Schistosoma mansoni: Glycogen content and utilization of glucose, pyruvate, glutamate, and citric acid cycle intermediates by cercariae and schistosomules

EXPERIMENTAL

PARASITOLOGY

26, 29-40

mansoni:

Schistosoma

Glucose,

( 1969)

Glycogen

Pyruvate, Cycle

J. I. Bruce,* Department

of

Glutamate,

intermediates and E. Weiss,

Content

and

and

Utilization

Citric

Acid

by Cercariae

Schistosomulesl M. A. Stirewalt,

and

D. R. Lincicome

Zoology, Howard University, Washington, D.C. 20001; Medical Research Institute, Bethesda, Maryland 20014 (Submitted

of

for publication,

6 November

and Naval

1968)

BRUCE, J. I., WEISS, E., STIREWALT, M. A., AND LINCICOME, D. R. 1969. Schistosoma mansoni: Glycogen content and utilization of glucose, pyruvate, glutamate, and citric acid cycle intermediates by cercariae and schistosomules. Experimental Parasitology 26, 29-40. We investigated the metabolism of three larval stages of Schistosoma mansoni: the developing cercaria infecting Biomphalaria glabrata, the freeliving cercaria, and the schistosomule collected after penetration through rat skin membrane. The metabolism of the developing cercaria was studied by exposing infected snails to 14C-glucose and thereafter determining the distribution of 1% in the emerging cercariae. In one experiment, the radioactivity in the cercariae increased to a high peak on day 6 after 14C exposure, declined rapidly to day 12, and less rapidly to day 30. In a second experiment, increase and decrease were more moderate and the peak was reached on day 13. Protein, nucleic acid, lipid, and glycogen fractions were labeled with 1%. Radioactivity was highest during the first few days after infection and declined rapidly thereafter in most fractions, but the nucleic acid fraction retained a relatively high level of radioactivity for 19 days. When I%-labeled cercariae were maintained free-living overnight, most of the radioactivity of the glycogen fraction disappeared. Free-living cercariae metabolized exogenous glucose to a very slight degree shortly after emergence, but activity increased after 18 hours of free-living existence and depletion of endogenous glycogen. Pyruvate was utilized very rapidly under both conditions resulting in production of CO, from all three carbons and incorporation of a small fraction of the pyruvate carbon into the principal macromolecules. a-Ketoglutarate and acetate, and, to a small extent, glutamate, glutamine, and some of the intermediates of the citric acid cycle were also catabolized. The metabolic activity of the schistosomule appeared to differ considerably from that of the cercaria-the most pronounced difference being a great reduction in the level of pyruvate catabolism. We concluded that the developing cercaria is primarily engaged in synthesis and ducted according to the principles enunciated in “Guide for Laboratory Animal Facilities and Care” prepared by the Committee on the Guide for Laboratory Animal Resources, National Academy of Sciences-National Research Council. 2 Present address: Department of Medical Zoology, 406 Medical Laboratory, Department of the Army, APO San Francisco 96343. Supported by the Secretary of the Army’s Research and Study Fellowship.

1 This work was supported in part by U.S. Public Health Service Grant No. 5TOl-A100040 to D. R. Lincicome, and in part by the Bureau of Medicine and Surgery, Navy Department, Research Task MF12 524 009 1004. The opinions or assertions contained herein are those of the authors and are not to be construed as official or reflecting the views of the Navy Department or the Naval service at large. The experiments reported herein were con29

30

mucx

et al.

storage of glycogen. The free-living cercaria is geared to the production of energy either from its stores of glycogen or from an exogenous substrate such as pyruvate. The schistosomule loses its adaptation to rapid production of energy and, presumably, is again primarily engaged in synthesis. INDEX

labeling; Glucose; Glycogen

DESCRIPTORS:Schistosoma

munsoni; Cercaria; Schistosomule; Radioisotope Carbon-14; Metabolism; Synthesis of macromolecules; CO, production; Pvruvate; ‘Glutamate; Citric acidcycle intermediates; Glycogen content; utilization.

