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The potential role of mucus in the depuration of copper from the mussels Perna viridis (L.) and Septifer virgatus (Wiegmann)
Pergamon
0025-326X(95)00140-9
Marine Pollution Bulletin, Vol. 31, Nos 4-12, pp. 390--393, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0025-326X/95 $9.50 + 0.00
The Potential Role of Mucus in the Depuration of Copper from the Mussels Perna viridis (L.) and Septifer virgatus (Wiegnmnn) P. W. C. SZE and S. Y. LEE Department of Ecology & Biodiversity and The Swire Institute of Marine Science, The University of Hong Kong, Hong Kong
Experiments were conducted to measure the mucus secretion rate in two common mussels in Hong Kong, Perna viridis (L.) and Septifer virgatus (Wiegmann), when chronically exposed to Cu (50 pg !-1). After a 3month exposure period, the mucus production rate of P. viridis at 25°C in the metal treatment was 2.65 times that of the control (10.7 vs 4.0 mg g-1 dry wt h - l ) , while S. virgatus showed a 1.85 times difference (4.41 vs 2.38 mg g-1 dry wt h - l ) . Mucus secretion by P. viridis under acute Cu exposure (0.5 mg !-1) was significantly higher in the metal treatment than the control (13.43 vs 9.16 mg g-1 dry flesh wt). Metal contents of the mucus secreted was about 18 limes those in the control and 6 times in the soft tissues. Therefore, mucus appears to be an effective agent for Cu depuration in the mussel. The significance of these results to the local distribution and abundance of the mussels is discussed.
Trace metals occur in natural marine ecosystems in very low concentrations (Fergusson, 1990) but are capable of exerting biological effects. All such metals are considered as pollutants because of their toxicity if present above threshold concentration. Bivalve molluscs are capable of concentrating trace metals from solution, food and particulate matter in the marine environment. Enrichments over ambient seawater levels is common (e.g. Bryan, 1976; Phillips & Yim, 1981; Tan & Lim, 1984). The bio-accumulation of metals is dependent on size of the organism, chemical speciation, routes and mechanisms of uptake, intracellular eompartmentation and other aspects of cellular metal homeostasis (see review by Eisler (1979)). Elevated body burdens of trace metals have been shown to elicit changes in physiological, behavioural and cellular responses (Poulsen et al., 1982; Mathew & Menon, 1983; Calabrese et aL, 1984; Baby & Menon, 1986; Krishnakumar et al., 1990; Patel & Anthony, 1991). Recently, it has been demonstrated that an increase in mucus secretion is a significant response to heightened trace metal exposure (Scott & Major, 1972; 390
D'Silva & Kureishy, 1978; Krishnakumar et al., 1987). However, few quantitative measurements have been made on the relation between mucus secretion and metal exposure. Because of the common response of increased mucus secretion, it has been suggested that metals may be excreted in mucus and/or faecal materials as a means of detoxification (Scott & Major, 1972), but no confirmation of these excretory pathways have been published to date. The two mussels, Perna viridis and Septifer virgatus, have complementary but overlapping distributions in Hong Kong waters. Perna viridis is the dominant species recorded in Victoria Harbour and Tolo Harbour (Huang et al., 1985), both of which are highly polluted (Morton & Wu, 1975; Thompson & Shin, 1983). The population in these harbours exhibit stunted growth (Lee & Morton, 1985) and poor physiological conditions (Lee, 1985). Septifer virgatus forms a conspicuous mid-littoral band on the outer wave-swept coasts of Hong Kong (Coombes & Seed, 1992). These areas are relatively clean and free of pollution. Cape D'Aguilar, where S. virgatus dominates, for example, is considered to be one of the most metal-free sites in Hong Kong (Rainbow, 1993). Whereas wave exposure is probably the dominant factor influencing the horizontal distribution patterns of these two mussels, it is also of interest to investigate the effects of pollution on their performance. In the present study, quantitative measurements of mucus secretion in P. viridis and S. virgatus in response to Cu exposure were made. In addition, the Cu concentration in the secreted mucus from the two mussels was measured to assess the potential importance of mucus as an agent for the depuration of the metal.
