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Effects of removing symbiotic green algae on the response of Hydra viridissima (Pallas 1776) to metals
ARTICLE IN PRESS
Ecotoxicology and Environmental Safety 60 (2005) 301–305
Effects of removing symbiotic green algae on the response of Hydra viridissima (Pallas 1776) to metals W. Karntanut1 and D. Pascoe* School of Biosciences, Cardiff University, Main Building, P.O. Box 915, Cardiff CF10 3TL, UK Received 4 August 2003; received in revised form 10 March 2004; accepted 8 April 2004 Available online 28 May 2004
Abstract Hydra viridissima is distinctively green due to symbiotic algae within the endodermal cells. The current investigation was designed to see if these algae influenced the response of Hydra to pollutants, by comparing the toxicity of copper, cadmium, and zinc to both symbiotic and aposymbiotic (free of their endosymbiotic algae) H. viridissima. The results demonstrated that the toxicity of the metals was generally similar for both groups of Hydra. However, at the lowest copper concentrations there was a difference between the two group of polyps, with aposymbiotic animals dying at concentrations where symbiotic Hydra survived. The lowest observed effect concentrations were 0.0068 and 0.016 mg/L for aposymbiotic and symbiotic Hydra, respectively. It is suggested that the symbiotic Hydra derive benefits from the association that enable them to better tolerate the toxicant. This work demonstrated that experimental manipulation of symbionts can help to explain their complex interactions and the ways in which they respond to pollutants. r 2004 Elsevier Inc. All rights reserved. Keywords: Hydra viridissima; Symbiosis; Aposymbiosis; Green algae; Toxicity; Copper; Cadmium; Zinc
1. Introduction Several investigators have demonstrated that the cnidarian Hydra can be useful as an indicator of freshwater pollutants (Hyne et al., 1993; Pollino and Holdway, 1999; Pascoe et al., 2002). In a recent study, Karntanut and Pascoe (2002) examined the comparative sensitivity of four different Hydra to three metals (copper, cadmium, and zinc) that are important as pollutants in freshwater. The species used were Hydra vulgaris1 (Zurich strain, male clone), Hydra vulgaris2 (a dioecious strain reproducing sexually and asexually), H. oligactis (dioecious, reproducing sexually and asexually depending on environmental conditions), and H. viridissima (a monecious species, distinctively green due to its symbiotic green algae). The study demonstrated that all the Hydra responded to zinc in a similar way but that *Corresponding author. Fax: +44-29-20874305. E-mail address: [email protected] (D. Pascoe). 1 Current address: Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand. 0147-6513/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.04.001
the green H. viridissima was significantly more sensitive to copper and cadmium than the other species. Several other workers have studied toxicant effects upon H. viridissima (Benson and Boush, 1983; Kalafatic et al., 2001; Kovacevic et al., 2001) and some made similar observations. For example, Pollino and Holdway (1999) found that the 96-h LC50 of copper for H. viridissima was three times lower than that for H. vulgaris, while Pyatt and Dodd (1986) reported that Chlorohydra viridissima was far more vulnerable to heavy metal pollution than H. oligactis. However, Holdway et al. (2001) found that H. viridissima was more sensitive than H. vulgaris to zinc as well as cadmium. Several possible explanations, including a lack of metallothionein (Andersen et al., 1988) or a restricted gene base due to asexual laboratory culture (Pyatt and Dodd, 1986), have been discussed and discounted (Karntanut and Pascoe, 2002) as reasons for the enhanced metal sensitivity of the green Hydra. An alternative explanation could be related to the presence, and consequent physiological effects, of the zoochlorellae symbiotic within the endodermal cells of
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H. viridissima. The benefits provided by this relationship have been well documented, with photosynthetically fixed carbon translocated to Hydra from the algae largely as maltose (Pardy and White, 1977; Kessler et al., 1988; McAuley et al., 1996) and amino acids and other metabolites released to the algae from Hydra’s digestive processes (Thorington and Margulis, 1981; McAuley, 1987). The capacity for sugar release is common to all symbiotic Chlorella (Douglas and Huss, 1986) and may account for up to 69% of the host’s caloric requirements (Pardy and White, 1977). Aposymbiotic H. viridissima were first described by Whitney (1907), but this condition has never been recorded in nature (Bossert and Dunn, 1986). Pardy (1983) reported that culturing H. viridissima in a medium containing glycerol resulted in the ejection of their algae so that the polyps became ‘‘bleached.’’ However, these aposymbiotic Hydra survived and reproduced as well as symbiotic animals, providing they were fed regularly (Muscatine and Lenhoff, 1965). The purpose of this study therefore was to compare the toxicity of copper, cadmium, and zinc to symbiotic and aposymbiotic (free of their endosymbiotic algae) H. viridissima in an attempt to determine whether the presence of the algae modifies the response of Hydra, making it more sensitive to toxicants.
