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14C Uptake by Schistosoma mansoni from Biomphalaria glabrata exposed to 14C-glucose
JOURNAL
OF INVERTEBRATE
14C Uptake
PATHOLOGY
23,
297-302
by Schistosoma
mansoni
Exposed JOHN D. CHRISTIE,~ Bureau
of Biological
(1974)
Biomphalaria
glabrata
to 14C-GIucose1 B. FOSTER,
WILLIAM
Research, Rutgers-The New Brunswick, New Received
from
September
State Jersey
AND
LESLIE
Uniejersity OS9OS
A. STAUBER of New
Jersey,
17, 1973
Exposure of Biomphalaria glabrata infected with Schistosoma mansoni to “C-glucose results in a greater uptake of original total snail label by the parasitized digestive gland-gonad, site of the developing daughter sporocysts and cereariae, than by the digestive gland-gonad of control animals. As a consequence of this greater uptake by the infected digestive gland-gonad, the albumen gland and remainder of the carcass of parasitized snails receive less label than do those areas in normal snails. Emergence of cercariae from the snail and daughter sporocyst mass account for a diversion of 12.6% of original total label from the infected snail itself. This diversion of label from the snail to the parasite may explain carbohydrate depletion in parasitized snails.
Lewert and Para (1966) showed autoradiographically that daughter sporocysts and cercariae of Schi.stosoma mansoni, derived from Biomphalaria glabrata exposed to I%-glucose, took up 14C. The radioactive carbon was incorporated into cercarial nucleic acids, protein, glycogen, and, to some extent, in the lipids (Bruce et al., 1969). We now report that the “C is shunted from B. glabrata to the parasites, and also that more label is incorporated in the digestive gland-gonad (DGG) of infected than of uninfected snails. This diversion of labelled glucose from host to parasite may account for the depletion of glycogen in infected animals (Christie et al., 1974). MATERIALS
AND
METHODS
The maintenance, stock, infection, and dissection of B. glabrata has already been ‘This research was supported by NIAID training grant No. A100192. * Present address: Department of Tropical Public Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115.
described (Christie et al., 1974). Relative weight of each organ is the percentage of the organ to the carcass (soft parts minus digestive tract) of the snail. Groups of 23-day-infected and control snails were separately placed in distilled water containing 25 &i of ‘“C-(U) -Dglucose (New England Nuclear, Inc.). After 48 hr, the animals were removed and thereafter kept in dechlorinated tap water. We crushed and removed the shells, weighed the soft parts, and then solubilized the DGG, albumen gland, and remainder of the carcass in Protosol (New England Nuclear, Inc.) at 50°C for 12-18 hr. To the solubilized tissues, we added a liquid scintillation “cocktail,” Aquasol (New England Nuclear, Inc.), and we then measured radioactivity incorporated in the tissues with a Packard Tri-Carb spectrometer. Tissue radioactivity was counted for 20,000 events or 1000 set, whichever came first. We determined counting efficiency by the channels ratio method (Bush, 1963). Labeled cercariae were obtained from infected snails exposed to the *4C-glucose. Aliquots of the cercarial suspension were
297 Copyright All rights
0 1974 by Academic Press, Inc. of reproduction in any form reserved
298
CHRISTIE,
FOSTER
AND
STAUBER
On a relative weight basis, distribution of the label is lower in the DGG of infected animals than in the DGG from normal snails (Fig. 2). However, only at day 39 postinfection, after the start of cercarial shedding, are the values in the DGG of infected snails significantly lower than those in the DGG of control animals. Label in Albumen Gland
23
30
39
DAYS POSTINFECTION
FIG. 1. Distribution of “C label in DGG of normal and infected Biomphalnria glabruta. The vertical bars represent 90% confidence limits about the sample mean. Each point is the mean of 8 or more determinations.