Materials and Methods Mucus secretion rate Samples of P. viridis and S. virgatus were collected from Ma Liu Shui and Cape D'Aguilar, respectively. Mussels of shell length 20-30 mm were separated from the clumps by carefully cutting the byssus and all
Volume 31/Numbers 4-12/April-December 1995
epibionts were removed. The mussels were acclimatized in filtered seawater for 7 days before metal exposure. Mussels of the two species were separately exposed to 50 I~g 1-1 Cu at 25°C for 3 months in a recirculated system in the laboratory. Control sets with no Cu addition were maintained simultaneously. Seawater was changed daily to maintain the respective levels of metal exposure. The mussels were fed daily with a mixture of Thalassiorsira and lsochrysis. To measure mucus production, mussels were transfeted into a plastic tray filled with filtered seawater. A thin glass microscope slide was inserted into the mantle cavity gently when the valves were opened. The mussels were then held in air to allow the collection of the mucus produced. The slides on which mucus was deposited were rinsed with distilled water carefully and the mucus was scraped off and transferred into preweighed aluminium foil. The dry weight was obtained after drying at 80°C to constant weight. Mucus production rate was measured at 0, 0.5, 1, 2 and 3 months of metal exposure. The procedure was repeated for another set of mussels at 17°C to estimate mucus secretion when exposed to a typical winter temperature.
~5 ~6
20
..--.
16
1 "7
.25oc
12
~0 8" O
4 [
0
1
2
Time (Month) Fig. 1 Variations in mucus secretion in Perna viridis when exposed to 50 I~g I - l Cu.
20
16 Cu in secreted mucus Perna viridis of shell length 60-70 mm were collected from Ma Liu Shui, Hong Kong, using the same sampling procedure described above. After acclimatization, the mussels were acutely exposed to 0.5 mg 1-1 Cu for 24 h. To collect the mucus, mussels were transferred into plastic trays containing filtered and clean seawater in which they were allowed to recover. Mucus subsequently secreted was insoluble in the seawater (Grenon & Walker, 1980) and was picked up using a pair of forceps and rinsed in distilled water before transferring to microscope slides. The slides were dried at 80°C to constant weight for the determination of dry weight. Dry samples of mucus were removed, using stainless-steel razor blades, from the glass slides for the measurement of Cu content. Mucus samples from 15 mussels were pooled and ground to a homogeneous powder to provide an adequate quantity for analysis. The homogenate was digested in a mixture of 2 ml distilled water and 5 ml concentrated nitric acid using a microwave digestor (CEM, Charlotte, NC, USA). This method allows the acid digestion of mucus in a closed vessel using pressure-controlled microwave heating in multiple steps. The Cu content in the digested solutions was determined by flame atomic absorption spectroscopy (Varian, Mulgrave, Victoria, Australia). The concentration was read off from a calibration curve obtained from standard solutions (copper sulphate; Sigma, St. Louis, USA). Throughout the analysis, certified reference materials of known metal concentrations were used for quality control (NBS Standard Reference Material Oyster Tissues, National Bureau of Standard, Gaithersburg, MD, USA).
Results Mucus secretion rate in P. viridis and S. virgatus after different periods of Cu exposure at the two
Q 25°C 0 17°C
12 E
0
J
p
0
1 2 Time (Month)
3
Fig. 2 Variationsin mucussecretionin Septifer virgatuswhenexposed to 50 ~tg1-1 Cu. temperatures are shown in Figs 1 and 2. A similar pattern is observed for both species with only differences in magnitude. There was a pronounced increase in mucus secretion in P. viridis on exposure to Cu. A rapid increase (114%) was observed 1 month after metal had been added. The concomitant increase for S. virgatus was around 34%. At the end of the experiments, P. viridis exposed to Cu still maintained a secretion rate 58% higher than that of its counterpart in the control set. All experiments showed significantly higher (p<0.05) mucus secretion rate in Cu-exposed individuals than those in the corresponding controls. Perna viridis had a much higher mucus secretion rate than S. virgatus (p<0.005), often indicated by the foaming and frothing of the medium resulting from the vast amount of mucus produced. This was never recorded from the S. virgatus tanks. Temperature did not have a significant effect on mucus secretion (/7>0.05) in either mussels except over the first month in P. viridis. The Cu content of the mucus excreted by the mussels acutely exposed to Cu was about 18-fold greater than 391
Marine Pollution Bulletin TABLE 1 Cu concentration (ttgg- i dry wt) of secretedmucus and soft tissuesin Perna viridisunder acute C u exposure.*
0.5 mg 1-l Cu
Control
Mucus
Tissues
247.9 251.4 235.6 240.4
37.5 35.8 40.4 42.2
14.0 14.3 13.2 14.1
15.1 15.1 14.4 14.7
*Values are mean of three replicates from pooled samplesfrom 15 individuals.
that excreted by the control mussels (p <~0.001, Table 1). The mucus also contained significantly more Cu than the soft tissues of the mussels exposed to Cu.
