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Effects of toxicants on developing oocytes and embryos of the blue crab, Callinectes sapidus
Marine Environmenral Research Vol. 42, No. 14, pp. 125-128, 1996 Copyright 0 1996 Elsevier Science Ltd Printed-in &eat Britain. All rights reserved 0141-1136(95)00079-8 0141-1136/96/$15.00+0.00 ELSEVfER
Effects of Toxicants on Developing Oocytes and Embryos of the Blue Crab, Callinectes sapidus Richard F. Lee,’ Kristen O’Malley” & Yugi Oshimab “Skidaway Institute of Oceanography, 10 Ocean Science Circle, Savannah, Georgia 31411, USA *Department of Fisheries, Faculty of Agriculture, Kyushy University 46, Fukuoka 812, Japan
ABSTRACT Oocytes (90 urn in diameter) were isolated from ovaries of 14-day post-molt adult female crabs and maintained in culture media. The addition of cadmium (20 pg/ liter) or tributyltin (2 uglliter) to developing oocytes resulted in decreases in the growth of oocytes as measured by protein and lipovitellin accumulation relative to controls. Crab embryos isolated from yellow ‘sponge’ of female blue crabs were maintained in seawater until hatching (6-8 days). Toxicants tested included cadmium, copper, tributyltin and endosulfan. Some of the processes or events which were followed included water uptake, lipovitellin utilization rate, formation of appendages, formation of a heart, formation of eyespots and hatching to zoea stage. The primary eflects from addition of toxicants were deformed eyespots and reduced hatching success. The hatching ECso (concentration at which 50% of embryosfailed to hatch) for copper, cadmium, tributyltin and endosulfan were 3.1, 0.25, 0.047 and 450 pglliter, respectively. Crab embryos appear to be suitable for testing of the eflects of a variety of toxicants with advantages including low cost, reproducibility, low variability and sensitivity. Copyright 0 1996 Elsevier Science Ltd
During oocyte development in blue crabs, there is an accumulation of protein and lipid. The major protein of crab yolk granules are lipovitellins, which are high-density lipoproteins (Lee & Puppione, 1988). After fertilization, the eggs leave the ovaries and remain in a fibrous ‘sponge’ attached to the female’s abdomen. The embryos develop in egg sacs for 14-20 days after which they ‘hatch’ from the egg sacs into the swimming zoea stage. The large stores of accumulated lipovitellins provide energy and amino acids for the development of the non-feeding embryos. The presence of cadmium or the juvenile hormone antagonist, precocene II, in food given to adult female blue crabs, resulted in oocytes with lower lipovitellin concentrations than oocytes from control crabs (Lee & Noone, 1995). Since embryos rely on lipovitellin for nutrition, low lipovitellin concentrations in oocytes would likely result in poor embryo survival. 125
126
R. F. Lee et al.
In work reported here, we determined the effects of cadmium and an organometallic compound, tributyltin, on the growth of the crab oocytes maintained in culture. We also determined the effects of toxicants on crab embryos in egg sacs which were isolated from sponging blue crabs and maintained in cell culture plates until hatching took place. Earlier studies by Fisher & Foss (1993) showed that detached embryos from the grass shrimp, Palaemonetes pugio, could be used to test a variety of toxicants. Immature female crabs were fed and allowed to molt in tanks with flowing seawater (salinity 28%). Ovaries were dissected from adult female crabs which were 12-14 days post-molt. Oocytes were washed out of minced ovaries with filtered seawater, followed by passage through nylon filters of different sizes to obtain a clean oocyte preparation. It was necessary to be extremely careful during the isolation and culture of oocytes to prevent fungus from entering oocyte cultures. Crabs were opened in a sterile hood under ultraviolet lamps, and all dissecting equipment and filters used for oocyte isolation were sterile. Light microscopy was used to determine oocyte diameters. After homogenization of oocyte preparations, the protein in oocyte extracts was determined by the