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Alterations in the osmoregulation of the pulmonate gastropod Biomphalaria glabrata due to copper
JOURNAL
OF INVERTEBRATE
Alterations
PATHOLOGY
29, lol-lt)4
(1977)
in the Osmoregulation Biomphalaria
glabrata
of the Pulmonate Due to Copper1
Gastropod
THOMAS C.CHENG AND JOHN T. SULLIVAN~ Institute
for Pathobiology, Center for Health Bethlehem, Pennsylvania
Sciences, 18015
Lehigh
University,
Received July 29, 1976 Specimens of Biomphalaria glabrata were exposed to 0.06 ppm of copper in the form of CuSO,, and the resulting changes in the wet and dry weights of the soft tissues and in the osmolality of the hemolymph were measured. The wet weights of snails exposed to copper increased as a function of time, while those of the controls decreased. The dry weights of both the experimental and control snails decreased equally. Finally, the ratio of wet weight to dry weight of the experimental snails was significantly higher than that of the controls after 24 and 48 hr of exposure to copper. In addition, the osmolality of the hemolymph of snails exposed to copper was significantly lower than that of the controls after 12. 24, and 36 hr of exposure. These data have led to the conclusion that exposure of B. glabrata to copper results in an osmotic influx of water into its tissues and thereby causes death.
INTRODUCTION
On the basis of electron microscopical evidence, Sullivan and Cheng (1975) advanced the hypothesis that the molluscitidal action of copper on the pulmonate gastropod Biomphalaria glabrata is due to disruption of the osmoregulatory physiology of the snail. Specifically, our earlier fine structural studies revealed that there is a distention of the loose vascular connective tissue and epithelium of the rectal ridge, which is apparently the result of an osmotic influx of water. In this paper we report the effects of this accumulation of water on the ratio of wet weight to dry weight of the soft tissues and on the osmolality of the hemolymph in snails that had been exposed to copper. ’ This research was supported in part by a grant (INCRA-193) from the International Cooper Research Association and in part by a grant (AI 12355-02-Al) from the National Institute of Allergy and Infectious Diseases. Some of the materials used in this study were provided by the U.S.-Japan Cooperative Medical Science Program-NIAID. ’ Present address: University of California International Center for Medical Research, Kuala Lumpur, Malaysia.
MATERIALS
The specimens of B. glabrata used in this study were of the NIH albino strain (Newton, 1955). This strain of snails had been maintained in this laboratory for over 4 years in aerated glass aquaria containing deionized water to which 1.58 ml/liter of Nolan and Carriker’s (1946) salt solution were added. The snails were fed iceburg lettuce ad libitum. Ratio of wet to dry weight. A total of 116 B. gfabrata, each measuring 9 to 11 mm in shell diameter, were placed in separate Stendor dishes (6.35 cm in diameter), each containing 25 ml of 0.06 ppm of copper as CuSO,. Subsequent to 24 hr of incubation in this solution, 12 snails had died, and 30 of the survivors were employed in measurements of the ratio of wet weight to dry weight. After 48 hr of incubation, 48 of the remaining 74 snails had died, while the surviving 26 snails were employed for measurements of wet weight/dry weight ratios. The ratio of wet weight to dry weight for the surviving snails was determined in the following manner. Upon removal from the CuSO, solution, each specimen was dissected from its 101
Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
AND METHODS
102
CHENG AND SULLIVAN