Discussion Mucus is indispensable to many important metabolic and behavioural processes in marine molluscs. Mucus is produced and used in ingestion, including the trapping of food particles on ctenidia or palp proboscises and the transportation of food from the ctenidia to the mouth (Prezant, 1990). It is also involved in egestion, for the packing and binding of faecal materials as well as pseudofaeces. Mucus helps reduce desiccation when animals are exposed to air (Grenon & Walker, 1980). Recently, pedal mucus production by gastropods has been a subject of much interest (Davies et al., 1990, 1992a,b). In contrast, few studies have been made on mucus production in bivalves (Aiello et al., 1988). Tan & Lim (1984) reported that the first visible symptom of the toxic effect of Pb on P. viridis was an increase in mucus production to result in foaming and frothing of the water. A similar response was observed in the present study. Scott & Major (1972) recorded that 0.3 mg 1-1 Cu stimulated copious mucus secretion in Mytilus edulis, while D'Silva & Kureishy (1978) reported upon a similar response in M. viridis (--P. viridis) at even 0.01 mg 1-l Cu. The increase in mucus production in treated mussels is probably due to an increase in the number of mucus glands (Tan & Lim, 1984). However, there are wide interspecific differences in the level of response. Septifer virgatus, in the present study, was found to produce significantly less mucus compared with P. viridis, in spite of its higher body burden of Cu than the latter species; Morton (1987) has commented that P. viridis has an unusually high mucus secretion rate. This may be explained by the smaller number of mucus glands in S. virgatus than P. viridis. High tissue Cu concentration may cause destructive damage on the mucus glands. Hietanen et al. (1988) reported that a high concentration of Zn caused swelling and degeneration of mucus-secreting ceils in M. edulis. Temperature did not have a significant effect on mucus secretion in the present study, only P. viridis showed a 20% increase when temperature rose from 17 392
to 25°C. Metabolic rate and accumulation of metal from seawater probably increases with temperature and may also cause an increase in the number of mucus glands and the amount of mucus secreted per gland. Phillips (1976a,b) suggested that M. edulis should not be used as an indicator for Cu in the marine environment because its accumulation of Cu is influenced by many factors, such as water depth, temperature and salinity. Bryan (1976) pointed out that some species are able to excrete a higher proportion of the metal taken in and thereby regulate their concentrations in the body to a fairly normal level. Sivalingam & Bhaskaran (1980) found that peak Cu accumulation in P. viridis was attained after 12 h of exposure followed by a regulation mechanism leading to stable levels on prolonged exposure. Scott & Major (1972) indicated that Cu accumulated by M. edulis was excreted in the mucus in a non-soluble form. The hypothesis that an increase in mucus secretion with increasing metal exposure is an attempt by the mussels to ameliorate pollutant effects was doubted in the past as little information was available on the metal content of the secreted mucus. Scott & Major (1972) tried to analyse the level of Cu in secreted mucus but failed because of the formation of a stable emulsion during extraction. The present study found that Cu content of the mucus secreted by the treated mussels was about 6fold higher than that of the tissues, while there was no difference between the two in the control mussels. This suggests that Cu ions were either pre-concentrated in the mucus before transportation to other parts of the body, as described by Bryan (1976), or the absorbed Cu ions in the tissues were accumulated in mucus which acts as a storage site. The former pathway seems to be more plausible, as mucus has been suggested as the substance responsible for sequestering both particulate and soluble forms of metals from solution (Romeril, 1971; Hameed & Mohan Raj, 1990) and the mussels were only exposed to Cu for 24 h in the present experiment. Pentreath (1973) recorded that Fe 59 was associated with the mucus covering of the gills in M. edulis, while Koringa (1952) found that cations can be absorbed on the mucus secreted by the gills of Crassostrea virginica. Bryan (1976) suggested that pre-concentration of metals by attachment to mucus may promote their diffusion through the body surface or may enhance absorption if the mucus is eventually ingested or allowed to stay on the surface of tissues without being removed. In the present study, the secreted mucus also contained significantly higher Cu content ratios than the control. Therefore, increase in mucus secretion and subsequent loss of the mucus back to the water will help regulate and maintain metal levels in the tissues. Mucus, then, appears to be an effective agent in the depuration of metals in the mussels, especially P. viridis. Such differences in the mucus secretion response may partly account for the difference in horizontal distribution of the two mussels in relation to pollution intensity. This research is supported by a grant from the Universityof Hong Kong Committeeon Researchand ConferenceGrants.