method of Bradford (1976). Lipovitellin concentrations in oocyte extracts was determined by competitive enzyme-linked immunosorbent assays (ELISA) using a monoclonal antibody to one of the lipovitellin peptides. The procedures for the preparation of the anti-lipovitellin antibodies and the quantitation of lipovitellin by the ELISA method has been described (Lee & Walker, 1995). Isolated oocytes were cultured in 24 well tissue culture plates. The culture media was Grace’s Insect Media supplemented with 10% hybridoma fetal bovine serum, antibiotics and other nutrients recommended by Luedeman & Lightner (1992). Toxicants tested included cadmium (2 and 20 pg/liter) and tributyltin (0.1 and 2 pg/liter). For the embryo studies, we used sponging blue crabs and staged the embryos by examination under a dissecting microscope. The color of the sponge was also indicative of the embryo stage with the color going from bright orange to yellow to reddish brown and, finally, dark brown to black just before hatching. We used embryos which were 68 days from hatching. Clusters of embryos were dissected from the sponge followed by separation of single embryos with 10 embryos added to each well of a 24 well culture plate. Each well contained 1 ml of filtered seawater. Various concentrations of tributyltin (10, 30 and 50 ng/liter), cadmium (50, 100, 200 and 300 ng/liter) and endosulfan (70,000, 100,000 and 300,000 ng/liter) were tested. Each concentration was run in triplicate. Metal concentrations for both oocyte and embryo studies were determined by inductively coupled plasma mass spectrometry with ultrasonic nebulization (Beauchemin et al., 1987). Tributyltin concentrations were determined by the hydride derivative method with atomic absorption detection (Valkirs et al., 1986). Endosulfan concentrations were determined by extraction from water by hexane and analysis by gas chromatography with electron capture detector using a 30 m DB-1701 fused capillary column. After the addition of toxicants or an equal volume of seawater or carrier solvent (3 pl), the embryos were observed each day under a dissecting microscope. The lipovitellin concentrations per embryo were determined using procedures referred to above. The percent of embryos which hatched was the primary focus of our observations. In addition, we noted the time when appendages, heart and eye spots appeared and any abnormalities associated with their appearance. Smaller oocytes (less than 90 pm) isolated from small stage 1 ovaries ( < 1 g) could be maintained in cell culture, but did not increase in size. Larger oocytes (more than 90 pm) increased in diameter and showed increases in both protein and lipovitellin over a 4-day incubation period. Exposure of these developing oocytes to cadmium (20 pg/liter) or
Effects of toxicants on the blue crab
127
TABLE 1
Effects of Toxicants on Oocyte Development Incubation time (days) 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4
Toxicant added
None None None Cadmium Cadmium Cadmium Cadmium Cadmium Cadmium Tributyltin Tributyltin Tributyltin Tributyltin Tributyltin Tributyltin
Concentration of toxicant @g/titer) 20 20 20 2 2 2 2 2 2 0.1 0.1 0.1
of the Blue Crab, Callinectes sapidus’ Oocyte diameter (pm) 90*4 94*3 110*5 90+4 92zt4 105*6 90*4 93+3 106zt4 90*4 91*3 9Ok5 90*4 93*3 107*5
Protein concentration @g/oocyte) 410*35 420 f 27 505 zt 30 410*35 44oIk40 480 f 35 41ozt35 420 f 34 500 f 30 410*35 400 f 30 420 f 50 410*35 430 f 32 500 f 30
Lipovitellin concentration @g/oocyte) 6zk4 lOIt 55*9 654 8*3 25zt 10 6zt4 10*3 60*12 6%4 8*4 11+5 6+4 12*3 50* 17
*Ovaries were disected from 1Zday post-molt adult female crabs. Oocytes were isolated from minced ovaries by passage through a series of nylon filters. Oocytes were cultured in 24 well tissue culture plates in Grace’s insect media supplemented with fetal bovine serum and other nutrients. Concentrations of cadmium were determined by inductively coupled plasma spectrometry with ultrasonic nebulization and tributyltin by hydride derivative method with atomic absorption detection. Data expressed as mean f standard deviation where n = 3.