shell, gently blotted for 2 min on wet filter paper in a covered Petri dish in order to standardize the amount of moisture on the body surface and within the mantle cavity, and then immediately weighed. Each snail was subsequently dried at 100°C for 24 hr, after which it was reweighed. The ratio of wet weight to dry weight for each snail was then calculated. Ratios of wet to dry weight for 30 snails that had been exposed to deionized water without food for 0, 24, or 48 hr served as the controls. Mean values of the ratio of wet weight to dry weight for experimental and control snails were compared with the use of the two-tailed Student’s t test (Snedecor and Cochran, 1967). Osmolality. Each of a group of 15 to 20 snails measuring 9 to 11 mm in shell diameter was placed in a separate Stendor dish (6.35 cm in diameter) containing 25 ml of 0.06 ppm of copper as CuSO,. Subsequent to 12, 24, or 36 hr of exposure to this concentration of copper, during which approximately 0, 10, or 20% mortality had occurred, respectively, the osmolality of the pooled hemolymph of the surviving snails was measured in the following manner. Each specimen was removed from the Stendor dish, blotted dry, and wiped clean on the left side with tissue paper. The snail was then forced back into its shell with a wad of tissue paper stuffed into the shell aperture. A small hole was chipped in the left side of the shell over the hemocoel, which lies between the mantle cavity and the digestive gland and in which the gizzard is located. A microcapillary, 75 mm in length by 0.5 to 0.9 mm in diameter, was then pushed through the mantle into the hemocoel and was allowed to fill by capillary action. Once the tube was at least 90% filled with hemolymph, it was sealed at one end and placed in a Petri dish in an ice bath until a total of at least 13 microcapillary tubes had been similarly prepared. The tubes were then centrifuged in a microcapillary centrifuge at approximately 11,900 rpm for 2 min, and the supernatants were
pooled in a 0.25ml sample tube. Finally, the osmolality of the pooled hemolymph sample was determined on a Model 3W freezing point osmometer (Advanced Instruments, Inc., Needham Heights, Massachusetts). Osmolalities of pooled serum samples from snails which had been exposed to deionized water without food for 0, 12, 24, or 36 hr served as the controls. Some difficulty was encountered in bleeding certain snails, particularly those that had been exposed to 0.06 ppm of copper for 36 hr. However, the hemolymph from these specimens, which often filled no more than one-third of the microcapillary tube, was included in the pooled samples. Among the experimental snails, three pooled hemolymph samples were measured at each time period, i.e., after 12, 24, and 36 hr of exposure to copper. For the nonstarved controls, three pooled hemolymph samples were measured, while for the controls starved for 12, 24, or 36 hr, two pooled samples were employed. Osmolalities of the hemolymph of the experimental and control snails were compared with the use of the two-tailed Student’s t test (Snedecor and Cochran, 1967). RESULTS
Ratio of Wet to Dry Weight The wet weight of snails exposed to 0.06 ppm of copper as CuSO, increased as a function of time, while that of controls decreased (Fig. 1). The dry weight of both categories of snails decreased as a function of time, presumably due to starvation (Fig. 2). Finally. the ratio of wet weight to dry weight of snails exposed to copper was significantly greater (P < 0.001) than that of the controls after 24 and 48 hr of exposure (Fig. 3). Osmolality The osmolality of the hemolymph of snails exposed to copper was significantly
COPPER AND OSMOREGULATION
IN EIOMPHALARIA
103
6 1 2 i
1. Graph showing wet weights of Biomphalaria (shell diameter = 10.0 k 0.5 mm) exposed to 0.06 ppm of copper as CuSO, as a function of time. Vertical lines represent standard deviations. (@), Experimental, n = 26: (Cl), nonstarved control, n = 30; (O), starved control, n = 30.
2. Graph showing dry weights of Biomphalaria (shell diameter = 10.0 2 0.5 mm) exposed to 0.06 ppm of copper as CuSO, as a function of time. Vertical lines represent standard deviations. (O), Experimental, n = 26; (Cl), nonstarved control, n = 30; (0), starved control, n = 30.
lower (P < 0.01) than that of the hemolymph of control snails after 12, 24, and 36 hr of exposure (Fig. 4). The osmolality of the control snails remained essentially unchanged throughout the experiment.
the environment, is primarily responsible for the decreased osmolality. Although the dry weights of experimental snails are not significantly different from those of the starved controls, leakage of solutes from the hemolymph of experimental snails may also partially account for the decrease in osmolality.