The review of von Brand (1966) and the recent publications by Conte-de1 Pino et al. (1966, 1968) indicate that considerable information is available on the nature of enzymes of adult trematodes. Our knowledge of substrate utilization of the freeliving forms and other larval stages, however, is inadequate. The investigation on Schistosoma mansoni here described represents an effort to bridge this gap in our knowledge and to elucidate the following aspects of larval metabolism. (1) What is the fate of assimilated or stored constituents obtained by the developing cercariae from its snail host? (2) What are the potential endogenous or exogenous substrates of energy metabolism of the free-living cercaria? (3) To what extent does the pattern of substrate utilization change when the cercaria becomes a schistosomule (postpenetration larva) ? The results provide partial answers to these questions and permit cautious speculations regarding the changing metabolic pattern of schistosome larvae. MATERIALS AND METHODS

Reagents Sterile siliconized glassware (Fife et al., 1967) was used for all procedures involving cercariae and schistosomules. 14Carbonlabeled substrates were obtained from Calbiochem, Los Angeles, California, and New England Nuclear Corporation, Boston, Massachusetts. “Hepes” and “Tes” buffers were obtained from Calbiochem. The following buffers were used: “Hepes,” (N-2hydroxyethylpiperazine-N’-2-ethanesulfonic

acid), pH 7.6 (Good et al., 1966) ; “Tes,” N-Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid, pH 7.6 (Good et al., 1966); Ringer’s phosphate (Umbreit et al., 1964); Dulbecco’s buffer (Dulbecco and Vogt, 1954); Hanks’ balanced salt solution (HBSS) (Hanks and Wallace, 1949). The first three buffers were prepared in sterile dechlorinated tap water, the latter two in triple-distilled water. Substrates, including those labeled with ‘*C were stored at -70°C when not used immediately. The labeled compounds were diluted to the desired specific activity and molar@ with their respective unlabeled compounds. Experimental

Ankmab

The Schistosoma mansoni used was a laboratory strain maintained in Biomphaluria glabrata and in Swiss albino mice for 20 years. Both schistosomes and snails were of Puerto Rican origin. Stock snails were maintained on boiled lettuce in charcoalfiltered tap water under constant aeration at variable room temperature. They were exposed individually to four to six miracidia, after which they were maintained in the dark at 28°C and were used for the collection of cercariae 35 days later. Cercariae were collected in dechlorinated tap water by exposing 400-500 infected snails to light for 3 hours. The cercarial suspensions were passed through a Millipore wire grid (Millipore Filter Corporation, Bedford, Massachusetts) to remove snail feces and other debris. The number of cercariae in this and subsequent suspensions

METABOLIC ACTIVITY OF S. munsoni LARVAE was determined by repeated counts. Bacterial contamination was reduced by maintaining the cercariae for 80 minutes in 200 units of penicillin and streptomycin per milliliter. The suspension was then transferred to an 0.45-y Millipore suction filter unit, the cercariae were washed four times and suspended in 10 ml of “Hepes” buffer (Stirewalt and Uy, 1969). Cercariae thus obtained were defined as newly emerged, or, more accurately, free-living for a mean of 3 hours. Other lots of cercariae were maintained overnight at room temperature (used 18 hours after emergence) and again exposed to antibiotics, washed, and concentrated as described above. When plated on blood agar, these cercarial suspensions yielded only an occasional bacterial colony. For the labeling of snails and developing cercariae, infected or uninfected snails weighing 0.6 gm, their shells washed with 70% ethanol and rinsed in five changes of water, were placed in dechlorinated water containing glucose-U14C. The snails were kept in the dark under an exhaust hood at 26°C. After 48 hours, they were removed from the 14C-glucose solution, washed free of radioactive material, and maintained in dechlorinated tap water. They were washed again prior to stimulation of emergence of cercariae. Schistosomules were collected in HBSS, containing 400 units/ml each of penicillin and streptomycin, after penetration through a prepared rat skin membrane as describ,ed by Stirewalt et al. ( 1966) and Stirewalt and Uy ( 1969). Th e schistosomules were sedimented at 15OOg for 5 minutes, washed thrice with HBSS and twice with Dulbecco’s buffer. The final suspension produced only an occasional colony on blood agar plates. &physical

and Biochemical

Radioactivity clear Chicago

Procedures

was measured in a Nuliquid scintillation counter.