prepared in triplicate, and each suspension was passed through a wire grid to remove snail feces and other debris. After centrifuging the suspension at 1200 g for 15 min at 5”C, the cercariae were resuspended in distilled water and counted. The cercariae were solubilized, and their label was counted in the same manner as that of snail tissue. RESULTS
Initially, the albumen gland from normal snails incorporated 2.5 times more label than did the albumen gland of infected snails (Fig. 3). By day 30 postinfection, this difference had widened so that the albumen gland of control animals contained 12% of the total carcass label while the albumen gland of infected animals contained 4% of total label. However, after onset of egglaying by normal animals, the total label in the normal albumen gland dropped to the levels in the albumen gland of parasitized snails. At the same time, the amount of label in the gland from infected snails slowly approached normal levels. On a relative weight basis, the gland of the control snail has a larger percentage of total label than does that of the parasitized animal (Fig. 4). By day 30, the percentage of label is almost twice as great in glands
Label in DGG We found no difference in label removed from water by infected or control snails. However, because of variability in label taken up by individual snails, we converted the counts in the DGG, albumen gland, and remainder of the carcass to a percentage of total label incorporated by the whole animal. Throughout the experiment, the DGG of infected animals contained a significantly greater percentage of total body radioactivity than did the DGG from normal snails (Fig. 1). Furthermore, although the amount of label decreased with time in the DGG of both control and parasitized animals, differences between the two groups remained constant at 12 percentage points.
23 DAYS
30
39
POSTINFECTION
FIG. 2. Distribution of 14C label weight basis in the DGG of normal Biomphalaria glnbarta. The vertical 90% confidence limits about the Each point is the mean of determinations.
on a relative and infected bars represent sample mean. S or more
LABELING
OF
fi.
of normal snails as in the albumen gland of infected snails. With the onset of egglaying, the amount of label in albumen glands of control animals drops to values found in glands of infected animals. Label in the Remainder
299
mCLnSOni
15 0
12 -
of Carcass
At first, the remainder of the carcass of normal animals contained 12 percentage points more label than did the remainder of the carcass of infected snails (Fig. 5). However, by day 30, the percentage of total label found in the remainder of the carcass of control snails had dropped, bringing the two groups together. But, by the end of the experiment, the remainder of the carcass of normal animals once more has a significantly larger percentage of total label than that of parasitized animals. At no time during the experiment is there any difference, on a relative weight basis, between amounts of label found in the remainder of the carcass from control or infected animals (Fig. 6).
Control Infected
23
39
30t Egg-Laymg
DAYS POSTINFECTION FIG.
gland brata. limits mean
3. Distribution of “C label in albumen of normal and infected BiomphaEaria glaThe vertical bars represent 90% confidence about the sample mean. Each point is the of 8 or more determinations.
Label in S. mansoni Cercariae Twenty B. glabrata infected for 26 days were exposed for 48 hr to 25 microcuries of ‘C-labeled glucose. The snails did not start to shed until 3 days after they were removed from the water containing the labeled glucose. Peak incorporation of the label into the cercariae occurred at 5 days after initial exposure. From the initial activity of the snails and the cercarial activity per snail (Table l), it can be seen that cercarial production accounts for 7.5% of the total label incorporated by the snail. In terms of overall loss of label, cercarial shedding is responsible for 10.3% of the loss. Label in Eggs of Normal
B. glabrata
In order to calculate the amount of label set aside for the demands of egg production, eggs produced by normal snails, which had taken up the labeled glucose, were counted for activity. Eggs were not laid until 11 days after exposure of the
Control Infected
DAYS
Egg-laying POSTINFECTION
FIG. 4. Distribution of “C label on a relative weight basis in the albumen gland of normal and infected Biomphalaria glabrata. The vertical bars represent 90% confidence limits about the sample mean. Each point is the mean of 8 or more determinations.
snails to the isotope, and peak activity was not reached until day 13 (Table 2). From the initial activity of the snails and the egg activity per snail, it can be calculated that egg production takes a minimum of 4.2% of the labeled glucose. In terms of overall loss of label, egg production accounts for a minimum of 14.3% of label lost by the snail.
300
CHRISTIE,
FOSTER
COWOl Infected
23
30
39
DAYS POSTINFECTION
FIG. 5. Distribution of 72 label in remainder of carcass of normal and infected Biomphalaria glabrata. The vertical bars represent 90% confidence limits about the sample mean. Each point is the mean of 8 or more determinations.