2. Materials and methods 2.1. Test organisms and their culture Symbiotic H. viridissima, originally collected from a pond in Songkla Province, Thailand, were cultured and reared routinely as described previously (Beach and Pascoe, 1998; Karntanut and Pascoe, 2000, 2002) using the well-established methods of Lenhoff (1983). A modification of Pardy’s (1983) procedure was used to prepare and maintain aposymbiotic H. viridissima, free of their endosymbiotic algae for this investigation. A solution of 0.5% (v/v) glycerol was made up using Hydra medium as diluent and 30 green Hydra were then transferred to a glass beaker containing 50 mL of this 0.5% glycerol solution. All animals were fed daily with excess Artemia nauplii. After feeding, the culture was cleared of food debris and the glycerol medium was renewed. Within 3 weeks of commencing this regime the Hydra became visibly paler and they were free of algal symbionts within 5 weeks. This was confirmed microscopically. These aposymbiotic Hydra, free of algae, were then placed in normal Hydra medium (without glycerol). Individual polyps were kept separately in each 2-mL well of multiwell Repli dishes (Sterilin Ltd., UK) in order to observe the offspring and to continue feeding according to the normal schedule. New Hydra buds were transferred to another Repli dish and observation was
continued. As a result it was possible to ensure that the parental polyp and its offspring did not become green, which would have indicated reinfection. The aposymbiotic offspring were then ready to use for mass culture in aquaria at 2071 C with 16:8 h light:dark, following normal culture procedures. This aposymbiotic culture has subsequently remained algae-free for over 2 years. 2.2. Test chemicals A range of seven copper, five cadmium, and nine zinc concentrations was prepared using Hydra medium as diluent from 100 mg/L stock solutions of copper sulfate (CuSO4 5H2O), cadmium chloride ðCdCl2 2 12 H2 OÞ; and zinc sulfate (ZnSO4 7H2O) obtained from SigmaAldrich, UK. 2.3. Experimental design Toxicity tests were carried out in glass vials (2 2.5 cm) previously equilibrated with toxicant. A single aposymbiotic Hydra polyp without bud was transferred to 3 mL of fresh metal solution. Ten such animals were used as replicates for each metal concentration and for the control without metal. The same test procedure was carried out with symbiotic Hydra, i.e., a total of 220 symbiotic and 220 aposymbiotic polyps were used. The experiment lasted for 19 days and was carried out under the same environmental conditions as the culture of Hydra, i.e., 2071 C with 16:8 h light: dark. 2.4. Water quality and toxicant analysis Test solutions were renewed every 4 days and the samples were measured for actual metal concentrations by inductively coupled plasma mass spectrometry. Water quality (hardness, conductivity, dissolved oxygen, and pH) of the Hydra medium was measured throughout the study using standard procedures. Toxicity was assessed by recording mortality as described previously (Karntanut and Pascoe, 2000, 2002). 2.5. Data analysis Mortality data were examined by time–response analysis (Litchfield, 1949) to determine median lethal times (LT50) and median lethal concentrations (LC50). In addition, the slope functions and reaction times (LT50s) derived from the probit response lines were compared at each concentration. Toxicity data analyses were based upon measured, rather than nominal, metal concentrations.
ARTICLE IN PRESS W. Karntanut, D. Pascoe / Ecotoxicology and Environmental Safety 60 (2005) 301–305
3. Results All the control aposymbiotic and symbiotic Hydra survived. Water quality parameters (7SD) recorded for the Hydra medium during the study were hardness (20770.5 mg/L as CaCO3), conductivity (49070.4 mS/ cm), pH (7.770.26), and DO (7.270.7 mg/L). The pH of test solutions was 7.8 for copper and cadmium and 6.2 for zinc solutions. Recorded metal concentrations are shown in Table 1. A comparison of toxicity curves relating LT50 and exposure concentrations (Fig. 1) clearly shows that copper is the most toxic of the metals, followed by cadmium and then zinc. It is also evident that the patterns of mortality are very similar for symbiotic and aposymbiotic animals. Time–response analysis, comparing median lethal times (LT50) and slope functions of the probit response lines at each concentration (Table 1), shows no consistent significant difference between aposymbiotic and symbiotic Hydra exposed to the three metals. LC50 values (Table 2) extrapolated from the time–response toxicity curves for symbiotic and aposymbiotic Hydra at various exposure times confirm that the responses of symbiotic and aposymbiotic animals are similar and also that the relative toxicity is copper4cadmium4zinc. However, in the case of copper, it can be seen from Fig. 1 and Table 1 that at the lower concentrations there is a difference between the two group of polyps, with aposymbiotic animals dying at concentrations where symbiotic Hydra survived. The lowest observed effect concentrations were 0.0068 and 0.016 mg/L for aposymbiotic and symbiotic Hydra, respectively.