31

Bray’s solution (1960) was added to specimens that contained appreciable amounts of water. Fifteen milliliters of toluene, containing 6 gm of 2,Sdiphenyloxazole ( PPO) and 0.25 gm of p-2( 5-phenyloxazolyl) -benzene ( POPOP) per liter, plus 2.5 ml of methanol were added to specimens containing very little or no water. The results were expressed in terms of disintegrations per minute (dpm, counts per minute divided by efficiency of counting), nmoles CO2 produced or mnoles carbon incorporated. These calculations were based on the specific activity of the substrates and the number of carbon atoms labeled. The radioactivity of the solution used for the labeling of snails was tested at intervals using filtered aliquots, in order to eliminate radioactivity incorporated into bacteria or protozoa. Uninfected snails previously exposed to 14C-glucose were dissected at intervals, the hepatopancreas was excised, blotted on filter paper, weighed, and divided into two portions. Both were digested at 100°C for 20 minutes with 0.6 ml of 35% KOH and one was transferred directly to a counting vial for determination of radioactivity. Carrier glycogen (0.25 mg in 0.05 ml) was added to the other portion prior to digestion and glycogen was isolated by the following modification (Weiss, unpublished) of the method of Good, Kramer, and Somogyi (1933). After digestion, 1.8 ml of 95% ethanol were added and the mixture boiled for 2 minutes, cooled and stored overnight at 4°C. After centrifugation at 4000g for 30 minutes, the supernate was decanted. The precipitated glycogen was dissolved in 1.1 ml of 67% ethanol containing 3 X lop3 M Hepes and passed through a 0.3~ Millipore filter. The filters were washed five times with the Hepes-ethanol mixture, dried at 70°C for 20 minutes, and tested for radioactivity. Labeled cercariae were assayed for

32

BRUCE

et al.

radioactivity after centrifuging triplicate cipitated by TCA was determined as for specimens of 4000 cercariae at 7OOOg for labeled cercariae. Most experiments were 0.5 hours and washing three times with done with approximately 4000 cercariae or “Hepes.” They were then tested directly, 2,500 schistosomules. The total volumes of or indirectly after processing for the sepa- the reaction mixtures were 2 and 1 ml, reration of glycogen, or after precipitating spectively. A 5-minute period was allowed them with trichloroacetic acid (TCA). for thermal equilibration and cercariae The TCA precipitate was further fractionwere incubated at 26°C and schistosomules ated into hot TCA-insoluble and soluble at 32°C for 2 hours. In vitro tests were done fractions and a chloroform-methanol soluble in triplicate with controls consisting of fraction as described by Weiss et al., (1964). boiled organisms or reagents without orThe glycogen content of unlabeled cer- ganisms. The dpm of the controls were cariae was determined by the method of subtracted from the dpm of the test group. Neptune and Foreman (1959) using lO,OOO- Variation among triplicate tests was usually 25,000 cercariae and calculated from the less than 5%. amount of glucose recovered. RESULTS In vitro substrate utilization by cercariae and schistosomules was studied as de- Uptake of l”C Glucose by B. glabrata and Developing Cercariae of S. mansoni scribed by Weiss (1967) using 25-ml When infected or uninfected snails were Erlenmeyer flasks. CO2 was trapped by water containing Hyamine-saturated filter paper wicks in placed in dechlorinated r”C-glucose, the radioactivity disappeared plastic cups suspended above the reaction very rapidly, as shown in Fig. 1. Approximixture. Incorporation into fractions pre-

0-

16

1 24

30

1 36

48

HOURS

FIG. 1. Disappearance of radioactivity from snail medium after addition of glucose-U-l% (specific activity 110 pCi/pmole). In Exp. I, II, III, 100, 80, and 20 &i were added, respectively, to 1400, 700, and 350 ml sterile charcoal filtered tap-water containing 80, 40, and 20 snails.

METAROLI~ ACTIVITY OF S. munsoni

mately 40-60% had disappeared by 4 hours, 65-85% by 20-24 hours, and 85-95% by 48 hours. Differences among three groups were small. An attempt was made to reduce the bacterial population associated with the snails, but it has not been determined to what extent bacteria contributed to the removal of radioactive carbon from the water. Two days after exposure to 14C-glucose, the snails were washed and suspended in dechlorinated water free of radioactive carbon. The 14C content of the hepatopancreas (the organ in which most sporocysts produce cercariae) of two’ uninfected snails was determined immediately after washing and two were tested at each of several intervals thereafter. The results are shown in Fig. 2. In whole tissue the radioactivity was relatively high at 2 days after exposure to 14C-glucose, about 6860 dpm/mg wet weight, but there was a