Of
’ 23 DAYS
I 30
I 39
POSTINFECTION
FIG. 6. Distribution of “C label on a relative weight basis in the remainder of the carcass of normal and infected Biomphalaria glabrata. The vertical bars represent 90% confidence limits about the sample mean. Each point is the mean of 8 or more determinations.
DISCUSSION
This study supports the idea that nutrient utilization by the parasite may be responsible for the depletion of glycogen noted in infected B. glabrata (Christie et al., 1974). Cercarial shedding alone represents a loss of 7.5% of 14C-glucose administered to infected snails. The I%-labeled glucose is utilized by the parasite for nucleic acid, protein, glycogen, and lipid pro-
AND
STAUBER
duction (Bruce et al., 1969). If we accept Becker’s (1968) estimate that an infection with 5 miracidia produces 15-18 mg of daughter sporocyst tissue in the DGG, then, in our experimental situation, there is approximat’ely 15 mg of sporocyst tissue in the DGG. We can then multiply the amount of sporocyst tissue by the amount of label found in each milligram of DGG after cercarial shedding (6873 dpm). This calculation indicates that 5.1% (6873 X 15/404297) of total initial label may be found in the daughter sporocysts. Thus, the developing parasites, cercariae and sporocysts, utilize a minimum of 12.6% of the initial label picked up by the snail. This estimate does not include loss of label due to sporocyst metabolism or to the high cost of cercarial synthesis. There is also some autoradiographic evidence (Lewert and Para, 1966) that there is unequal partitioning of the label between the host and the parasite, with the parasite getting the better of the bargain. Thus, the estimate of 5.1 % of initial label found in the daughter sporocysts may be low. Accompanying the diversion of label from host to parasite are major changes in the intramolluscan localization of the ‘%-label. The increase in amount of label found in the DGG of infected snails seems to be due to the change in relative weight contributed by the mass of the daughter sporocysts. When calculated on a relative weight basis, there is no difference in amount of label going to the DGG of control or infected animals except for day 39 postinfection. At this time, the decline in label in the DGG of infected snails may be due to loss of label associated with cercarial shedding. With the diversion of label to the DGG in infected animals, there is less total label available for the remainder of the carcass and the albumen gland. Although there is a significant diversion of label from the albumen gland to the DGG in infected snails, there is no significant decrease in galactogen levels in the albumen gland during infection (Christie
LABELING
INCORPORATION OF 1% LABEL DERIVED FROM Biomphalaria
Time (days)
OF
301
TABLE 1 IN CERCARIAE OF Schistosoma mansoni glabrata EXPOSED TO 1% GLUCOSE
Activity of cercariae (dpm) a
No. of cercariae per snail
3.69 1.67 2.50 1.93
4100 3600 900 3600
28 31 33 35 37
S. mansoni
Cercarial activity per snail (dpd
Activity per snail (dpm) 404297
15129 6012 2250 6948 Total
30339 110111
44 Activity a dpm
= disintegrations
per
INCORPORATION
OF 1% glabrata
(days)
TABLE 2 LABEL IN EGGS OF NORMAL EXPOSED TO 1% GLUCOSE
294186
Biomphalaria
Activity
Activity of eggs (dpd
Time
lost
minute.