Volume 31/Numbers 4-12/April-December 1995 Aiello, E., Hager, E., Laing, K. & Meenan, G. (1988). Opioid modulation of a dopaminergic system for stimulating mucus secretion and transport in the mussel Mytilus edulis. Physiol. Zool. 61, 41-49. Baby, K. V. & Menon, N. R. (1986). Oxygen uptake in the brown mussel, Perna indica (Kuriakose & Nair) under sublethal stress of Hg, Cd and Zn. Indian J. Mar. Sci. 15, 127-128. Bryan, G. W. (1976). Some aspects of heavy metal tolerance in aquatic organisms. In Effects of Pollutants on Aquatic Organisms (A. P. M. Lockwood, ed.), pp. 7-34. Cambridge University Press, London, UK. Calabrese, A., Maclnnes, J. R., Nelson, D. A., Greig, R. A. & Yevich, P. P. (1984). Effects of long-term exposure to silver or copper on growth, bioaccumulation and histopathology in the blue mussel. Mytilus edulis. Mar. Environ. Res. 11, 253-274. Coombes, M. R. A. & Seed, R. (1992). Predation of the black mussel Septifer virgatus by the red-eyed crab Eriphia laeviormana smithii (Xanthidae). Asian Mar. Biol. 9, 245-258. Davies, M. S., Hawkins, S. J. & Jones, H. D. (1990). Mucus production and physiological energetics in Patella vulgata L. J. Moll. Stud. 56, 499-503. Davies, M. S., Hawkins, S. J. & Jones, H. D. (1992a). Pedal mucus and its influence on the microbial food supply of two intertidal gastropod, Patella vulgata L. and Littorina littorea (L). J. Exp. Mar. Biol. EcoL 161, 57-77. Davies, M. S., Jones, H. D. & Hawkins, S. J. (1992b). Physical factors affecting the fate of pedal mucus produced by the common limpet. Patella vulgata. J. Mar. Biol. Assoc. UK 72, 633--643. D'Silva, C. & Kureishy, T. W. (1978). Experimental studies on the accumulation of copper and zinc in the green mussel. Mar. Pollut. Bull. 9, 187-190. Eisler, R. (1979). Copper accumulations in coastal and marine biota. In Copper in the Environment (J. O. Niragn, ed.), pp. 383-449. Wiley, New York, USA. Fergusson, J. E. (1990). The Heavy Elements: Chemistry, Environmental Impacts and Health Effects, pp. 243-328. Pergamon Press, Oxford, UK. Grenon, J. F. & Walker, G. (1980). Biochemical and rheological properties of the pedal mucus of the limpets, Patella vulgata L. Comp. Biochem. PhysioL 66, 451-458. Hameed, P. S. & Mohan Raj, A. I. (1990). Freshwater mussel, Lamellidens marginalis (Lamarch) (Mollusca: Bivalvia: Unionidae) as an indicator of river pollution. Chem. Ecol. 4, 57-64. Hietanen, B., Sunila, I. & Kristoffersson, R. (1988). Toxic effects of zinc on the common mussels Mytilus edulis L. (Bivalvia) in brackish water. I. Physiological and histopathological studies. Ann. Zool. Fenn. 25, 341-347. Huang, Z. G., Lee, S. Y. & Mak, P. M. S. (1985). The distribution and population structure of Perna viridis (Bivalvia: Mytilaeea) in Hong Kong waters. In Proceedings of the Second International Workshop on the Malacofauna of Hong Kong and Southern China (B. S. Morton & D. Dudgeon, eds), pp. 465--472. Hong Kong University Press, Hong Kong. Koringa, P. (1952). Recent advances in oyster biology. Quart. Rev. Biol. 27, 266-308. Krishnakumar, P. K., Damodaran, R. & Nambisan, P. N. K. (1987). Acute toxicity of selected heavy metals to green mussel Perna viridis (L). Indian J. Mar. Sci. 16, 363-264.