FIG.
glabrara
DISCUSSION
The higher ratio of wet weight to dry weight in B. glabrata that had been exposed to copper relative to control snails could be due to one or both of two phenomena: (1) an accumulation of water in the tissues, and (2) a loss of blood, necrosis of tissues, or increased catabolism of metabolic reserves. However, since the dry weights of both the experimental and control snails decreased in parallel fashion, it can be concluded that the higher ratio of wet weight to dry weight of the former must be due to an accumulation of water in the tissues. The decreased osmolality of the hemolymph of snails that had been exposed to copper could be due to dilution of the hemolymph with water and/or leakage of osmotically active solutes from the hemolymph. It is inferred from the wet weight/dry weight ratio that the first explanation, i.e., dilution of the hemolymph with water from
FIG.
glabrata
FIG. 3. Graph showing ratio of wet weight to dry weight of Biomphalaria glabrata (shell diameter = 10.0 k 0.5 mm) exposed to 0.06 ppm of copper as CuSO, as a function of time. Vertical lines represent standard deviations. (O), Experimental, n = 26; (Cl), nonstarved control, n = 30: (0) starved control, n = 30.
104
CHENG AND SULLIVAN
30 20 10 F I
I 0
I 1.7
I
TIME [HI31
24
I
36
FIG. 4. Graph showing the osmolahty of the hemolymph of Biomphalaria glabrata exposed to 0.06 ppm of copper as CuSO, as a function of time. Vertical lines represent standard deviations. (0). Experimental, n = three pooled samples; (G’), nonstarved control, n = three pooled samples; (O), starved control, n = two pooled samples.
The data presented support those resulting from fine structural studies (Sullivan and Cheng, 1975), with all implying that an osmotic influx of water occurs in the tissues of B. glabrata which have been exposed to copper. This accumulation of water in turn results in fine structural histopathological changes in the rectal ridge, which include a distention of the mantle epithelium and underlying basal lamina and the lysis of the pigment cells in the loose vascular connective tissue of the rectal ridge. The osmolality of the hemolymph of B. glabrata measuring 10 mm in shell diameter is approximately 100 mosM/liter, which is hyperosmotic to the surrounding aquatic environment. Hence, the snail is vulnerable to the entry of water by osmosis and loss of
salts. In order to maintain a constant osmotic pressure in the hemolymph, freshwater molluscs utilize mechanisms of “anisosmotic extracellular regulation” (Schoffeniels and Gilles, 1972), which include reduced permeability to water, uptake of ions from the external environment and from food, and excretion of a hypoosmotic urine. Until further evidence becomes available, it cannot be determined whether the accumulation of water in the tissues is due to an increased permeability of the epithelial surfaces of the snail or to disruption of the excretory functions of the kidney as a result of exposure to copper. Furthermore, it is not known what effects copper has on ion uptake in B. glabrata. Nevertheless, it is clear that exposure to copper results in a disruption of the osmoregulatory physiology of B. glabrata, and it is quite possible that this disruption is responsible for the molluscicidal effect of copper. REFERENCES NEWTON. W. L. 1955. The establishment of a strain of Australorbis glabratus which combines albinism and a high susceptibility to infection with Schistosoma
mansoni.
J. Parasitol..
41, 526-528.
M. R. 1946. Observations on the biology of the snail Lymnaea stagnalis aspersa during twenty years in laboratory culture.
NOLAN,
Amer.
L. E.,
AND
Midl.
Nat.,
CARRIKER,
36, 467-493.