4. Discussion It has been known since the work of Goetsch (1924) and confirmed by Muscatine and Lenhoff (1965) that symbiotic algae aid the growth of stressed H. viridissima because when the available food supply is limited, Hydra from which the algae have been removed will survive for a much shorter time than green animals. However, wellfed albino Hydra grow as well as green animals. When Hydra are grown in continuous darkness with less maltose available from photosynthesis, the algae depend more upon Hydra-derived metabolites and so polyps containing the symbionts grow more slowly than aposymbiotic Hydra, which do not have to support the growth of algae (Douglas and Smith, 1983). Under normal conditions the algal population within H. viridissima remains relatively constant but with environmental change the polyp may regulate the number of algae per digestive cell either by ejection of excess algae or more likely by controlling their proliferation rate within the cells, possibly through inhibition of algal
303
mitosis (Pardy and Muscatine, 1973; McAuley, 1981). In the present study no algal expulsion was observed from symbiotic Hydra after exposure to heavy metals. It is possible that the metabolic balance between the two symbionts could also be disrupted by the presence of pollutants, resulting in toxic effects not seen in other Hydra without symbiotic algae. For example, Karntanut and Pascoe (2002) argued that damage to the symbiotic algae, caused by metals such as copper and cadmium, which are known to be toxic to algae, could deprive Hydra of some of the benefits normally derived from its symbionts. This may have resulted in a reallocation of resources within the polyp’s energy budget and a reduced ability to deal with the toxicant itself leading to the greater metal sensitivity, compared to other Hydra species, recorded in their study (i.e., the metal would damage/remove the algae, effectively creating aposymbiotic animals, and these would therefore be more sensitive). The results from the current investigation, though, do not initially appear to provide evidence in support of such an hypothesis, since there is generally no significant difference in reaction times (LT50s) between polyps with and without endosymbiotic algae. However, the data do show that when Hydra are exposed to low concentrations of copper, the absence of algae (i.e., in the aposymbiotic animals) does appear to increase toxicity, since animals die at concentrations where symbiotic polyps do not. At low concentrations the copper taken up by symbiotic Hydra may be sequestered by the algae (as shown for free-living Chlorella by Tien, 2002), providing a degree of protection for the polyp itself. Therefore the greater sensitivity of aposymbiotic Hydra to copper at low concentrations could be attributed to their lack of symbiotic algae. At the higher concentrations it is probable that any defence systems are overwhelmed by the toxicant effect and any slight benefit derived from the endosymbiotic algae is of little consequence. Such an effect would not be expected with cadmium, which plays no essential role in either species, and in the case of zinc it seems that the concentrations examined were all too high for such a protective mechanism to operate. The physiological interrelationships occurring in symbioses are complex and subject to disturbance by stressors such as toxic chemicals. However, experimental manipulation of the symbionts, such as described in this study, can help to explain their interaction and the ways in which they respond to pollutants.
5. Conclusions Experimental manipulation to remove symbiotic green algae from the endodermal cells of H. viridissima made it possible to compare the toxicity of the heavy metals copper, cadmium, and zinc to the same species of
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Table 1 A comparison of slope functions and reaction times (LT50) from Fig. 1 at each concentration of copper, cadmium, and zinc for aposymbiotic and symbiotic Hydra Actual concentration7SD (mg/L) Copper 0.006870.001 0.009670.001 0.01670.000 0.03370.004 0.0570.009 0.0970.007 0.1670.008 Cadmium 0.2870.006 0.3570.021 0.8170.021 1.8970.035 3.8770.424 Zinc 0.6670.454 0.8170.599 1.0470.865 1.7170.391 4.2270.863 21.4871.990 28.7371.672 36.7771.686 54.9070.728
Slope function for aposymbiotic Hydra (95% confidence limits in parentheses)
Slope function for symbiotic Hydra (95% confidence limits in parentheses)
Statistical significance for slope function comparison
Statistical significance for LT50 comparison
1.11 1.87 1.55 1.21 1.15 2.58
(1.17–1.06) (2.50–1.40) (1.93–1.25) (1.33–1.11) (1.23–1.08) (3.99–1.66)
1.39 1.11 1.21 3.86
(1.61–1.19) (1.17–1.06) (1.32–1.11) (7.21–2.07)
1.16 1.05 1.12 1.04 1.25
(1.24–1.08) (1.08–1.03) (1.17–1.06) (1.05–1.02) (1.38–1.13)
1.37 1.09 1.08 1.14 1.29
(1.59–1.19) (1.14–1.05) (1.13–1.04) (1.22–1.07) (1.46–1.15)
1.16 1.10 1.11 1.58 1.25 1.37 1.31 1.18 1.29
(1.25–1.09) (1.01–1.00) (1.16–1.06) (1.95–1.28) (1.39–1.13) (1.58–1.18) (1.49–1.16) (1.28–1.09) (1.45–1.15)
1.07 1.04 1.08 1.61 2.09 1.77 1.72 1.64 1.47
(1.11–1.04) (1.06–1.02) (1.12–1.04) (2.01–1.29) (2.94–1.49) (2.30–1.36) (2.21–1.34) (2.05–1.30) (1.75–1.23)
10% mortality, so no LT50 or slope function could be recorded. No mortality, so no LT50 or slope function could be recorded. Significant difference Pp0:05 (Litchfield, 1949).
1000 Copper Zinc
100 LT50 (h)
Cadmium
10 Aposymbiotic Hydra Symbiotic Hydra
1 0.001
0.01
0.1
1
10
100
Concentration (mg/L)
Fig. 1. A comparison of toxicity curves, relating median lethal time (LT50) and metal concentration, for aposymbiotic and symbiotic Hydra exposed to copper, cadmium, and zinc.
Hydra under symbiotic and aposymbiotic conditions. It was found that the symbiotic Hydra were more resistant to copper, though not to the other metals, and it is
suggested that they derive some benefit from the association, which enables them to better tolerate the toxicant.
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Table 2 Median lethal concentrations (LC50) of copper, cadmium, and zinc extrapolated from time-response toxicity curves Metal
LC50 (mg/L) 24 h
Copper Cadmium Zinc a
48 h
72 h
96 h
Aposymbiotic Hydra
Symbiotic Hydra
Aposymbiotic Hydra
Symbiotic Hydra
Aposymbiotic Hydra
Symbiotic Hydra
Aposymbiotic Hydra
Symbiotic Hydra
0.064 0.36 54
0.058 0.43 33
0.043 0.14 12
0.038 0.16 7.4
0.035 —a 5.2
0.032 —a 3.8
0.031 —a 3.5
0.028 —a 2.5
No observations at these times.