6

33

LARVAE

marked decrease, with some fluctuation, to day 15. From days 15-30, the radioactivity remained approximately constant at 500 dpm. The glycogen fraction of the hepatopancreas contained approximately onesixth of the total tissue radioactivity at day 2 and about one-half at day 5. By day 8, it had declined to relatively low levels and, although there were some fluctuations, it remained approximately constant. Between days 15 and 30, the glycogen fraction accounted for about one-third of the tissue radioactivity. The total radioactivity of cercariae collected at intervals from two groups of snails exposed to 14C-glucose is illustrated in Fig. 3. In Exp. I, radioactivity increased rapidly from cercariae collected on day 2, 6 to a peak of 12 dpm per cercaria and then declined rapidly to day 12 and at a somewhat slower rate to day 30. In Exp. II, both increase and decrease were more mod-

i’,

I

5

IO DAYS AFTER

15 20 ADDITION OF “C-GLUCOSE

25

30

FIG. 2. Radioactivity in hepatopancreas of uninfected snails exposed to glucose-U-l%, in Fig. 1, Exp. III. At day 2, the snails were washed and maintained in water containing glucose. Each circle represents the mean of two determinations.

as shown no added

34

BHUCEI

I 5

c?t (Il.

I 10

I 15

DAYS AFTER

ADDITION

I 20

I 25

/ 30

OF “C-GLUCOSE

FIG. 3. Radioactivity of cercariae emerging - - from snails exposed to glucose-U-l%. resents the mean of tri&ate samples.

erate and a peak of 7 dpm per cercaria was reached on day 13. Despite the difference in the initial intervals of the two experiments, the radioactivity of the cercariae was similar from days 10 to 22, the last day of Exp. II. Newly emerged cercariae collected in Exp. I were fractionated and the radioactivity of four fractions is illustrated in Fig. 4. The fraction designated as “nucleic acid” (soluble in hot trichloroacetic acid) contained the highest amount of radioactivity, about 3.p4.0 dpm per cercaria at days 2-6 and 2.0 dpm during the period of 8-19 days. One appreciably lower value, obtained at day 10, was probably the result of a technical error. After day 19, th’e radioactivity declined rapidly. The radioactivity of the fraction designated as “protein” (insoluble in trichloroacetic acid) was as high as that of the nucleic acid during the initial period of days 2-6, but declined to about 0.4 dpm by day 12, remained at that level for 10 days, and then further declined. The “lipid” fraction (soluble in chloroform-methanol) was also labeled,

Each circle

rep-

0.6-1.0 dpm per cercaria at days 2-6, and about 0.3 dpm thereafter. The pattern of the glycogen fraction was similar to that of the protein, but uniformly lower, with a peak of 2.7 dpm per cercaria at day 4, 0.30.5 dpm at days 8 and 10, and usually 0.1 dpm thereafter. Fractionations were also carried out with cercariae maintained free-living for 18 hours after emergence from the labeled snails. With the exception of glycogen, the results were identical to those depicted in Fig. 4. In the case of glycogen, the initial high radioactivity disappeared, and, except for minor fluctuations, was uniformly low, as in the later intervals of the newly emerged cercariae. This finding, that the initial high radioactivity in the glycogen fraction is eliminated during 18 hours of free-living existence, was fully confirmed in Exp. II, as shown in Fig. 5. LMetabolism of Erogenous mansoni Cercariae

Substrates by S.

The experiments described below were carried out with cercariae suspended in

METABOLIC

i

ACTIVITY

0~

S. munsoni

35

LARVAE

\ \ \ \ \ \

” \ L

-.

!

DAYS AFTER

FIG. 4. Radioactivity from Exp. I.

of various

cercarial

ADDITION

fractions.

dechlorinated water containing 0.01 M “Hepes” buffer, adjusted to pH 7.6 with NaOH. Under these conditions, no loss of cercarial motility was detected during a period of observation of 24 hours. When buffer was omitted, utilization of certain substrates was greatly reduced and a large percentage of the cercariae lost their motility during the course of the experiment. “Tes” or Ringer’s phosphate buffers, or “Hepes” adjusted to pH 6 or 8 with NaOH, or to pH 7.6 with KOH were unsatisfactory. As shown in Table I, exogenously supplied glucose failed to stimulate metabolism of newly emerged cercariae to any appreciable extent. Less CO2 was produced than with most other substrates tested (Table II) and glycogen formation was almost negligible. This apparent lack of activity was possibly due to the availability of endogenous glucose from the large