No. of eggs
Activity per snail
No. of snails
per egg (dpm)
(dpm)
_..-
142560
7 11 12 13 16
9026 20083 30311 5877 Total
67 55 72 65
134.7 365.1 419.6 90.4
11 11 11
11
65297
Activity Egg
activity
et al., 1974). In the normal albumen gland, the percentage of total label present is much greater than that in the albumen gland of infected snails until the control snails start laying eggs. At that time, the amount of total label in normal albumen gland drops precipitously to the levels found in the albumen gland of infected snails. Thus, the drain of egg-laying in normal animals may prevent galactogen concentrations from being significantly greater than in parasitized animals. Egg-laying accounts for a minimum of 4.2% of the total label found in the snail. Most of that label would probably come from the albumen gland since the secretions of the albumen gland seem to make up most of the egg clutch. If egg-laying utilizes a maximum of 20% of the galactogen in the albumen gland (McMahon et al., 1957), then the
per
snail
100961 lost
41599
5936
constant drain of egg-laying would keep the galactogen levels low enough and variable enough so that is would take a tremendous stress, such as complete starvation, before galactogen levels in the experimental animal would fall significantly below normal. Another aspect to be considered is the fact that egg-laying accounts for a minimum of 4.2% of the total label while parasitism is responsible for diversion of 12.5% of the label. It appears that parasitism is a more severe drain on the snail than egglaying, and that cessation of egg-laying in parasitic castration might have a nutritional basis. The diversion of nutrients from the albumen gland in parasitism might reflect the situation in the gonad itself. On the other hand, there seems to be a change in percentage of label taken up on a relative weight basis in the albumen gland of para-
302
CHRISTIE,
FOSTER
sitized snails compared to that of normal animals. This change may simply reflect the severity of the nutritional drain on the albumen gland during parasitism, or there may be a change in the hormone levels affecting galactogen synthesis. In ACdimax columbianus, an extract of the optic tentacles decreases galactogen synthesis and incorporation of I%-labeled glucose into galactogen while ablation of the optic tentacles causes the opposite effect (Meenakshi and Scheer, 1969). It is postulated that this extract affects the gonad, which in turn controls the albumen gland. A similar situation is found in Lymnaea stagnalis where the dorsal bodies of the dorsal complex of the brain secrete a factor which is necessary for vitellogenesis and growth and function of the accessory sex glands (Joosse and Geraerts, 1969). Perhaps the daughter sporocysts of S. mansoni affect the brain region of B. glabrata, causing inhibition or blocking of the dorsal bodies and cessation of gonad and accessory sex organ function. It is also possible that the gonad may fail to control the metabolism of the accessory sex organs due to the presence of daughter sporocysts. The whole question of hormonal changes in parasitized molluscs needs much more work. In conclusion, we can say that production of larval stages of 8. mansoni represents a nutritional drain on B. glabrata. This diversion of nutrients may cause major alterations in the host’s metabolism and reproductive capacity. ACKNOWLEDGMENTS The authors would like to thank Dr. Dan Ostland of Merck Institute of Therapeutic Research, Dr. Edward H. Michelson of the Harvard School of Public Health, and Dr. Henry van der Schalie of the University of Michigan for their help in supplying normal and infected B. glabrata. The
AND STATJBER snails from Dr. van der Schalie were obtained through the auspices of the U.S.-Japan ‘Cooperative Medical Science Program-KIAID. The senior author would also like personally to thank Dr. van der Schalie for his help, generosity, and encouragement to a beginner in the field. Funds for this investigation were provided in part by a grant from Rutger University Research Council, USPHS Parasitology Training Grant AI-187, BSSG funds from USPH RR 7058, and The Charles and Johanna Busch Memorial Fund Award, to the Bureau of Biological Research. REFERENCES W. 1968. Untersuchungen iiber die aus der Muttersporocysto auswandemden Tochtersporocysten von Schistosoma mansoni. I. Beitrage zum Kohlenhydratstoffwechsel dieser Stadien. Z. Parasitenk., 30, 233-251. 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. BECKER,
Exp.
Parasitol.,
26, 29-40.
BUSH, E. T. 1963. General applicability of the channels ratio method of measuring liquid scintillation counting efficiencies. Anal. Chem., 35, 1024-1029. CHRISTIE, J. D., FOSTER, W. B., AND STAUBER, L. A. 1974. The effect of parasitism and starvation on carbohydrate reserves of Boimphalaria glabrata. J. Invertebr. JOOSSE, J., AND GERAERTS,
Pathol.,
23, 55-62.
W. J. 1969. On the influence of the dorsal bodies and the adjacent neurosecretory cells on the reproduction and metabolism of Lymnaea stagnalis. Gen. Comp. Endocrinol., 13, 511. LEWERT, R. M., AND PARA, J. B. 1966. The physiological incorporation of carbon-14 in Schistosoma mansoni cercariae. J. Infect. Dis., 116, 171-182. MCMAHON, P., WON BRAND, T., AND NOLAN, M. 0. 1957. Observations on the poIysaccarides of aquatic snails. J. Cell. Comp. Physiol., 50, 219-239. MEENAKSHI, V. R., AND SCHEER, B. T. 1969. Regulation of galactogen synthesis in the slug Ariolimaz columbianus. Comp. Biochem. Physiol., 29, 841-845.