Acknowledgments This work was partly funded by the Royal Thai Government.
References Andersen, R.A., Wiger, R., Daae, H.L., Eriksen, K.D., 1988. Is the metal binding protein metallothionein present in the coelenterate Hydra attenuata? Comp. Biochem. Physiol. C 91 (2), 553–557. Beach, M.J., Pascoe, D., 1998. The role of Hydra vulgaris (Pallas) in assessing the toxicity of freshwater pollutants. Water Res. 32, 101–106. Benson, B., Boush, G.M., 1983. Effect of pesticides and PCBs on budding rates of green Hydra. Bull. Environ. Contam. Toxicol. 30, 344–350. Bossert, P., Dunn, K., 1986. Regulation of intracellular algae by various strains of the symbiotic Hydra viridissima. J. Cell Sci. 85, 187–195. Douglas, A.E., Huss, V.A.R., 1986. On the characteristics and taxonomic position of symbiotic Chlorella. Arch. Microbiol. 145, 80–84. Douglas, A.E., Smith, D.C., 1983. The cost of symbionts to their hosts in green Hydra. In: Schenk, H.E.A., Schwemmler, W. (Eds.), Endocytobiology. de Gruyter, Berlin, pp. 631–648. Goetsch, W., 1924. Die symbiose der su¨sswasser-hydroiden und ihre ku¨nstliche beeinflussung. Z. Morph. O¨kol. Tierre 1, 660–731. Holdway, D., Lok, K., Semaan, M., 2001. The acute and chronic toxicity of cadmium and zinc to two Hydra species. Environ. Toxicol. 16 (6), 557–565. Hyne, R., Rippon, G.D., Ellender, G., 1993. Investigation of uraniuminduced toxicity in freshwater Hydra. In: Dallinger, R., Rainbow, P. (Eds.), Ecotoxicology of Metals in Invertebrates. CRC Press, Boca Raton, FL, pp. 149–173. Kalafatic, M., Kovacevic, G., Ljubesic, N., Sunjic, H., 2001. Effects of ciprofloxacin on green Hydra and endosymbiotic alga. Periodic Biol. 103, 267–272. Karntanut, W., Pascoe, D., 2000. A comparison of methods for measuring acute toxicity to Hydra vulgaris. Chemosphere 41 (10), 149–154. Karntanut, W., Pascoe, D., 2002. The toxicity of copper, cadmium and zinc to four different Hydra. Chemosphere 47 (10), 1059–1064.
Kessler, E., Huss, V.A.R., Rahat, M., 1988. Species-specific ability of Chlorella strains (Chlorophyceae) to form stable symbioses with Hydra viridis. Plant Syst. Evol. 160, 241–246. Kovacevic, G., Kalafatic, M., Ljubesic, N., Sunjic, H., 2001. The effect of chloramphenicol on the symbiosis between alga and Hydra. Biologia 56, 605–610. Lenhoff, H.M., 1983. Culturing large numbers of Hydra. In: Lenhoff, H.M. (Ed.), Hydra Research Methods. Plenum, New York, pp. 53–62. Litchfield, J.T., 1949. A method for the rapid graphic solution of time–percentage effect curves. J. Pharm. Exp. Ther. 97, 399–408. McAuley, P.J., 1981. Ejection of algae in the green Hydra symbiosis. J. Exp. Zool. 217, 23–31. McAuley, P.J., 1987. Quantitative estimation of movement of an amino acid from host to Chlorella symbionts in green Hydra. Biol. Bull. 173, 504–512. McAuley, P.J., Dorling, M., Hodge, H., 1996. Effect of maltose release on uptake and assimilation of ammonium by symbiotic Chlorella (Chlorophyta). J. Phycol. 32, 839–846. Muscatine, L., Lenhoff, H.M., 1965. Symbiosis of Hydra and algae. II. Effects of limited food and starvation on growth of symbiotic and aposymbiotic Hydra. Biol. Bull. 129, 316–328. Pardy, R.L., 1983. Preparing aposymbiotic algae. In: Lenhoff, H.M. (Ed.), Hydra Research Methods. Plenum, New York, pp. 393–397. Pardy, R.L., Muscatine, L., 1973. Recognition of symbiotic algae by green Hydra. A quantitative study of the uptake of living algae by aposymbiotic H. viridis. Biol. Bull. 145, 565–579. Pardy, R.L., White, B.N., 1977. Metabolic relationships between green Hydra and its symbiotic algae. Biol. Bull. 153, 228–236. Pascoe, D., Carroll, K., Karntanut, W., Watts, M.M., 2002. Toxicity of 17a ethinylestradiol and bisphenol A to the freshwater cnidarian Hydra vulgaris. Arch. Environ. Contam. Toxicol. 43 (1), 56–63. Pollino, C.A., Holdway, D.A., 1999. Potential of two Hydra species as standard toxicity test animals. Ecotoxicol. Environ. Saf. 43, 309–316. Pyatt, F.B., Dodd, N.M., 1986. Some effects of metal ions on the freshwater organisms, Hydra oligactis and Chlorohydra viridissima. Ind. J. Exp. Biol. 24, 169–173. Thorington, G., Margulis, L., 1981. Hydra viridis: transfer of metabolites between Hydra and symbiotic algae. Biol. Bull. 160 (1), 175–188. Tien, C.-J., 2002. Biosorption of metal ions by freshwater algae with different surface characteristics. Process Biochem. 38, 605–613. Whitney, D.D., 1907. Artificial removal of the green bodies of Hydra viridis. Biol. Bull. 13, 291–299.