OF ‘%GLUCOSE

These

determinations

1

were

yRl*;

done with

samples

;\

5

IO

DAYS AFTER ADDITION OF “C-GLlJCOSE

FIG. 5. Radioactivity of cercarial glycogen fractions obtained at 3 and 18 hours after emergence from snails. These determinations were done with samples from Exp. II.

stores of glycogen of the cercariee. When the cercariae were maintained in a freeliving existence for 18 hours, their glycogen

36

BnucE et al. TABLE

Relationship

between

Glycogen

Content

I

of S. mansoni

Cercariae Metabolism

CO, produced ( nmoles ) Hours after emergence from snails” 3 18

Glycogen content (k%) 42 t

of incubation

into glycogen (dpm)

19c2

(12)

20 2 2 (6)

(11)

69t2

(12)

4523(B)

fraction0

at 26°C

2

3 (12)

12-c2

Glucosen

of added glucose

Incorporation Hours

2

of Added

and Metabolism

4

8

60&2

(6)

200&3(6)

17022

(6)

600-c

3 (6)

5 The glycogen content was determined from glucase formed after hydrolysis using 10,000 to 25,000 cercariae per determination. The metabolic experiments were performed in triplicate with approximately 4000 cercariae per flask suspended in 2.0 ml of “Hepes” buffer ( pH 7.6), containing final concentrations of 1 X 10-s M MgCl,, 2 X 10-4 M MnCl,, and 1 X 10-s M glucose-U-l%, 0.2 uCi/umole. The total numbers of determinations are indicated in parentheses. The data are expressed as the Mean k Standard Error glyco’gen content or metabolic activity of 10,000 cercariae. 6 At start of experiment. 0 It was assumed that the large carbohydrate fraction for radioactive glycogen identified by the method of Neptune and Foreman ( 1959).

assay was identical

to that

TABLE II was greatly reduced. CO2 and Catabolism of Selected Substrates by S. mansoni glycogen productions from added glucose, Cercariaea although still small, were correspondingly CO, produced from increased. labeled carbons ( nmoles ) In contrast to glucose, cercariae cataboTime (hr ) after lized pyruvate very rapidly and produced emergence from snails CO, from all three carbons (Table II ) . 3 18 Substrate -3 3 a-Ketoglutarate and acetate were also utilized, but to a smaller extent. Glutamate, 1784 1450 1265 l-175 Pyruvate-l-r% 797 1230 glutamine, and some of the intermediates 865 840 Pyruvate-2-r% 643 911 810 800 Pyruvate-3-r% of the citric acid cycle, other than a-keto273 264 u-Ketoglutarate-l-r% glutarate, were catabolized to a much 142 194 152 130 smaller degree. Cercariae maintained in a Acetate-U-r% 41 40 38 42 Succinate-1,4-r% free-living existence for 18 hours in most 30 32 Succinate-2,3-r‘% cases utilized these substrates more rapidly, 34 42 Glutamate-U-l% but differences were not as great as those 18 22 Fumarate-1,4-r% obtained with glucose. 14 12 48 16 Glutamine-U-r% Since pyruvate was catabolized most Malate-4-r% 10 12 4 3 12 rapidly of all substrates tested, an attempt 3 Citrate-1,5-r% was made to determine whether some of its a The experiments were performed as described carbon was incorporated into macromolein the legend of Table I. The specific activity of cules. The results are shown in Table III. the substrates ranged from 0.05 to 0.3 uCi/umole. Each number represents the mean of triplicate Only about 2.5% of the total carbon metabflasks and refers to the activity of 10,000 cercariae olized was incorporated (compare Tables incubated at 26°C for 2 hours. Cercariae tested at II and III). The nucleic acid fraction con- 18 hours after emergence from the snails (fourth tained the largest share of the radioactivity, column) were tested at 3 hours (third cohrmn) in the same experiment. followed by glycogen, protein, and lipid. content

mm4~oL1c

ACTIVITY

0~

S. mansoni

TABLE

Distribution

of

Incorporated

III

Incubation

l&C following

37

LARVAE

of

Percentage

S. mansoni radioactivity

Cercariae with Pyruvate-140 in various

fractions

Trichloroacetic Position of labeled carbon

Labeled carbon incorporated (nmoles)b

Ethanol insoluble (glyw34

acid Chloroformmethanol soluble (lipid)

Hot soluble (nucleic acids)

Hot insoluble (protein)

1 2 3

5 17 49

1 34 30

26 19 17

20 41 45

53 6 8

l-3

71

29

18

42

11

a At 26°C for 2 hours. b Per 10,000 cercariae, mean of triplicate

determinations.