Krishnakumar, P. K., Asokan, P. K. & Pillai, V. K. (1990). Physiological and cellular responses to copper and mercury in the green mussel Perna viridis (Linnaeus). Aquat. Toxicol, 18, 163-174. Lee, S. Y. (1985). The population dynamics, growth and reproduction of Perna viridis (Linnaeus, 1758) (Bivalvia: Mytilacea) in Hong Kong. MPhil thesis, The University of Hong Kong, Hong Kong. Lee, S. Y. & Morton, B. S. (1985). Hong Kong Mytilidae. In Proceedings of the Second International Workshop on the Malacofauna of Hong Kong and Southern China (13. S. Morton & D. Dudgeon, eds), pp. 49-76. Hong Kong UniversityPress, Hong Kong. Mathew, R. & Menon, N. R. (1983). Oxygen consumption in tropical bivalves Perna viridis (Lima.) and Meretrix easta (Chem.) exposed to heavy metal. Indian J. Mar. Sci. 12, 57-59. Morton, B. (1987). The functional morphology of the organs of the mantle cavity of Perna viridis (Linnaeus, 1758) (Bivalvia: Mytilaeea). Am. Malaeol. Bull. 5, 159-164. Morton, B. & Wu, S. S. (1975). The hydrology of the coastal waters of Hong Kong. Environ. Res. 10, 319-347. Patel, B. & Anthony, K. (1991). Uptake of cadmium in tropical marine lamellibranchs, and effects on physiological behaviour. Mar. Biol. 108, 457-470. Pentreath, R. J. (1973). The accumulation from water of Zn 65, Mn 54, Co 5s and Fe 59 by the mussel, Mytilus edulis. J. Mar. Biol. Assoc. UK 53, 127-143. Phillips, D. J. H. (1976a). The common mussel Mytilus edulis as an indicator of pollution by zinc, cadmium, lead and copper. I. Effects of environmental variables on uptake metals. Mar. Biol. 38, 59-69. Phillips, D. J. H. (1976b). The common mussel Mytilua edulis as an indicator of pollution by zinc, cadmium, lead and copper. II, Relationship of metals in the mussel to those discharged by industry. Mar. Biol. 38, 71-80. Phillips, D. J. H. & Yim, W. W. S. (1981). A comparative evaluation of oysters, mussels and sediments as indicators of trace metals in Hong Kong waters. Mar. Ecol. Progr. Ser. 6, 285-293. Poulsen, E., Riisgard, H. U. & Mohlenberg, F. M. (1982). Accumulation of cadmium and bioenergetics in the mussel Mytilus edulis. Mar. Biol. 68, 25-29. Prezant, R. S. (1990). Form, function and phylogeny of bivalve mucins. In The BivalviamThe Proceedings of a Memorial Symposium in Honour of Sir Charles Maurice Yonge, Edinburgh, 1986 (B. Morton, ed.), pp. 83-95. Hong Kong University Press, Hong Kong. Rainbow, P. S. (1993). The significance of trace metal concentrations in marine invertebrates. In Ecotoxicology of Metals in Invertebrates (R. Dallinger & P. S. Rainbow, eds), pp. 3-23. SETAC Special Publication Series, Lewis Publishers, Boca Raton, FL, USA. Romeril, M. G. (1971). The uptake and distribution of Zn 65 in oyster. Mar. Biol. 9, 347-354. Scott, D. M. & Major, C. W. (1972). The effect of copper (II) on survival, respiration, and heart rate in the common blue mussel, Mytilus edulis. Biol. Bull. 143, 679--688. Sivalingam, P. M. & Bhaskaran, B. (1980). Experimental insight of trace metal environmental pollution problems in mussel farming. Aquaculture 20, 291-303. Tan, W. H. & Lim, L. H. (1984). The tolerance to and uptake of lead in the green mussel, Perna viridis. Aquaculture 42, 317-332. Thompson, G. W. & Shin, P. K. S. (1983). Sewage pollution and the infannal benthos of Victoria Harbour, Hong Kong. J. Exp. Mar. Biol. Ecol. 67, 279-299.