Ecotoxicology and Environmental Safety 60 (2005) 301–305
Effects of removing symbiotic green algae on the response of Hydra viridissima (Pallas 1776) to metals W. Karntanut1 and D. Pascoe* School of Biosciences, Cardiff University, Main Building, P.O. Box 915, Cardiff CF10 3TL, UK Received 4 August 2003; received in revised form 10 March 2004; accepted 8 April 2004 Available online 28 May 2004
Abstract Hydra viridissima is distinctively green due to symbiotic algae within the endodermal cells. The current investigation was designed to see if these algae influenced the response of Hydra to pollutants, by comparing the toxicity of copper, cadmium, and zinc to both symbiotic and aposymbiotic (free of their endosymbiotic algae) H. viridissima. The results demonstrated that the toxicity of the metals was generally similar for both groups of Hydra. However, at the lowest copper concentrations there was a difference between the two group of polyps, with aposymbiotic animals dying at concentrations where symbiotic Hydra survived. The lowest observed effect concentrations were 0.0068 and 0.016 mg/L for aposymbiotic and symbiotic Hydra, respectively. It is suggested that the symbiotic Hydra derive benefits from the association that enable them to better tolerate the toxicant. This work demonstrated that experimental manipulation of symbionts can help to explain their complex interactions and the ways in which they respond to pollutants. r 2004 Elsevier Inc. All rights reserved. Keywords: Hydra viridissima; Symbiosis; Aposymbiosis; Green algae; Toxicity; Copper; Cadmium; Zinc
1. Introduction Several investigators have demonstrated that the cnidarian Hydra can be useful as an indicator of freshwater pollutants (Hyne et al., 1993; Pollino and Holdway, 1999; Pascoe et al., 2002). In a recent study, Karntanut and Pascoe (2002) examined the comparative sensitivity of four different Hydra to three metals (copper, cadmium, and zinc) that are important as pollutants in freshwater. The species used were Hydra vulgaris1 (Zurich strain, male clone), Hydra vulgaris2 (a dioecious strain reproducing sexually and asexually), H. oligactis (dioecious, reproducing sexually and asexually depending on environmental conditions), and H. viridissima (a monecious species, distinctively green due to its symbiotic green algae). The study demonstrated that all the Hydra responded to zinc in a similar way but that *Corresponding author. Fax: +44-29-20874305. E-mail address: [email protected] (D. Pascoe). 1 Current address: Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand. 0147-6513/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.04.001
the green H. viridissima was significantly more sensitive to copper and cadmium than the other species. Several other workers have studied toxicant effects upon H. viridissima (Benson and Boush, 1983; Kalafatic et al., 2001; Kovacevic et al., 2001) and some made similar observations. For example, Pollino and Holdway (1999) found that the 96-h LC50 of copper for H. viridissima was three times lower than that for H. vulgaris, while Pyatt and Dodd (1986) reported that Chlorohydra viridissima was far more vulnerable to heavy metal pollution than H. oligactis. However, Holdway et al. (2001) found that H. viridissima was more sensitive than H. vulgaris to zinc as well as cadmium. Several possible explanations, including a lack of metallothionein (Andersen et al., 1988) or a restricted gene base due to asexual laboratory culture (Pyatt and Dodd, 1986), have been discussed and discounted (Karntanut and Pascoe, 2002) as reasons for the enhanced metal sensitivity of the green Hydra. An alternative explanation could be related to the presence, and consequent physiological effects, of the zoochlorellae symbiotic within the endodermal cells of
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H. viridissima. The benefits provided by this relationship have been well documented, with photosynthetically fixed carbon translocated to Hydra from the algae largely as maltose (Pardy and White, 1977; Kessler et al., 1988; McAuley et al., 1996) and amino acids and other metabolites released to the algae from Hydra’s digestive processes (Thorington and Margulis, 1981; McAuley, 1987). The capacity for sugar release is common to all symbiotic Chlorella (Douglas and Huss, 1986) and may account for up to 69% of the host’s caloric requirements (Pardy and White, 1977). Aposymbiotic H. viridissima were first described by Whitney (1907), but this condition has never been recorded in nature (Bossert and Dunn, 1986). Pardy (1983) reported that culturing H. viridissima in a medium containing glycerol resulted in the ejection of their algae so that the polyps became ‘‘bleached.’’ However, these aposymbiotic Hydra survived and reproduced as well as symbiotic animals, providing they were fed regularly (Muscatine and Lenhoff, 1965). The purpose of this study therefore was to compare the toxicity of copper, cadmium, and zinc to symbiotic and aposymbiotic (free of their endosymbiotic algae) H. viridissima in an attempt to determine whether the presence of the algae modifies the response of Hydra, making it more sensitive to toxicants.