OF INVERTEBRATE
14C Uptake
PATHOLOGY
23,
297-302
by Schistosoma
mansoni
Exposed JOHN D. CHRISTIE,~ Bureau
of Biological
(1974)
Biomphalaria
glabrata
to 14C-GIucose1 B. FOSTER,
WILLIAM
Research, Rutgers-The New Brunswick, New Received
from
September
State Jersey
AND
LESLIE
Uniejersity OS9OS
A. STAUBER of New
Jersey,
17, 1973
Exposure of Biomphalaria glabrata infected with Schistosoma mansoni to “C-glucose results in a greater uptake of original total snail label by the parasitized digestive gland-gonad, site of the developing daughter sporocysts and cereariae, than by the digestive gland-gonad of control animals. As a consequence of this greater uptake by the infected digestive gland-gonad, the albumen gland and remainder of the carcass of parasitized snails receive less label than do those areas in normal snails. Emergence of cercariae from the snail and daughter sporocyst mass account for a diversion of 12.6% of original total label from the infected snail itself. This diversion of label from the snail to the parasite may explain carbohydrate depletion in parasitized snails.
Lewert and Para (1966) showed autoradiographically that daughter sporocysts and cercariae of Schi.stosoma mansoni, derived from Biomphalaria glabrata exposed to I%-glucose, took up 14C. The radioactive carbon was incorporated into cercarial nucleic acids, protein, glycogen, and, to some extent, in the lipids (Bruce et al., 1969). We now report that the “C is shunted from B. glabrata to the parasites, and also that more label is incorporated in the digestive gland-gonad (DGG) of infected than of uninfected snails. This diversion of labelled glucose from host to parasite may account for the depletion of glycogen in infected animals (Christie et al., 1974). MATERIALS
AND
METHODS
The maintenance, stock, infection, and dissection of B. glabrata has already been ‘This research was supported by NIAID training grant No. A100192. * Present address: Department of Tropical Public Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115.
described (Christie et al., 1974). Relative weight of each organ is the percentage of the organ to the carcass (soft parts minus digestive tract) of the snail. Groups of 23-day-infected and control snails were separately placed in distilled water containing 25 &i of ‘“C-(U) -Dglucose (New England Nuclear, Inc.). After 48 hr, the animals were removed and thereafter kept in dechlorinated tap water. We crushed and removed the shells, weighed the soft parts, and then solubilized the DGG, albumen gland, and remainder of the carcass in Protosol (New England Nuclear, Inc.) at 50°C for 12-18 hr. To the solubilized tissues, we added a liquid scintillation “cocktail,” Aquasol (New England Nuclear, Inc.), and we then measured radioactivity incorporated in the tissues with a Packard Tri-Carb spectrometer. Tissue radioactivity was counted for 20,000 events or 1000 set, whichever came first. We determined counting efficiency by the channels ratio method (Bush, 1963). Labeled cercariae were obtained from infected snails exposed to the *4C-glucose. Aliquots of the cercarial suspension were
297 Copyright All rights
0 1974 by Academic Press, Inc. of reproduction in any form reserved
298
CHRISTIE,
FOSTER
AND
STAUBER
On a relative weight basis, distribution of the label is lower in the DGG of infected animals than in the DGG from normal snails (Fig. 2). However, only at day 39 postinfection, after the start of cercarial shedding, are the values in the DGG of infected snails significantly lower than those in the DGG of control animals. Label in Albumen Gland
23
30
39
DAYS POSTINFECTION
FIG. 1. Distribution of “C label in DGG of normal and infected Biomphalnria glabruta. The vertical bars represent 90% confidence limits about the sample mean. Each point is the mean of 8 or more determinations.