tributyltin (2 pg/liter) resulted in decreases in the amount of lipovitellin and protein accumulated relative to controls (Table 1). Particularly pronounced was the effect of tributyltin (2 pg/liter), where there was no significant increase, as expected, in oocyte diameter, protein or lipovitellin over a 4-day incubation. Lower concentrations of cadmium chloride (2 pg/liter) and tributyltin (0.1 pg/liter) did not effect oocyte growth. Crab embryos isolated from the yellow ‘sponge’ of female crabs and transferred to filtered sterilized seawater, were found to hatch at the same time as embryos left in the sponge (6-8 days). Hatching success varied from 95 to 98%. Some of the events which occurred during the incubation were water uptake, lipovitellin utilization, formation of appendages, formation of heart, formation of eyespots and finally hatching to zoea stage. While all of these processes or events were followed, the primary effect we focused on for this study was how toxicants affected hatching. The toxicants tested included cadmium, copper, tributyltin and endosulfan. The pesticide, endosulfan, did not effect hatching until the concentrations were higher than 200 pg/liter. Concentrations of copper between 1 and 10 fig/liter resulted in effects on hatching and deformed eyespots. Cadmium concentrations between 0.1 and 0.3 pg/liter resulted in effects on hatching and deformed eyespots. Deformed eyespots, inhibition of heart formation, lipovitellin utilization and hatching inhibition were observed in crab embryos exposed to tributyltin concentrations between 0.01 and 0.05 pg/liter. Rodriguez & Pisano (1993) noted atrophy of eyes of the embryo of the crab, Chasmagnathus granulata, exposed to a pesticide, parathion, and herbicide, 2,4-D.
R. F. Lee et al.
128
0
6
1W Cadmium Cont. (ng/llter)
303
Fig. 1. The effect of cadmium on hatching of zoea from egg sacs of blue crab, Callinectes sapidus. The hatching EC50 (concentration at which 50% of embryos failed to hatch) for copper, cadmium (Fig. I), tributyltin and endosulfan were 3.1, 0.25, 0.047 and 450 pg/liter, respectively. These preliminary studies suggest that embryos are much more sensitive to toxicants than developing oocytes. Only high concentrations of the pesticide, endosulfan, resulted in effects on hatching of the blue crab embryos (hatching ECSo, 450 pg/liter). The 96-h LCso of adult P. pugio exposed to endosulfan was 0.25 ,ug/liter (Scott et al., 1987). Thus, endosulfan had a much greater effect on adult P. pugio than C. sapidus embryos. For the other compounds tested, the hatching of crab embryos showed high sensitivity. Other toxicants presently being tested include mercury, insect molting inhibitors and juvenile hormone antagonists. The use of crab embryo development appears to be suitable for testing of a variety of toxicants, both metals and organics, as well as mixtures, such as effluents and sediment pore waters. Advantages include low cost, reproducibility, low variability and sensitivity.
REFERENCES Beauchemin, D., McLaren, J. W., Mykytiuk, A. P. & Berman, S. S. (1987). Analyt. Chem., 59, 778-783. Bradford, M. M. (1976). Analyt. Biochem., 72,248-254. Fisher, W. S. & Foss, S. S. (1993). Mar. Pollut. Bull., 26, 385-391. Lee, R. F. & Noone, T. (1995). Mar. Environ. Res., 39, 151-154. Lee, R. F. & Puppione, D. L. (1988). J. Exp. Zool., 248, 278-289. Lee, R. F. &Walker, A. J. (1995). J. Exp. Zool., 271, 401-412. Luedeman, R. A. & Lightner, D. V. (1992). Aquaculture, 101, 205-211. Rodriguez, E. M. & Pisano, A. (1993). Comp. Biochem. Physiol., 104C, 71-78. Scott, G. I., Baughman, D. S., Trim, A. H. & Dee, J. C. (1987). In Vernberg, W. G., Calabrese, A., Thurberg, F. P. & Vemberg, F. J. (eds) Pollution Physiology of Estuarine Organisms. University of South Carolina Press, Columbia, pp. 251-273. Valkirs, A. O., Seligman, P. F., Stand, P. M., Homer, M., Lieberman, S. H., Vafa, G. & Dooley, C. A. (1986). Mar. Pollut. Bull., 17, 319-324.