2. Materials and methods 2.1. Test organisms and their culture Symbiotic H. viridissima, originally collected from a pond in Songkla Province, Thailand, were cultured and reared routinely as described previously (Beach and Pascoe, 1998; Karntanut and Pascoe, 2000, 2002) using the well-established methods of Lenhoff (1983). A modification of Pardy’s (1983) procedure was used to prepare and maintain aposymbiotic H. viridissima, free of their endosymbiotic algae for this investigation. A solution of 0.5% (v/v) glycerol was made up using Hydra medium as diluent and 30 green Hydra were then transferred to a glass beaker containing 50 mL of this 0.5% glycerol solution. All animals were fed daily with excess Artemia nauplii. After feeding, the culture was cleared of food debris and the glycerol medium was renewed. Within 3 weeks of commencing this regime the Hydra became visibly paler and they were free of algal symbionts within 5 weeks. This was confirmed microscopically. These aposymbiotic Hydra, free of algae, were then placed in normal Hydra medium (without glycerol). Individual polyps were kept separately in each 2-mL well of multiwell Repli dishes (Sterilin Ltd., UK) in order to observe the offspring and to continue feeding according to the normal schedule. New Hydra buds were transferred to another Repli dish and observation was
continued. As a result it was possible to ensure that the parental polyp and its offspring did not become green, which would have indicated reinfection. The aposymbiotic offspring were then ready to use for mass culture in aquaria at 2071 C with 16:8 h light:dark, following normal culture procedures. This aposymbiotic culture has subsequently remained algae-free for over 2 years. 2.2. Test chemicals A range of seven copper, five cadmium, and nine zinc concentrations was prepared using Hydra medium as diluent from 100 mg/L stock solutions of copper sulfate (CuSO4 5H2O), cadmium chloride ðCdCl2 2 12 H2 OÞ; and zinc sulfate (ZnSO4 7H2O) obtained from SigmaAldrich, UK. 2.3. Experimental design Toxicity tests were carried out in glass vials (2 2.5 cm) previously equilibrated with toxicant. A single aposymbiotic Hydra polyp without bud was transferred to 3 mL of fresh metal solution. Ten such animals were used as replicates for each metal concentration and for the control without metal. The same test procedure was carried out with symbiotic Hydra, i.e., a total of 220 symbiotic and 220 aposymbiotic polyps were used. The experiment lasted for 19 days and was carried out under the same environmental conditions as the culture of Hydra, i.e., 2071 C with 16:8 h light: dark. 2.4. Water quality and toxicant analysis Test solutions were renewed every 4 days and the samples were measured for actual metal concentrations by inductively coupled plasma mass spectrometry. Water quality (hardness, conductivity, dissolved oxygen, and pH) of the Hydra medium was measured throughout the study using standard procedures. Toxicity was assessed by recording mortality as described previously (Karntanut and Pascoe, 2000, 2002). 2.5. Data analysis Mortality data were examined by time–response analysis (Litchfield, 1949) to determine median lethal times (LT50) and median lethal concentrations (LC50). In addition, the slope functions and reaction times (LT50s) derived from the probit response lines were compared at each concentration. Toxicity data analyses were based upon measured, rather than nominal, metal concentrations.
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3. Results All the control aposymbiotic and symbiotic Hydra survived. Water quality parameters (7SD) recorded for the Hydra medium during the study were hardness (20770.5 mg/L as CaCO3), conductivity (49070.4 mS/ cm), pH (7.770.26), and DO (7.270.7 mg/L). The pH of test solutions was 7.8 for copper and cadmium and 6.2 for zinc solutions. Recorded metal concentrations are shown in Table 1. A comparison of toxicity curves relating LT50 and exposure concentrations (Fig. 1) clearly shows that copper is the most toxic of the metals, followed by cadmium and then zinc. It is also evident that the patterns of mortality are very similar for symbiotic and aposymbiotic animals. Time–response analysis, comparing median lethal times (LT50) and slope functions of the probit response lines at each concentration (Table 1), shows no consistent significant difference between aposymbiotic and symbiotic Hydra exposed to the three metals. LC50 values (Table 2) extrapolated from the time–response toxicity curves for symbiotic and aposymbiotic Hydra at various exposure times confirm that the responses of symbiotic and aposymbiotic animals are similar and also that the relative toxicity is copper4cadmium4zinc. However, in the case of copper, it can be seen from Fig. 1 and Table 1 that at the lower concentrations there is a difference between the two group of polyps, with aposymbiotic animals dying at concentrations where symbiotic Hydra survived. The lowest observed effect concentrations were 0.0068 and 0.016 mg/L for aposymbiotic and symbiotic Hydra, respectively.