E.. AND GILLES. R. 1972. Ionoregulation and osmoregulation in Mollusca. In “Chemical Zoology. Volume III: Mollusca” (M. Florkin and B. T. Scheer. eds.), pp. 393-420. Academic Press, New York. SNEDECOR, G. W., AND COCHRAN, W. G. 1967. “Statistical Methods.” Iowa State University Press. Ames, Iowa. SULLIVAN, .I. T., AND CHENG, T. C. 1975. Heavy metal toxicity to Biomphalaria glabrata (Mollusca: Pulmonata). Ann. N.Y. Acad. Sci., 266, 437-444. SCHOFFENIELS,
OF INVERTEBRATE
Alterations
PATHOLOGY
29, lol-lt)4
(1977)
in the Osmoregulation Biomphalaria
glabrata
of the Pulmonate Due to Copper1
Gastropod
THOMAS C.CHENG AND JOHN T. SULLIVAN~ Institute
for Pathobiology, Center for Health Bethlehem, Pennsylvania
Sciences, 18015
Lehigh
University,
Received July 29, 1976 Specimens of Biomphalaria glabrata were exposed to 0.06 ppm of copper in the form of CuSO,, and the resulting changes in the wet and dry weights of the soft tissues and in the osmolality of the hemolymph were measured. The wet weights of snails exposed to copper increased as a function of time, while those of the controls decreased. The dry weights of both the experimental and control snails decreased equally. Finally, the ratio of wet weight to dry weight of the experimental snails was significantly higher than that of the controls after 24 and 48 hr of exposure to copper. In addition, the osmolality of the hemolymph of snails exposed to copper was significantly lower than that of the controls after 12. 24, and 36 hr of exposure. These data have led to the conclusion that exposure of B. glabrata to copper results in an osmotic influx of water into its tissues and thereby causes death.
INTRODUCTION
On the basis of electron microscopical evidence, Sullivan and Cheng (1975) advanced the hypothesis that the molluscitidal action of copper on the pulmonate gastropod Biomphalaria glabrata is due to disruption of the osmoregulatory physiology of the snail. Specifically, our earlier fine structural studies revealed that there is a distention of the loose vascular connective tissue and epithelium of the rectal ridge, which is apparently the result of an osmotic influx of water. In this paper we report the effects of this accumulation of water on the ratio of wet weight to dry weight of the soft tissues and on the osmolality of the hemolymph in snails that had been exposed to copper. ’ This research was supported in part by a grant (INCRA-193) from the International Cooper Research Association and in part by a grant (AI 12355-02-Al) from the National Institute of Allergy and Infectious Diseases. Some of the materials used in this study were provided by the U.S.-Japan Cooperative Medical Science Program-NIAID. ’ Present address: University of California International Center for Medical Research, Kuala Lumpur, Malaysia.
MATERIALS
The specimens of B. glabrata used in this study were of the NIH albino strain (Newton, 1955). This strain of snails had been maintained in this laboratory for over 4 years in aerated glass aquaria containing deionized water to which 1.58 ml/liter of Nolan and Carriker’s (1946) salt solution were added. The snails were fed iceburg lettuce ad libitum. Ratio of wet to dry weight. A total of 116 B. gfabrata, each measuring 9 to 11 mm in shell diameter, were placed in separate Stendor dishes (6.35 cm in diameter), each containing 25 ml of 0.06 ppm of copper as CuSO,. Subsequent to 24 hr of incubation in this solution, 12 snails had died, and 30 of the survivors were employed in measurements of the ratio of wet weight to dry weight. After 48 hr of incubation, 48 of the remaining 74 snails had died, while the surviving 26 snails were employed for measurements of wet weight/dry weight ratios. The ratio of wet weight to dry weight for the surviving snails was determined in the following manner. Upon removal from the CuSO, solution, each specimen was dissected from its 101
Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
AND METHODS
102
CHENG AND SULLIVAN
