after surgical transfer (Crompton & Ward, 1967a). On the other hand, the results must still be interpreted with caution because of the many differences between the natural environment of P. minutus and the conditions to which it has been subjected during this work. For example, in vivo excretory products will be removed from the surroundings of the worms and not left to accumulate as has been the case during this work. It is not known how the continuous, vigorous movement of P. minutus during its first hours of maintenance in vitro affects its glucose consumption. This type of movement takes place when the worms’ proboscides are unattached. It has been found during this work that P. minutus readily became attached to Nylon netting; consequently the lowering in the rate of glucose uptake by acclimatized worms may have resulted from their having become attached to the Nylon baskets.

Evidence has been presented in this paper to show that the hypothesis for the absorption of glucose by a carrier system can be applied also to P. minutus. The worms have been found to absorb glucose against a concentration gradient and it is most likely that active transport is responsible for this process. It is also probable that the absorption of glucose occurs through the pores and canals located at the surface of P. minutus (Crompton, 1963; Crompton & Lee, 1965). More glucose can be expected to be absorbed through the metasomal surface than through the praesomal surface, per unit area, because the pores are larger and more numerous in the metasomal body wall. No morphological evidence exists to indicate that a particular region of the metasomal surface is better adapted for nutrient absorption than the other regions, although most pores are situated in the anterior part of the metasoma where the trunk spines project. The trunk spines increase the absorptive surface area of the worm, but there is no reason to believe that more pores are present here, per unit surface area, than elsewhere on the surface.

Radioactive glucose, glycogen, fumaric, malic, lactic and succinic acids, and amino acids have been found in extracts of M. dubius (Graff, 1964; Bryant & Nicholas, 1965), and lactic acid has been found to be an excretory product by Laurie (1959). It would appear, therefore, that the carbohydrate metabolism of P. minutus is similar to that of M. dubius. Recently, however,Crompton & Ward (1967 a, b) found that, while lactic and succinic acids are the major excretory products of P. minutus, ethanol is the chief excretory product of M. dubius, although this species also excreted small amounts of lactic and succinic acids. These findings suggest that the pathways involved in the energy production of these species may differ.

Trehalose could not be detected in any of the extracts of P. minutus prepared during this work. The sensitivity of the analytical methods used were tested not only on authentic trehalose but also on extracts of M. dubius in which trehalose was identified, and the results of Fairbairn (1958) were confirmed. If the absence of trehalose from P. minutus is as trustworthy a result as its presence is in Moniliformis dubius and Macracanthorhynchus hirudinaceus, it is conceivable that it reflects the evolutionary history of the species in the following manner. Trehalose is now known to be the most important blood sugar of insects (Gilmour, 1965), and the ancestral types of M. dubius and M. hirudinaceus may have acquired the ability to metabolize this disaccharide during their associations with the ancestors of cockroaches and beetles. Consequently, modern M. dubius and M. hirudinaceus metabolize trehalose while infecting their mammalian final hosts. P. minutus, in contrast to M. dubius and M. hirudinaceus, develops in crustaceans of the genus Gammarus, and glucose is considered to be the principal blood sugar of crustaceans (Florkin, 1960). Fairbairn (1958) reported the presence of trehalose in a few species of Crustacea, but it may be significant that he detected only a trace of the disaccharide in Gammarus fasciatus, and more recently, Butterworth (personal communication) has detected glucose alone in the haemolymph of G. pulex, the common intermediate host of P. minutus in the British Isles. Thus, it can be postulated that P. minutus does not possess the ability to metabolize trehalose because it has evolved from ancestors which, in their turn, evolved in association with hosts which had either lost or never developed the property of metabolizing trehalose.

We wish to thank Dr D. H. Northcote and Mr R. W. Stoddart for helpful discussions, and Dr J. E. G. Barnet for gifts of lyxose and galactosamine. Thanks are also due to Mr David Barnard for technical assistance. The use of equipment provided by the Royal Society for D.W.T.C., and D.S.I.R. (now S.R.C.) for A.P.M.L. is gratefully acknowledged.