4. Discussion It has been known since the work of Goetsch (1924) and confirmed by Muscatine and Lenhoff (1965) that symbiotic algae aid the growth of stressed H. viridissima because when the available food supply is limited, Hydra from which the algae have been removed will survive for a much shorter time than green animals. However, wellfed albino Hydra grow as well as green animals. When Hydra are grown in continuous darkness with less maltose available from photosynthesis, the algae depend more upon Hydra-derived metabolites and so polyps containing the symbionts grow more slowly than aposymbiotic Hydra, which do not have to support the growth of algae (Douglas and Smith, 1983). Under normal conditions the algal population within H. viridissima remains relatively constant but with environmental change the polyp may regulate the number of algae per digestive cell either by ejection of excess algae or more likely by controlling their proliferation rate within the cells, possibly through inhibition of algal
303
mitosis (Pardy and Muscatine, 1973; McAuley, 1981). In the present study no algal expulsion was observed from symbiotic Hydra after exposure to heavy metals. It is possible that the metabolic balance between the two symbionts could also be disrupted by the presence of pollutants, resulting in toxic effects not seen in other Hydra without symbiotic algae. For example, Karntanut and Pascoe (2002) argued that damage to the symbiotic algae, caused by metals such as copper and cadmium, which are known to be toxic to algae, could deprive Hydra of some of the benefits normally derived from its symbionts. This may have resulted in a reallocation of resources within the polyp’s energy budget and a reduced ability to deal with the toxicant itself leading to the greater metal sensitivity, compared to other Hydra species, recorded in their study (i.e., the metal would damage/remove the algae, effectively creating aposymbiotic animals, and these would therefore be more sensitive). The results from the current investigation, though, do not initially appear to provide evidence in support of such an hypothesis, since there is generally no significant difference in reaction times (LT50s) between polyps with and without endosymbiotic algae. However, the data do show that when Hydra are exposed to low concentrations of copper, the absence of algae (i.e., in the aposymbiotic animals) does appear to increase toxicity, since animals die at concentrations where symbiotic polyps do not. At low concentrations the copper taken up by symbiotic Hydra may be sequestered by the algae (as shown for free-living Chlorella by Tien, 2002), providing a degree of protection for the polyp itself. Therefore the greater sensitivity of aposymbiotic Hydra to copper at low concentrations could be attributed to their lack of symbiotic algae. At the higher concentrations it is probable that any defence systems are overwhelmed by the toxicant effect and any slight benefit derived from the endosymbiotic algae is of little consequence. Such an effect would not be expected with cadmium, which plays no essential role in either species, and in the case of zinc it seems that the concentrations examined were all too high for such a protective mechanism to operate. The physiological interrelationships occurring in symbioses are complex and subject to disturbance by stressors such as toxic chemicals. However, experimental manipulation of the symbionts, such as described in this study, can help to explain their interaction and the ways in which they respond to pollutants.
5. Conclusions Experimental manipulation to remove symbiotic green algae from the endodermal cells of H. viridissima made it possible to compare the toxicity of the heavy metals copper, cadmium, and zinc to the same species of
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Table 1 A comparison of slope functions and reaction times (LT50) from Fig. 1 at each concentration of copper, cadmium, and zinc for aposymbiotic and symbiotic Hydra Actual concentration7SD (mg/L) Copper 0.006870.001 0.009670.001 0.01670.000 0.03370.004 0.0570.009 0.0970.007 0.1670.008 Cadmium 0.2870.006 0.3570.021 0.8170.021 1.8970.035 3.8770.424 Zinc 0.6670.454 0.8170.599 1.0470.865 1.7170.391 4.2270.863 21.4871.990 28.7371.672 36.7771.686 54.9070.728
Slope function for aposymbiotic Hydra (95% confidence limits in parentheses)
Slope function for symbiotic Hydra (95% confidence limits in parentheses)
Statistical significance for slope function comparison
Statistical significance for LT50 comparison
1.11 1.87 1.55 1.21 1.15 2.58
(1.17–1.06) (2.50–1.40) (1.93–1.25) (1.33–1.11) (1.23–1.08) (3.99–1.66)
1.39 1.11 1.21 3.86
(1.61–1.19) (1.17–1.06) (1.32–1.11) (7.21–2.07)
1.16 1.05 1.12 1.04 1.25
(1.24–1.08) (1.08–1.03) (1.17–1.06) (1.05–1.02) (1.38–1.13)
1.37 1.09 1.08 1.14 1.29
(1.59–1.19) (1.14–1.05) (1.13–1.04) (1.22–1.07) (1.46–1.15)
1.16 1.10 1.11 1.58 1.25 1.37 1.31 1.18 1.29
(1.25–1.09) (1.01–1.00) (1.16–1.06) (1.95–1.28) (1.39–1.13) (1.58–1.18) (1.49–1.16) (1.28–1.09) (1.45–1.15)
1.07 1.04 1.08 1.61 2.09 1.77 1.72 1.64 1.47
(1.11–1.04) (1.06–1.02) (1.12–1.04) (2.01–1.29) (2.94–1.49) (2.30–1.36) (2.21–1.34) (2.05–1.30) (1.75–1.23)
10% mortality, so no LT50 or slope function could be recorded. No mortality, so no LT50 or slope function could be recorded. Significant difference Pp0:05 (Litchfield, 1949).
1000 Copper Zinc
100 LT50 (h)
Cadmium
10 Aposymbiotic Hydra Symbiotic Hydra
1 0.001
0.01
0.1
1
10
100
Concentration (mg/L)
Fig. 1. A comparison of toxicity curves, relating median lethal time (LT50) and metal concentration, for aposymbiotic and symbiotic Hydra exposed to copper, cadmium, and zinc.
Hydra under symbiotic and aposymbiotic conditions. It was found that the symbiotic Hydra were more resistant to copper, though not to the other metals, and it is
suggested that they derive some benefit from the association, which enables them to better tolerate the toxicant.
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Table 2 Median lethal concentrations (LC50) of copper, cadmium, and zinc extrapolated from time-response toxicity curves Metal
LC50 (mg/L) 24 h
Copper Cadmium Zinc a
48 h
72 h
96 h
Aposymbiotic Hydra
Symbiotic Hydra
Aposymbiotic Hydra
Symbiotic Hydra
Aposymbiotic Hydra
Symbiotic Hydra
Aposymbiotic Hydra
Symbiotic Hydra
0.064 0.36 54
0.058 0.43 33
0.043 0.14 12
0.038 0.16 7.4
0.035 —a 5.2
0.032 —a 3.8
0.031 —a 3.5
0.028 —a 2.5
No observations at these times.
Acknowledgments This work was partly funded by the Royal Thai Government.