Effects of Toxicants on Developing Oocytes and Embryos of the Blue Crab, Callinectes sapidus Richard F. Lee,’ Kristen O’Malley” & Yugi Oshimab “Skidaway Institute of Oceanography, 10 Ocean Science Circle, Savannah, Georgia 31411, USA *Department of Fisheries, Faculty of Agriculture, Kyushy University 46, Fukuoka 812, Japan
ABSTRACT Oocytes (90 urn in diameter) were isolated from ovaries of 14-day post-molt adult female crabs and maintained in culture media. The addition of cadmium (20 pg/ liter) or tributyltin (2 uglliter) to developing oocytes resulted in decreases in the growth of oocytes as measured by protein and lipovitellin accumulation relative to controls. Crab embryos isolated from yellow ‘sponge’ of female blue crabs were maintained in seawater until hatching (6-8 days). Toxicants tested included cadmium, copper, tributyltin and endosulfan. Some of the processes or events which were followed included water uptake, lipovitellin utilization rate, formation of appendages, formation of a heart, formation of eyespots and hatching to zoea stage. The primary eflects from addition of toxicants were deformed eyespots and reduced hatching success. The hatching ECso (concentration at which 50% of embryosfailed to hatch) for copper, cadmium, tributyltin and endosulfan were 3.1, 0.25, 0.047 and 450 pglliter, respectively. Crab embryos appear to be suitable for testing of the eflects of a variety of toxicants with advantages including low cost, reproducibility, low variability and sensitivity. Copyright 0 1996 Elsevier Science Ltd
During oocyte development in blue crabs, there is an accumulation of protein and lipid. The major protein of crab yolk granules are lipovitellins, which are high-density lipoproteins (Lee & Puppione, 1988). After fertilization, the eggs leave the ovaries and remain in a fibrous ‘sponge’ attached to the female’s abdomen. The embryos develop in egg sacs for 14-20 days after which they ‘hatch’ from the egg sacs into the swimming zoea stage. The large stores of accumulated lipovitellins provide energy and amino acids for the development of the non-feeding embryos. The presence of cadmium or the juvenile hormone antagonist, precocene II, in food given to adult female blue crabs, resulted in oocytes with lower lipovitellin concentrations than oocytes from control crabs (Lee & Noone, 1995). Since embryos rely on lipovitellin for nutrition, low lipovitellin concentrations in oocytes would likely result in poor embryo survival. 125
126
R. F. Lee et al.
In work reported here, we determined the effects of cadmium and an organometallic compound, tributyltin, on the growth of the crab oocytes maintained in culture. We also determined the effects of toxicants on crab embryos in egg sacs which were isolated from sponging blue crabs and maintained in cell culture plates until hatching took place. Earlier studies by Fisher & Foss (1993) showed that detached embryos from the grass shrimp, Palaemonetes pugio, could be used to test a variety of toxicants. Immature female crabs were fed and allowed to molt in tanks with flowing seawater (salinity 28%). Ovaries were dissected from adult female crabs which were 12-14 days post-molt. Oocytes were washed out of minced ovaries with filtered seawater, followed by passage through nylon filters of different sizes to obtain a clean oocyte preparation. It was necessary to be extremely careful during the isolation and culture of oocytes to prevent fungus from entering oocyte cultures. Crabs were opened in a sterile hood under ultraviolet lamps, and all dissecting equipment and filters used for oocyte isolation were sterile. Light microscopy was used to determine oocyte diameters. After homogenization of oocyte preparations, the protein in oocyte extracts was determined by the method of Bradford (1976). Lipovitellin concentrations in oocyte extracts was determined by competitive enzyme-linked immunosorbent assays (ELISA) using a monoclonal antibody