prepared in triplicate, and each suspension was passed through a wire grid to remove snail feces and other debris. After centrifuging the suspension at 1200 g for 15 min at 5”C, the cercariae were resuspended in distilled water and counted. The cercariae were solubilized, and their label was counted in the same manner as that of snail tissue. RESULTS
Initially, the albumen gland from normal snails incorporated 2.5 times more label than did the albumen gland of infected snails (Fig. 3). By day 30 postinfection, this difference had widened so that the albumen gland of control animals contained 12% of the total carcass label while the albumen gland of infected animals contained 4% of total label. However, after onset of egglaying by normal animals, the total label in the normal albumen gland dropped to the levels in the albumen gland of parasitized snails. At the same time, the amount of label in the gland from infected snails slowly approached normal levels. On a relative weight basis, the gland of the control snail has a larger percentage of total label than does that of the parasitized animal (Fig. 4). By day 30, the percentage of label is almost twice as great in glands
Label in DGG We found no difference in label removed from water by infected or control snails. However, because of variability in label taken up by individual snails, we converted the counts in the DGG, albumen gland, and remainder of the carcass to a percentage of total label incorporated by the whole animal. Throughout the experiment, the DGG of infected animals contained a significantly greater percentage of total body radioactivity than did the DGG from normal snails (Fig. 1). Furthermore, although the amount of label decreased with time in the DGG of both control and parasitized animals, differences between the two groups remained constant at 12 percentage points.
23 DAYS
30
39
POSTINFECTION
FIG. 2. Distribution of 14C label weight basis in the DGG of normal Biomphalaria glnbarta. The vertical 90% confidence limits about the Each point is the mean of determinations.
on a relative and infected bars represent sample mean. S or more
LABELING
OF
fi.
of normal snails as in the albumen gland of infected snails. With the onset of egglaying, the amount of label in albumen glands of control animals drops to values found in glands of infected animals. Label in the Remainder
299
mCLnSOni
15 0
12 -
of Carcass
At first, the remainder of the carcass of normal animals contained 12 percentage points more label than did the remainder of the carcass of infected snails (Fig. 5). However, by day 30, the percentage of total label found in the remainder of the carcass of control snails had dropped, bringing the two groups together. But, by the end of the experiment, the remainder of the carcass of normal animals once more has a significantly larger percentage of total label than that of parasitized animals. At no time during the experiment is there any difference, on a relative weight basis, between amounts of label found in the remainder of the carcass from control or infected animals (Fig. 6).
Control Infected
23
39
30t Egg-Laymg
DAYS POSTINFECTION FIG.
gland brata. limits mean
3. Distribution of “C label in albumen of normal and infected BiomphaEaria glaThe vertical bars represent 90% confidence about the sample mean. Each point is the of 8 or more determinations.
Label in S. mansoni Cercariae Twenty B. glabrata infected for 26 days were exposed for 48 hr to 25 microcuries of ‘C-labeled glucose. The snails did not start to shed until 3 days after they were removed from the water containing the labeled glucose. Peak incorporation of the label into the cercariae occurred at 5 days after initial exposure. From the initial activity of the snails and the cercarial activity per snail (Table l), it can be seen that cercarial production accounts for 7.5% of the total label incorporated by the snail. In terms of overall loss of label, cercarial shedding is responsible for 10.3% of the loss. Label in Eggs of Normal
B. glabrata
In order to calculate the amount of label set aside for the demands of egg production, eggs produced by normal snails, which had taken up the labeled glucose, were counted for activity. Eggs were not laid until 11 days after exposure of the
Control Infected
DAYS
Egg-laying POSTINFECTION
FIG. 4. Distribution of “C label on a relative weight basis in the albumen gland of normal and infected Biomphalaria glabrata. The vertical bars represent 90% confidence limits about the sample mean. Each point is the mean of 8 or more determinations.
snails to the isotope, and peak activity was not reached until day 13 (Table 2). From the initial activity of the snails and the egg activity per snail, it can be calculated that egg production takes a minimum of 4.2% of the labeled glucose. In terms of overall loss of label, egg production accounts for a minimum of 14.3% of label lost by the snail.
300
CHRISTIE,
FOSTER
COWOl Infected
23
30
39
DAYS POSTINFECTION
FIG. 5. Distribution of 72 label in remainder of carcass of normal and infected Biomphalaria glabrata. The vertical bars represent 90% confidence limits about the sample mean. Each point is the mean of 8 or more determinations.