References Andersen, R.A., Wiger, R., Daae, H.L., Eriksen, K.D., 1988. Is the metal binding protein metallothionein present in the coelenterate Hydra attenuata? Comp. Biochem. Physiol. C 91 (2), 553–557. Beach, M.J., Pascoe, D., 1998. The role of Hydra vulgaris (Pallas) in assessing the toxicity of freshwater pollutants. Water Res. 32, 101–106. Benson, B., Boush, G.M., 1983. Effect of pesticides and PCBs on budding rates of green Hydra. Bull. Environ. Contam. Toxicol. 30, 344–350. Bossert, P., Dunn, K., 1986. Regulation of intracellular algae by various strains of the symbiotic Hydra viridissima. J. Cell Sci. 85, 187–195. Douglas, A.E., Huss, V.A.R., 1986. On the characteristics and taxonomic position of symbiotic Chlorella. Arch. Microbiol. 145, 80–84. Douglas, A.E., Smith, D.C., 1983. The cost of symbionts to their hosts in green Hydra. In: Schenk, H.E.A., Schwemmler, W. (Eds.), Endocytobiology. de Gruyter, Berlin, pp. 631–648. Goetsch, W., 1924. Die symbiose der su¨sswasser-hydroiden und ihre ku¨nstliche beeinflussung. Z. Morph. O¨kol. Tierre 1, 660–731. Holdway, D., Lok, K., Semaan, M., 2001. The acute and chronic toxicity of cadmium and zinc to two Hydra species. Environ. Toxicol. 16 (6), 557–565. Hyne, R., Rippon, G.D., Ellender, G., 1993. Investigation of uraniuminduced toxicity in freshwater Hydra. In: Dallinger, R., Rainbow, P. (Eds.), Ecotoxicology of Metals in Invertebrates. CRC Press, Boca Raton, FL, pp. 149–173. Kalafatic, M., Kovacevic, G., Ljubesic, N., Sunjic, H., 2001. Effects of ciprofloxacin on green Hydra and endosymbiotic alga. Periodic Biol. 103, 267–272. Karntanut, W., Pascoe, D., 2000. A comparison of methods for measuring acute toxicity to Hydra vulgaris. Chemosphere 41 (10), 149–154. Karntanut, W., Pascoe, D., 2002. The toxicity of copper, cadmium and zinc to four different Hydra. Chemosphere 47 (10), 1059–1064.
Kessler, E., Huss, V.A.R., Rahat, M., 1988. Species-specific ability of Chlorella strains (Chlorophyceae) to form stable symbioses with Hydra viridis. Plant Syst. Evol. 160, 241–246. Kovacevic, G., Kalafatic, M., Ljubesic, N., Sunjic, H., 2001. The effect of chloramphenicol on the symbiosis between alga and Hydra. Biologia 56, 605–610. Lenhoff, H.M., 1983. Culturing large numbers of Hydra. In: Lenhoff, H.M. (Ed.), Hydra Research Methods. Plenum, New York, pp. 53–62. Litchfield, J.T., 1949. A method for the rapid graphic solution of time–percentage effect curves. J. Pharm. Exp. Ther. 97, 399–408. McAuley, P.J., 1981. Ejection of algae in the green Hydra symbiosis. J. Exp. Zool. 217, 23–31. McAuley, P.J., 1987. Quantitative estimation of movement of an amino acid from host to Chlorella symbionts in green Hydra. Biol. Bull. 173, 504–512. McAuley, P.J., Dorling, M., Hodge, H., 1996. Effect of maltose release on uptake and assimilation of ammonium by symbiotic Chlorella (Chlorophyta). J. Phycol. 32, 839–846. Muscatine, L., Lenhoff, H.M., 1965. Symbiosis of Hydra and algae. II. Effects of limited food and starvation on growth of symbiotic and aposymbiotic Hydra. Biol. Bull. 129, 316–328. Pardy, R.L., 1983. Preparing aposymbiotic algae. In: Lenhoff, H.M. (Ed.), Hydra Research Methods. Plenum, New York, pp. 393–397. Pardy, R.L., Muscatine, L., 1973. Recognition of symbiotic algae by green Hydra. A quantitative study of the uptake of living algae by aposymbiotic H. viridis. Biol. Bull. 145, 565–579. Pardy, R.L., White, B.N., 1977. Metabolic relationships between green Hydra and its symbiotic algae. Biol. Bull. 153, 228–236. Pascoe, D., Carroll, K., Karntanut, W., Watts, M.M., 2002. Toxicity of 17a ethinylestradiol and bisphenol A to the freshwater cnidarian Hydra vulgaris. Arch. Environ. Contam. Toxicol. 43 (1), 56–63. Pollino, C.A., Holdway, D.A., 1999. Potential of two Hydra species as standard toxicity test animals. Ecotoxicol. Environ. Saf. 43, 309–316. Pyatt, F.B., Dodd, N.M., 1986. Some effects of metal ions on the freshwater organisms, Hydra oligactis and Chlorohydra viridissima. Ind. J. Exp. Biol. 24, 169–173. Thorington, G., Margulis, L., 1981. Hydra viridis: transfer of metabolites between Hydra and symbiotic algae. Biol. Bull. 160 (1), 175–188. Tien, C.-J., 2002. Biosorption of metal ions by freshwater algae with different surface characteristics. Process Biochem. 38, 605–613. Whitney, D.D., 1907. Artificial removal of the green bodies of Hydra viridis. Biol. Bull. 13, 291–299.