About two-thirds of the carbon incorporated was derived from carbon 3 and the least amount from carbon 1. A large share of carbon 1 was recovered as lipid, which suggests the possibility that some of this carbon was incorporated after it was released as COO. Metabolism of Exogenous Substrates by S. mansoni Schistosomuh Schistosomules were studied under conditions considerably different than those used for cercariae: The suspending medium was Dulbecco’s buffer and the temperature of incubation was 32°C (instead of 26’C). Furthermore, results in both cases are presented on the basis of 10,666 organisms, and the schistosomules contain fewer cells than the cercariae. Despite these differences, a few cautious comparisons can be made from the results shown in Table IV. Of the substrates tested, pyruvate possibly still ranked first as a substrate for CO2 production. This is true if the assumption is made that as much COZ was produced from carbon 3 (not shown in Table IV) as from carbon 2. However, the degradation of pyruvate by schistosomules was only a fraction of that obtained with cercariae. a-Ketoglutarate was also catabohzed at a much slower rate. The rates of utilization

of acetate and of some of the intermediates of the citric acid cycle were comparable to those obtained with cercariae. Glutamate, glutamine, and glucose were utilized at somewhat higher rates. The more rapid utilization of glucose is possibly related to the lower glycogen content of schistosoTABLE

Catabolism

IV

Selected Substrates by S. mansoni Schistosomulesa

Substrate Pyruvate-1-14C Pyruvate-2-r% Acetate-U-i% Glutamate-V-i4C Glutamine-U-r% Glucose-U-r4C Fumarate-1,4-14C Succinate-1,4-i4C Succinate-2,3-i4C Malate-4-r% a-Ketoglutarate-IJ-r4C Citrate-1,5-14C

of

CO, produced from labeled carbons (nmoles/lO,OOD schistosomules) 50 64 117 68 57 42 28 20 17 18 10 4

a The experiments were performed with approximately 2500 schistosomules suspended in 1.0 ml of Dulbecco’s buffer (pH 7.3) containing 1 X 10-s M MgCl,, 2 X 10-4 M MnCl,, and 1 X 10-s M substrate, ranging in specific activity from 0.05 to 0.3 uCi/umole. The organisms were incubated for 2 hours at 32°C. The numbers shown are the means of two experiments, each carried out in triplicate.

~mcx

38

mules. In four determinations not shown in Table IV, the glycogen content was found to be 2.5 * 2 (SE) ug per 10,000 schistosomules. DISCUSSION

The experiments in which snails and cercariae were labeled with W-glucose yielded results similar to those reported by Lewert and Para (1966), even though several details of procedure were different. Lewert and Para provided a more complete diet to the snails-calcium alginate inst’ead of boiled lettuce-and assayed infected instead of uninfected snails. In both series of experiments, added glucose disappeared rapidly from the water in which the snails were maintained, the level of radioactivity of the hepatopancreas of the snails was highest during thte first few days and there was some suggestion of fluctuation in radioactivity during subsequent days. In Exp. I, as in the experiments of Lewert and Para ( 1966), there was a rapid initial uptake and elimination of radioactive compounds. In Exp. 2 this pattern was essentially repeated though the initial uptake was not as rapid. Evans and Stirewalt (1951) showed that the physiological condition of the snail directly affected the infectivity of the cercariae, and thus undoubtedly, their metabolism. It is conceivable, therefore, that minor fluctuations in temperature or in other factors affected the endogenous supply of low and high molecular compounds of the snails at the precise time of exposure to 14C-glucose. It is obvious from Fig. 2 that an appreciable portion of the added glucose was converted to glycogen by the snail and considerable amounts of radioactive glycogen were found in the newly emerged cercariae. It is not known whether the bulk of the radioactivity was acquired by the cercariae as glycogen or glucose. The results shown in Tables I and II indicate that

et al.