Of
’ 23 DAYS
I 30
I 39
POSTINFECTION
FIG. 6. Distribution of “C label on a relative weight basis in the remainder of the carcass of normal and infected Biomphalaria glabrata. The vertical bars represent 90% confidence limits about the sample mean. Each point is the mean of 8 or more determinations.
DISCUSSION
This study supports the idea that nutrient utilization by the parasite may be responsible for the depletion of glycogen noted in infected B. glabrata (Christie et al., 1974). Cercarial shedding alone represents a loss of 7.5% of 14C-glucose administered to infected snails. The I%-labeled glucose is utilized by the parasite for nucleic acid, protein, glycogen, and lipid pro-
AND
STAUBER
duction (Bruce et al., 1969). If we accept Becker’s (1968) estimate that an infection with 5 miracidia produces 15-18 mg of daughter sporocyst tissue in the DGG, then, in our experimental situation, there is approximat’ely 15 mg of sporocyst tissue in the DGG. We can then multiply the amount of sporocyst tissue by the amount of label found in each milligram of DGG after cercarial shedding (6873 dpm). This calculation indicates that 5.1% (6873 X 15/404297) of total initial label may be found in the daughter sporocysts. Thus, the developing parasites, cercariae and sporocysts, utilize a minimum of 12.6% of the initial label picked up by the snail. This estimate does not include loss of label due to sporocyst metabolism or to the high cost of cercarial synthesis. There is also some autoradiographic evidence (Lewert and Para, 1966) that there is unequal partitioning of the label between the host and the parasite, with the parasite getting the better of the bargain. Thus, the estimate of 5.1 % of initial label found in the daughter sporocysts may be low. Accompanying the diversion of label from host to parasite are major changes in the intramolluscan localization of the ‘%-label. The increase in amount of label found in the DGG of infected snails seems to be due to the change in relative weight contributed by the mass of the daughter sporocysts. When calculated on a relative weight basis, there is no difference in amount of label going to the DGG of control or infected animals except for day 39 postinfection. At this time, the decline in label in the DGG of infected snails may be due to loss of label associated with cercarial shedding. With the diversion of label to the DGG in infected animals, there is less total label available for the remainder of the carcass and the albumen gland. Although there is a significant diversion of label from the albumen gland to the DGG in infected snails, there is no significant decrease in galactogen levels in the albumen gland during infection (Christie
LABELING
INCORPORATION OF 1% LABEL DERIVED FROM Biomphalaria
Time (days)
OF
301
TABLE 1 IN CERCARIAE OF Schistosoma mansoni glabrata EXPOSED TO 1% GLUCOSE
Activity of cercariae (dpm) a
No. of cercariae per snail
3.69 1.67 2.50 1.93
4100 3600 900 3600
28 31 33 35 37
S. mansoni
Cercarial activity per snail (dpd
Activity per snail (dpm) 404297
15129 6012 2250 6948 Total
30339 110111
44 Activity a dpm
= disintegrations
per
INCORPORATION
OF 1% glabrata
(days)
TABLE 2 LABEL IN EGGS OF NORMAL EXPOSED TO 1% GLUCOSE
294186
Biomphalaria
Activity
Activity of eggs (dpd
Time
lost
minute.