shell, gently blotted for 2 min on wet filter paper in a covered Petri dish in order to standardize the amount of moisture on the body surface and within the mantle cavity, and then immediately weighed. Each snail was subsequently dried at 100°C for 24 hr, after which it was reweighed. The ratio of wet weight to dry weight for each snail was then calculated. Ratios of wet to dry weight for 30 snails that had been exposed to deionized water without food for 0, 24, or 48 hr served as the controls. Mean values of the ratio of wet weight to dry weight for experimental and control snails were compared with the use of the two-tailed Student’s t test (Snedecor and Cochran, 1967). Osmolality. Each of a group of 15 to 20 snails measuring 9 to 11 mm in shell diameter was placed in a separate Stendor dish (6.35 cm in diameter) containing 25 ml of 0.06 ppm of copper as CuSO,. Subsequent to 12, 24, or 36 hr of exposure to this concentration of copper, during which approximately 0, 10, or 20% mortality had occurred, respectively, the osmolality of the pooled hemolymph of the surviving snails was measured in the following manner. Each specimen was removed from the Stendor dish, blotted dry, and wiped clean on the left side with tissue paper. The snail was then forced back into its shell with a wad of tissue paper stuffed into the shell aperture. A small hole was chipped in the left side of the shell over the hemocoel, which lies between the mantle cavity and the digestive gland and in which the gizzard is located. A microcapillary, 75 mm in length by 0.5 to 0.9 mm in diameter, was then pushed through the mantle into the hemocoel and was allowed to fill by capillary action. Once the tube was at least 90% filled with hemolymph, it was sealed at one end and placed in a Petri dish in an ice bath until a total of at least 13 microcapillary tubes had been similarly prepared. The tubes were then centrifuged in a microcapillary centrifuge at approximately 11,900 rpm for 2 min, and the supernatants were
pooled in a 0.25ml sample tube. Finally, the osmolality of the pooled hemolymph sample was determined on a Model 3W freezing point osmometer (Advanced Instruments, Inc., Needham Heights, Massachusetts). Osmolalities of pooled serum samples from snails which had been exposed to deionized water without food for 0, 12, 24, or 36 hr served as the controls. Some difficulty was encountered in bleeding certain snails, particularly those that had been exposed to 0.06 ppm of copper for 36 hr. However, the hemolymph from these specimens, which often filled no more than one-third of the microcapillary tube, was included in the pooled samples. Among the experimental snails, three pooled hemolymph samples were measured at each time period, i.e., after 12, 24, and 36 hr of exposure to copper. For the nonstarved controls, three pooled hemolymph samples were measured, while for the controls starved for 12, 24, or 36 hr, two pooled samples were employed. Osmolalities of the hemolymph of the experimental and control snails were compared with the use of the two-tailed Student’s t test (Snedecor and Cochran, 1967). RESULTS
Ratio of Wet to Dry Weight The wet weight of snails exposed to 0.06 ppm of copper as CuSO, increased as a function of time, while that of controls decreased (Fig. 1). The dry weight of both categories of snails decreased as a function of time, presumably due to starvation (Fig. 2). Finally. the ratio of wet weight to dry weight of snails exposed to copper was significantly greater (P < 0.001) than that of the controls after 24 and 48 hr of exposure (Fig. 3). Osmolality The osmolality of the hemolymph of snails exposed to copper was significantly
COPPER AND OSMOREGULATION
IN EIOMPHALARIA
103
6 1 2 i
1. Graph showing wet weights of Biomphalaria (shell diameter = 10.0 k 0.5 mm) exposed to 0.06 ppm of copper as CuSO, as a function of time. Vertical lines represent standard deviations. (@), Experimental, n = 26: (Cl), nonstarved control, n = 30; (O), starved control, n = 30.
2. Graph showing dry weights of Biomphalaria (shell diameter = 10.0 2 0.5 mm) exposed to 0.06 ppm of copper as CuSO, as a function of time. Vertical lines represent standard deviations. (O), Experimental, n = 26; (Cl), nonstarved control, n = 30; (0), starved control, n = 30.
lower (P < 0.01) than that of the hemolymph of control snails after 12, 24, and 36 hr of exposure (Fig. 4). The osmolality of the control snails remained essentially unchanged throughout the experiment.
the environment, is primarily responsible for the decreased osmolality. Although the dry weights of experimental snails are not significantly different from those of the starved controls, leakage of solutes from the hemolymph of experimental snails may also partially account for the decrease in osmolality.