393
0025-326X(95)00140-9
Marine Pollution Bulletin, Vol. 31, Nos 4-12, pp. 390--393, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0025-326X/95 $9.50 + 0.00
The Potential Role of Mucus in the Depuration of Copper from the Mussels Perna viridis (L.) and Septifer virgatus (Wiegnmnn) P. W. C. SZE and S. Y. LEE Department of Ecology & Biodiversity and The Swire Institute of Marine Science, The University of Hong Kong, Hong Kong
Experiments were conducted to measure the mucus secretion rate in two common mussels in Hong Kong, Perna viridis (L.) and Septifer virgatus (Wiegmann), when chronically exposed to Cu (50 pg !-1). After a 3month exposure period, the mucus production rate of P. viridis at 25°C in the metal treatment was 2.65 times that of the control (10.7 vs 4.0 mg g-1 dry wt h - l ) , while S. virgatus showed a 1.85 times difference (4.41 vs 2.38 mg g-1 dry wt h - l ) . Mucus secretion by P. viridis under acute Cu exposure (0.5 mg !-1) was significantly higher in the metal treatment than the control (13.43 vs 9.16 mg g-1 dry flesh wt). Metal contents of the mucus secreted was about 18 limes those in the control and 6 times in the soft tissues. Therefore, mucus appears to be an effective agent for Cu depuration in the mussel. The significance of these results to the local distribution and abundance of the mussels is discussed.
Trace metals occur in natural marine ecosystems in very low concentrations (Fergusson, 1990) but are capable of exerting biological effects. All such metals are considered as pollutants because of their toxicity if present above threshold concentration. Bivalve molluscs are capable of concentrating trace metals from solution, food and particulate matter in the marine environment. Enrichments over ambient seawater levels is common (e.g. Bryan, 1976; Phillips & Yim, 1981; Tan & Lim, 1984). The bio-accumulation of metals is dependent on size of the organism, chemical speciation, routes and mechanisms of uptake, intracellular eompartmentation and other aspects of cellular metal homeostasis (see review by Eisler (1979)). Elevated body burdens of trace metals have been shown to elicit changes in physiological, behavioural and cellular responses (Poulsen et al., 1982; Mathew & Menon, 1983; Calabrese et aL, 1984; Baby & Menon, 1986; Krishnakumar et al., 1990; Patel & Anthony, 1991). Recently, it has been demonstrated that an increase in mucus secretion is a significant response to heightened trace metal exposure (Scott & Major, 1972; 390
D'Silva & Kureishy, 1978; Krishnakumar et al., 1987). However, few quantitative measurements have been made on the relation between mucus secretion and metal exposure. Because of the common response of increased mucus secretion, it has been suggested that metals may be excreted in mucus and/or faecal materials as a means of detoxification (Scott & Major, 1972), but no confirmation of these excretory pathways have been published to date. The two mussels, Perna viridis and Septifer virgatus, have complementary but overlapping distributions in Hong Kong waters. Perna viridis is the dominant species recorded in Victoria Harbour and Tolo Harbour (Huang et al., 1985), both of which are highly polluted (Morton & Wu, 1975; Thompson & Shin, 1983). The population in these harbours exhibit stunted growth (Lee & Morton, 1985) and poor physiological conditions (Lee, 1985). Septifer virgatus forms a conspicuous mid-littoral band on the outer wave-swept coasts of Hong Kong (Coombes & Seed, 1992). These areas are relatively clean and free of pollution. Cape D'Aguilar, where S. virgatus dominates, for example, is considered to be one of the most metal-free sites in Hong Kong (Rainbow, 1993). Whereas wave exposure is probably the dominant factor influencing the horizontal distribution patterns of these two mussels, it is also of interest to investigate the effects of pollution on their performance. In the present study, quantitative measurements of mucus secretion in P. viridis and S. virgatus in response to Cu exposure were made. In addition, the Cu concentration in the secreted mucus from the two mussels was measured to assess the potential importance of mucus as an agent for the depuration of the metal.
Materials and Methods Mucus secretion rate Samples of P. viridis and S. virgatus were collected from Ma Liu Shui and Cape D'Aguilar, respectively. Mussels of shell length 20-30 mm were separated from the clumps by carefully cutting the byssus and all
Volume 31/Numbers 4-12/April-December 1995
epibionts were removed. The mussels were acclimatized in filtered seawater for 7 days before metal exposure. Mussels of the two species were separately exposed to 50 I~g 1-1 Cu at 25°C for 3 months in a recirculated system in the laboratory. Control sets with no Cu addition were maintained simultaneously. Seawater was changed daily to maintain the respective levels of metal exposure. The mussels were fed daily with a mixture of Thalassiorsira and lsochrysis. To measure mucus production, mussels were transfeted into a plastic tray filled with filtered seawater. A thin glass microscope slide was inserted into the mantle cavity gently when the valves were opened. The mussels were then held in air to allow the collection of the mucus produced. The slides on which mucus was deposited were rinsed with distilled water carefully and the mucus was scraped off and transferred into preweighed aluminium foil. The dry weight was obtained after drying at 80°C to constant weight. Mucus production rate was measured at 0, 0.5, 1, 2 and 3 months of metal exposure. The procedure was repeated for another set of mussels at 17°C to estimate mucus secretion when exposed to a typical winter temperature.