to one of the lipovitellin peptides. The procedures for the preparation of the anti-lipovitellin antibodies and the quantitation of lipovitellin by the ELISA method has been described (Lee & Walker, 1995). Isolated oocytes were cultured in 24 well tissue culture plates. The culture media was Grace’s Insect Media supplemented with 10% hybridoma fetal bovine serum, antibiotics and other nutrients recommended by Luedeman & Lightner (1992). Toxicants tested included cadmium (2 and 20 pg/liter) and tributyltin (0.1 and 2 pg/liter). For the embryo studies, we used sponging blue crabs and staged the embryos by examination under a dissecting microscope. The color of the sponge was also indicative of the embryo stage with the color going from bright orange to yellow to reddish brown and, finally, dark brown to black just before hatching. We used embryos which were 68 days from hatching. Clusters of embryos were dissected from the sponge followed by separation of single embryos with 10 embryos added to each well of a 24 well culture plate. Each well contained 1 ml of filtered seawater. Various concentrations of tributyltin (10, 30 and 50 ng/liter), cadmium (50, 100, 200 and 300 ng/liter) and endosulfan (70,000, 100,000 and 300,000 ng/liter) were tested. Each concentration was run in triplicate. Metal concentrations for both oocyte and embryo studies were determined by inductively coupled plasma mass spectrometry with ultrasonic nebulization (Beauchemin et al., 1987). Tributyltin concentrations were determined by the hydride derivative method with atomic absorption detection (Valkirs et al., 1986). Endosulfan concentrations were determined by extraction from water by hexane and analysis by gas chromatography with electron capture detector using a 30 m DB-1701 fused capillary column. After the addition of toxicants or an equal volume of seawater or carrier solvent (3 pl), the embryos were observed each day under a dissecting microscope. The lipovitellin concentrations per embryo were determined using procedures referred to above. The percent of embryos which hatched was the primary focus of our observations. In addition, we noted the time when appendages, heart and eye spots appeared and any abnormalities associated with their appearance. Smaller oocytes (less than 90 pm) isolated from small stage 1 ovaries ( < 1 g) could be maintained in cell culture, but did not increase in size. Larger oocytes (more than 90 pm) increased in diameter and showed increases in both protein and lipovitellin over a 4-day incubation period. Exposure of these developing oocytes to cadmium (20 pg/liter) or
Effects of toxicants on the blue crab
127
TABLE 1
Effects of Toxicants on Oocyte Development Incubation time (days) 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4
Toxicant added
None None None Cadmium Cadmium Cadmium Cadmium Cadmium Cadmium Tributyltin Tributyltin Tributyltin Tributyltin Tributyltin Tributyltin
Concentration of toxicant @g/titer) 20 20 20 2 2 2 2 2 2 0.1 0.1 0.1
of the Blue Crab, Callinectes sapidus’ Oocyte diameter (pm) 90*4 94*3 110*5 90+4 92zt4 105*6 90*4 93+3 106zt4 90*4 91*3 9Ok5 90*4 93*3 107*5
Protein concentration @g/oocyte) 410*35 420 f 27 505 zt 30 410*35 44oIk40 480 f 35 41ozt35 420 f 34 500 f 30 410*35 400 f 30 420 f 50 410*35 430 f 32 500 f 30
Lipovitellin concentration @g/oocyte) 6zk4 lOIt 55*9 654 8*3 25zt 10 6zt4 10*3 60*12 6%4 8*4 11+5 6+4 12*3 50* 17
*Ovaries were disected from 1Zday post-molt adult female crabs. Oocytes were isolated from minced ovaries by passage through a series of nylon filters. Oocytes were cultured in 24 well tissue culture plates in Grace’s insect media supplemented with fetal bovine serum and other nutrients. Concentrations of cadmium were determined by inductively coupled plasma spectrometry with ultrasonic nebulization and tributyltin by hydride derivative method with atomic absorption detection. Data expressed as mean f standard deviation where n = 3.