FIG.
glabrara
DISCUSSION
The higher ratio of wet weight to dry weight in B. glabrata that had been exposed to copper relative to control snails could be due to one or both of two phenomena: (1) an accumulation of water in the tissues, and (2) a loss of blood, necrosis of tissues, or increased catabolism of metabolic reserves. However, since the dry weights of both the experimental and control snails decreased in parallel fashion, it can be concluded that the higher ratio of wet weight to dry weight of the former must be due to an accumulation of water in the tissues. The decreased osmolality of the hemolymph of snails that had been exposed to copper could be due to dilution of the hemolymph with water and/or leakage of osmotically active solutes from the hemolymph. It is inferred from the wet weight/dry weight ratio that the first explanation, i.e., dilution of the hemolymph with water from
FIG.
glabrata
FIG. 3. Graph showing ratio of wet weight to dry weight of Biomphalaria glabrata (shell diameter = 10.0 k 0.5 mm) exposed to 0.06 ppm of copper as CuSO, as a function of time. Vertical lines represent standard deviations. (O), Experimental, n = 26; (Cl), nonstarved control, n = 30: (0) starved control, n = 30.
104
CHENG AND SULLIVAN
30 20 10 F I
I 0
I 1.7
I
TIME [HI31
24
I
36
FIG. 4. Graph showing the osmolahty of the hemolymph of Biomphalaria glabrata exposed to 0.06 ppm of copper as CuSO, as a function of time. Vertical lines represent standard deviations. (0). Experimental, n = three pooled samples; (G’), nonstarved control, n = three pooled samples; (O), starved control, n = two pooled samples.
The data presented support those resulting from fine structural studies (Sullivan and Cheng, 1975), with all implying that an osmotic influx of water occurs in the tissues of B. glabrata which have been exposed to copper. This accumulation of water in turn results in fine structural histopathological changes in the rectal ridge, which include a distention of the mantle epithelium and underlying basal lamina and the lysis of the pigment cells in the loose vascular connective tissue of the rectal ridge. The osmolality of the hemolymph of B. glabrata measuring 10 mm in shell diameter is approximately 100 mosM/liter, which is hyperosmotic to the surrounding aquatic environment. Hence, the snail is vulnerable to the entry of water by osmosis and loss of
salts. In order to maintain a constant osmotic pressure in the hemolymph, freshwater molluscs utilize mechanisms of “anisosmotic extracellular regulation” (Schoffeniels and Gilles, 1972), which include reduced permeability to water, uptake of ions from the external environment and from food, and excretion of a hypoosmotic urine. Until further evidence becomes available, it cannot be determined whether the accumulation of water in the tissues is due to an increased permeability of the epithelial surfaces of the snail or to disruption of the excretory functions of the kidney as a result of exposure to copper. Furthermore, it is not known what effects copper has on ion uptake in B. glabrata. Nevertheless, it is clear that exposure to copper results in a disruption of the osmoregulatory physiology of B. glabrata, and it is quite possible that this disruption is responsible for the molluscicidal effect of copper. REFERENCES NEWTON. W. L. 1955. The establishment of a strain of Australorbis glabratus which combines albinism and a high susceptibility to infection with Schistosoma
mansoni.
J. Parasitol..
41, 526-528.
M. R. 1946. Observations on the biology of the snail Lymnaea stagnalis aspersa during twenty years in laboratory culture.
NOLAN,
Amer.
L. E.,
AND
Midl.
Nat.,
CARRIKER,
36, 467-493.
E.. AND GILLES. R. 1972. Ionoregulation and osmoregulation in Mollusca. In “Chemical Zoology. Volume III: Mollusca” (M. Florkin and B. T. Scheer. eds.), pp. 393-420. Academic Press, New York. SNEDECOR, G. W., AND COCHRAN, W. G. 1967. “Statistical Methods.” Iowa State University Press. Ames, Iowa. SULLIVAN, .I. T., AND CHENG, T. C. 1975. Heavy metal toxicity to Biomphalaria glabrata (Mollusca: Pulmonata). Ann. N.Y. Acad. Sci., 266, 437-444. SCHOFFENIELS,