~5 ~6
20
..--.
16
1 "7
.25oc
12
~0 8" O
4 [
0
1
2
Time (Month) Fig. 1 Variations in mucus secretion in Perna viridis when exposed to 50 I~g I - l Cu.
20
16 Cu in secreted mucus Perna viridis of shell length 60-70 mm were collected from Ma Liu Shui, Hong Kong, using the same sampling procedure described above. After acclimatization, the mussels were acutely exposed to 0.5 mg 1-1 Cu for 24 h. To collect the mucus, mussels were transferred into plastic trays containing filtered and clean seawater in which they were allowed to recover. Mucus subsequently secreted was insoluble in the seawater (Grenon & Walker, 1980) and was picked up using a pair of forceps and rinsed in distilled water before transferring to microscope slides. The slides were dried at 80°C to constant weight for the determination of dry weight. Dry samples of mucus were removed, using stainless-steel razor blades, from the glass slides for the measurement of Cu content. Mucus samples from 15 mussels were pooled and ground to a homogeneous powder to provide an adequate quantity for analysis. The homogenate was digested in a mixture of 2 ml distilled water and 5 ml concentrated nitric acid using a microwave digestor (CEM, Charlotte, NC, USA). This method allows the acid digestion of mucus in a closed vessel using pressure-controlled microwave heating in multiple steps. The Cu content in the digested solutions was determined by flame atomic absorption spectroscopy (Varian, Mulgrave, Victoria, Australia). The concentration was read off from a calibration curve obtained from standard solutions (copper sulphate; Sigma, St. Louis, USA). Throughout the analysis, certified reference materials of known metal concentrations were used for quality control (NBS Standard Reference Material Oyster Tissues, National Bureau of Standard, Gaithersburg, MD, USA).
Results Mucus secretion rate in P. viridis and S. virgatus after different periods of Cu exposure at the two
Q 25°C 0 17°C
12 E
0
J
p
0
1 2 Time (Month)
3
Fig. 2 Variationsin mucussecretionin Septifer virgatuswhenexposed to 50 ~tg1-1 Cu. temperatures are shown in Figs 1 and 2. A similar pattern is observed for both species with only differences in magnitude. There was a pronounced increase in mucus secretion in P. viridis on exposure to Cu. A rapid increase (114%) was observed 1 month after metal had been added. The concomitant increase for S. virgatus was around 34%. At the end of the experiments, P. viridis exposed to Cu still maintained a secretion rate 58% higher than that of its counterpart in the control set. All experiments showed significantly higher (p<0.05) mucus secretion rate in Cu-exposed individuals than those in the corresponding controls. Perna viridis had a much higher mucus secretion rate than S. virgatus (p<0.005), often indicated by the foaming and frothing of the medium resulting from the vast amount of mucus produced. This was never recorded from the S. virgatus tanks. Temperature did not have a significant effect on mucus secretion (/7>0.05) in either mussels except over the first month in P. viridis. The Cu content of the mucus excreted by the mussels acutely exposed to Cu was about 18-fold greater than 391
Marine Pollution Bulletin TABLE 1 Cu concentration (ttgg- i dry wt) of secretedmucus and soft tissuesin Perna viridisunder acute C u exposure.*
0.5 mg 1-l Cu
Control
Mucus
Tissues
247.9 251.4 235.6 240.4
37.5 35.8 40.4 42.2
14.0 14.3 13.2 14.1
15.1 15.1 14.4 14.7
*Values are mean of three replicates from pooled samplesfrom 15 individuals.
that excreted by the control mussels (p <~0.001, Table 1). The mucus also contained significantly more Cu than the soft tissues of the mussels exposed to Cu.