cercariae have the ability to synthesize glycogen from glucose or pyruvate. For other trematode larvae there is evidence for either event, transport of glycogen across the sporocyst wall (Cheng and Snyder, 1963) and glycogen synthesis ( Cheng, 1963). During the Is-hour period of free-living existence, most of the newly synthesized glycogen disappeared (Fig. 5) and the same was found to be true when total glycogen was measured (Table I). These results and the retention of other newly synthesized compounds, clearly suggest that endogenous glucose is the chief source of energy for the free-living cercaria. A small portion of the glycogen, however, was not rapidly metabolized. This might have been the content of the penetration glands, as suggested by Lewert and Para (1966). There is good evidence from the results presented in Fig. 4 that glucose, or its products of snail metabolism, was also rapidly metabolized by the cercariae for the synthesis of protein, nucleic acids, and lipid. Surprising is the contrast between the rapid decline of the radioactivity of the protein fraction, between the sixth and the twelfth day after the addition of llCglucose, and the relatively high level of the radioactivity of the nucleic acid between days 8 and 19 (Fig. 4). The results obtained with the nucleic acid fractions also contrast with the general decline in radioactivity in the snail and in the cercaria. They indicate that the snail makes available to the cercariae for an extended period labeled compounds that are preferentially utilized for nucleic acid synthesis. Possibly, breakdown products of snail nucleic acid metabolism become valuable precursors of cercarial nucleic acid metabolism, at the time when the supply of other labeled compounds wanes. There is no indication that constituents other than endogenous glucose or glycogen,

METABOLIC

ACTIVITY

not even lipid, are utilized by the freeliving cercaria as a ready source of energy during its free-living existence. The radioactivity of the protein, nucleic acid, and lipid fractions of the cercariae maintained free-living for 18 hours was identical to that shown in Fig. 4. Cercariae utilize exogenous glucose to a very limited extent. This is not surprising because endogenous glucose is available to them from their large stores of glycogen. A small but significant increase in exogenous glucose utilization was demonstrated after the glycogen supply had been depleted (Table I). The metabolism of cercariae, however, can be stimulated by exogenous compounds. From the data presented in Table II and from unpublished measurements by one of us (M.A.S.) that the dry weight of 10,ooO cercariae is approximately 25 to .28 mg, it can be calculated that cercariae produced a total of 16-12 umoles of CO, from pyruvate per milligram dry weight in 2 hours at 26°C. This amount increased to 15-18 umoles when the cercariae were maintained free-living overnight. Although synthesis of the major types of macromolecules is not likely to be a major function of the free-living cercaria, it is clear that some synthesis can take place with a rapidly metabolized ‘exogenous substrate, such as pyruvate (Table III ) . Permeability factors very likely played a role in the rate of utilization of the substrates listed in Table II. a-Ketoglutarate and acetate were catabolized to a moderate extent, while other compounds were utilized to a much smaller degree. The results are consistent with the assumption that free-living cercariae have a functional citric acid cycle. The short step leading to the transformation of the free-living cercaria into a schistosomule was accompanied by obvious changes in exogenous metabolism. The most dramatic of these changes, shown in

OF

S.

?WXl.SOni

LARVAE

39

Table IV, was the reduction in the level of COZ production from pyruvate by more than one order of magnitude. Some of these changes may be due to the loss of the cells of the tail or stores of glycogen. The most pronounced alterations of metabolic pattern must be attributed, however, to changes in permeability, to enzyme regulation, or to both. Stirewalt (1963) showed that the metamorphosis of the cercaria into a schistosomule was accompanied by a marked change in permeability to different media. The schistosomule, in marked contrast to the water-adapted cercaria, became immobile and vesiculated immediately upon contact with water. Furthermore, schistosomules remained active in saline and serum, which were detrimental to cercariae. Although the three larval stages (the developing cercaria, the free-living cercaria, and the schistosomule) were not studied by identical methods, a few cautious remarks can be made regarding their comparative metabolism. The chief function of the developing cercaria is synthesis of the principal macromolecules and accumulation of glycogen. If the turn-over of glucose is typical of exogenous substrates available to the snail, it can be concluded that the developing cercaria rapidly metabolized compounds taken up from the environment by the snail. One possible exception is nucleic acid metabolism, which appears to involve compounds acquired by the snail over a more prolonged period of time. The chief function of the free-living cercaria is energy metabolism which is sustained by its endogenous glycogen, but can also be stimulated by exogenous pyruvate. The schistosomule reverts to a type of metabolism which is less likely to provide immediate energy and, it can be surmised, is better adapted to synthesis. Thlese studies have provided a general outline of this changing metabolic pattern, and have paved the way for more detailed investigations.

BRUCE et (11.

40 ACKNOWLEDGMENTS

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