tributyltin (2 pg/liter) resulted in decreases in the amount of lipovitellin and protein accumulated relative to controls (Table 1). Particularly pronounced was the effect of tributyltin (2 pg/liter), where there was no significant increase, as expected, in oocyte diameter, protein or lipovitellin over a 4-day incubation. Lower concentrations of cadmium chloride (2 pg/liter) and tributyltin (0.1 pg/liter) did not effect oocyte growth. Crab embryos isolated from the yellow ‘sponge’ of female crabs and transferred to filtered sterilized seawater, were found to hatch at the same time as embryos left in the sponge (6-8 days). Hatching success varied from 95 to 98%. Some of the events which occurred during the incubation were water uptake, lipovitellin utilization, formation of appendages, formation of heart, formation of eyespots and finally hatching to zoea stage. While all of these processes or events were followed, the primary effect we focused on for this study was how toxicants affected hatching. The toxicants tested included cadmium, copper, tributyltin and endosulfan. The pesticide, endosulfan, did not effect hatching until the concentrations were higher than 200 pg/liter. Concentrations of copper between 1 and 10 fig/liter resulted in effects on hatching and deformed eyespots. Cadmium concentrations between 0.1 and 0.3 pg/liter resulted in effects on hatching and deformed eyespots. Deformed eyespots, inhibition of heart formation, lipovitellin utilization and hatching inhibition were observed in crab embryos exposed to tributyltin concentrations between 0.01 and 0.05 pg/liter. Rodriguez & Pisano (1993) noted atrophy of eyes of the embryo of the crab, Chasmagnathus granulata, exposed to a pesticide, parathion, and herbicide, 2,4-D.
R. F. Lee et al.
128
0
6
1W Cadmium Cont. (ng/llter)
303
Fig. 1. The effect of cadmium on hatching of zoea from egg sacs of blue crab, Callinectes sapidus. The hatching EC50 (concentration at which 50% of embryos failed to hatch) for copper, cadmium (Fig. I), tributyltin and endosulfan were 3.1, 0.25, 0.047 and 450 pg/liter, respectively. These preliminary studies suggest that embryos are much more sensitive to toxicants than developing oocytes. Only high concentrations of the pesticide, endosulfan, resulted in effects on hatching of the blue crab embryos (hatching ECSo, 450 pg/liter). The 96-h LCso of adult P. pugio exposed to endosulfan was 0.25 ,ug/liter (Scott et al., 1987). Thus, endosulfan had a much greater effect on adult P. pugio than C. sapidus embryos. For the other compounds tested, the hatching of crab embryos showed high sensitivity. Other toxicants presently being tested include mercury, insect molting inhibitors and juvenile hormone antagonists. The use of crab embryo development appears to be suitable for testing of a variety of toxicants, both metals and organics, as well as mixtures, such as effluents and sediment pore waters. Advantages include low cost, reproducibility, low variability and sensitivity.
REFERENCES Beauchemin, D., McLaren, J. W., Mykytiuk, A. P. & Berman, S. S. (1987). Analyt. Chem., 59, 778-783. Bradford, M. M. (1976). Analyt. Biochem., 72,248-254. Fisher, W. S. & Foss, S. S. (1993). Mar. Pollut. Bull., 26, 385-391. Lee, R. F. & Noone, T. (1995). Mar. Environ. Res., 39, 151-154. Lee, R. F. & Puppione, D. L. (1988). J. Exp. Zool., 248, 278-289. Lee, R. F. &Walker, A. J. (1995). J. Exp. Zool., 271, 401-412. Luedeman, R. A. & Lightner, D. V. (1992). Aquaculture, 101, 205-211. Rodriguez, E. M. & Pisano, A. (1993). Comp. Biochem. Physiol., 104C, 71-78. Scott, G. I., Baughman, D. S., Trim, A. H. & Dee, J. C. (1987). In Vernberg, W. G., Calabrese, A., Thurberg, F. P. & Vemberg, F. J. (eds) Pollution Physiology of Estuarine Organisms. University of South Carolina Press, Columbia, pp. 251-273. Valkirs, A. O., Seligman, P. F., Stand, P. M., Homer, M., Lieberman, S. H., Vafa, G. & Dooley, C. A. (1986). Mar. Pollut. Bull., 17, 319-324.