Discussion Mucus is indispensable to many important metabolic and behavioural processes in marine molluscs. Mucus is produced and used in ingestion, including the trapping of food particles on ctenidia or palp proboscises and the transportation of food from the ctenidia to the mouth (Prezant, 1990). It is also involved in egestion, for the packing and binding of faecal materials as well as pseudofaeces. Mucus helps reduce desiccation when animals are exposed to air (Grenon & Walker, 1980). Recently, pedal mucus production by gastropods has been a subject of much interest (Davies et al., 1990, 1992a,b). In contrast, few studies have been made on mucus production in bivalves (Aiello et al., 1988). Tan & Lim (1984) reported that the first visible symptom of the toxic effect of Pb on P. viridis was an increase in mucus production to result in foaming and frothing of the water. A similar response was observed in the present study. Scott & Major (1972) recorded that 0.3 mg 1-1 Cu stimulated copious mucus secretion in Mytilus edulis, while D'Silva & Kureishy (1978) reported upon a similar response in M. viridis (--P. viridis) at even 0.01 mg 1-l Cu. The increase in mucus production in treated mussels is probably due to an increase in the number of mucus glands (Tan & Lim, 1984). However, there are wide interspecific differences in the level of response. Septifer virgatus, in the present study, was found to produce significantly less mucus compared with P. viridis, in spite of its higher body burden of Cu than the latter species; Morton (1987) has commented that P. viridis has an unusually high mucus secretion rate. This may be explained by the smaller number of mucus glands in S. virgatus than P. viridis. High tissue Cu concentration may cause destructive damage on the mucus glands. Hietanen et al. (1988) reported that a high concentration of Zn caused swelling and degeneration of mucus-secreting ceils in M. edulis. Temperature did not have a significant effect on mucus secretion in the present study, only P. viridis showed a 20% increase when temperature rose from 17 392
to 25°C. Metabolic rate and accumulation of metal from seawater probably increases with temperature and may also cause an increase in the number of mucus glands and the amount of mucus secreted per gland. Phillips (1976a,b) suggested that M. edulis should not be used as an indicator for Cu in the marine environment because its accumulation of Cu is influenced by many factors, such as water depth, temperature and salinity. Bryan (1976) pointed out that some species are able to excrete a higher proportion of the metal taken in and thereby regulate their concentrations in the body to a fairly normal level. Sivalingam & Bhaskaran (1980) found that peak Cu accumulation in P. viridis was attained after 12 h of exposure followed by a regulation mechanism leading to stable levels on prolonged exposure. Scott & Major (1972) indicated that Cu accumulated by M. edulis was excreted in the mucus in a non-soluble form. The hypothesis that an increase in mucus secretion with increasing metal exposure is an attempt by the mussels to ameliorate pollutant effects was doubted in the past as little information was available on the metal content of the secreted mucus. Scott & Major (1972) tried to analyse the level of Cu in secreted mucus but failed because of the formation of a stable emulsion during extraction. The present study found that Cu content of the mucus secreted by the treated mussels was about 6fold higher than that of the tissues, while there was no difference between the two in the control mussels. This suggests that Cu ions were either pre-concentrated in the mucus before transportation to other parts of the body, as described by Bryan (1976), or the absorbed Cu ions in the tissues were accumulated in mucus which acts as a storage site. The former pathway seems to be more plausible, as mucus has been suggested as the substance responsible for sequestering both particulate and soluble forms of metals from solution (Romeril, 1971; Hameed & Mohan Raj, 1990) and the mussels were only exposed to Cu for 24 h in the present experiment. Pentreath (1973) recorded that Fe 59 was associated with the mucus covering of the gills in M. edulis, while Koringa (1952) found that cations can be absorbed on the mucus secreted by the gills of Crassostrea virginica. Bryan (1976) suggested that pre-concentration of metals by attachment to mucus may promote their diffusion through the body surface or may enhance absorption if the mucus is eventually ingested or allowed to stay on the surface of tissues without being removed. In the present study, the secreted mucus also contained significantly higher Cu content ratios than the control. Therefore, increase in mucus secretion and subsequent loss of the mucus back to the water will help regulate and maintain metal levels in the tissues. Mucus, then, appears to be an effective agent in the depuration of metals in the mussels, especially P. viridis. Such differences in the mucus secretion response may partly account for the difference in horizontal distribution of the two mussels in relation to pollution intensity. This research is supported by a grant from the Universityof Hong Kong Committeeon Researchand ConferenceGrants.
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