No. of eggs
Activity per snail
No. of snails
per egg (dpm)
(dpm)
_..-
142560
7 11 12 13 16
9026 20083 30311 5877 Total
67 55 72 65
134.7 365.1 419.6 90.4
11 11 11
11
65297
Activity Egg
activity
et al., 1974). In the normal albumen gland, the percentage of total label present is much greater than that in the albumen gland of infected snails until the control snails start laying eggs. At that time, the amount of total label in normal albumen gland drops precipitously to the levels found in the albumen gland of infected snails. Thus, the drain of egg-laying in normal animals may prevent galactogen concentrations from being significantly greater than in parasitized animals. Egg-laying accounts for a minimum of 4.2% of the total label found in the snail. Most of that label would probably come from the albumen gland since the secretions of the albumen gland seem to make up most of the egg clutch. If egg-laying utilizes a maximum of 20% of the galactogen in the albumen gland (McMahon et al., 1957), then the
per
snail
100961 lost
41599
5936
constant drain of egg-laying would keep the galactogen levels low enough and variable enough so that is would take a tremendous stress, such as complete starvation, before galactogen levels in the experimental animal would fall significantly below normal. Another aspect to be considered is the fact that egg-laying accounts for a minimum of 4.2% of the total label while parasitism is responsible for diversion of 12.5% of the label. It appears that parasitism is a more severe drain on the snail than egglaying, and that cessation of egg-laying in parasitic castration might have a nutritional basis. The diversion of nutrients from the albumen gland in parasitism might reflect the situation in the gonad itself. On the other hand, there seems to be a change in percentage of label taken up on a relative weight basis in the albumen gland of para-
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sitized snails compared to that of normal animals. This change may simply reflect the severity of the nutritional drain on the albumen gland during parasitism, or there may be a change in the hormone levels affecting galactogen synthesis. In ACdimax columbianus, an extract of the optic tentacles decreases galactogen synthesis and incorporation of I%-labeled glucose into galactogen while ablation of the optic tentacles causes the opposite effect (Meenakshi and Scheer, 1969). It is postulated that this extract affects the gonad, which in turn controls the albumen gland. A similar situation is found in Lymnaea stagnalis where the dorsal bodies of the dorsal complex of the brain secrete a factor which is necessary for vitellogenesis and growth and function of the accessory sex glands (Joosse and Geraerts, 1969). Perhaps the daughter sporocysts of S. mansoni affect the brain region of B. glabrata, causing inhibition or blocking of the dorsal bodies and cessation of gonad and accessory sex organ function. It is also possible that the gonad may fail to control the metabolism of the accessory sex organs due to the presence of daughter sporocysts. The whole question of hormonal changes in parasitized molluscs needs much more work. In conclusion, we can say that production of larval stages of 8. mansoni represents a nutritional drain on B. glabrata. This diversion of nutrients may cause major alterations in the host’s metabolism and reproductive capacity. ACKNOWLEDGMENTS The authors would like to thank Dr. Dan Ostland of Merck Institute of Therapeutic Research, Dr. Edward H. Michelson of the Harvard School of Public Health, and Dr. Henry van der Schalie of the University of Michigan for their help in supplying normal and infected B. glabrata. The
AND STATJBER snails from Dr. van der Schalie were obtained through the auspices of the U.S.-Japan ‘Cooperative Medical Science Program-KIAID. The senior author would also like personally to thank Dr. van der Schalie for his help, generosity, and encouragement to a beginner in the field. Funds for this investigation were provided in part by a grant from Rutger University Research Council, USPHS Parasitology Training Grant AI-187, BSSG funds from USPH RR 7058, and The Charles and Johanna Busch Memorial Fund Award, to the Bureau of Biological Research. REFERENCES W. 1968. Untersuchungen iiber die aus der Muttersporocysto auswandemden Tochtersporocysten von Schistosoma mansoni. I. Beitrage zum Kohlenhydratstoffwechsel dieser Stadien. Z. Parasitenk., 30, 233-251. 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. BECKER,
Exp.
Parasitol.,
26, 29-40.
BUSH, E. T. 1963. General applicability of the channels ratio method of measuring liquid scintillation counting efficiencies. Anal. Chem., 35, 1024-1029. CHRISTIE, J. D., FOSTER, W. B., AND STAUBER, L. A. 1974. The effect of parasitism and starvation on carbohydrate reserves of Boimphalaria glabrata. J. Invertebr. JOOSSE, J., AND GERAERTS,
Pathol.,
23, 55-62.
W. J. 1969. On the influence of the dorsal bodies and the adjacent neurosecretory cells on the reproduction and metabolism of Lymnaea stagnalis. Gen. Comp. Endocrinol., 13, 511. LEWERT, R. M., AND PARA, J. B. 1966. The physiological incorporation of carbon-14 in Schistosoma mansoni cercariae. J. Infect. Dis., 116, 171-182. MCMAHON, P., WON BRAND, T., AND NOLAN, M. 0. 1957. Observations on the poIysaccarides of aquatic snails. J. Cell. Comp. Physiol., 50, 219-239. MEENAKSHI, V. R., AND SCHEER, B. T. 1969. Regulation of galactogen synthesis in the slug Ariolimaz columbianus. Comp. Biochem. Physiol., 29, 841-845.