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Field application of lysosomal destabilisation indices in the mussel Mytilus galloprovincialis: biomonitoring and transplantation in the Lagoon of Venice (north-east Italy)
Marine Environmental Research 54 (2002) 817–822 www.elsevier.com/locate/marenvrev
Field application of lysosomal destabilisation indices in the mussel Mytilus galloprovincialis: biomonitoring and transplantation in the Lagoon of Venice (north-east Italy) L. Da Ros*, F. Meneghetti, C. Nasci Istituto di Biologia del Mare, Consiglio Nazionale delle Ricerche, Castello 1364/A, I-30122 Venezia, Italy
Abstract A field study was carried out in the Lagoon of Venice (north-east Italy) with the aim of evaluating the potential use of lysosomal destabilisation as a biomarker of anthropogenic stress in the autochthonous mussel Mytilus galloprovincialis. Two different approaches were adopted in biomonitoring six sites in the Lagoon, evaluating indigenous populations of mussels and organisms transplanted from a reference site and checked at several points in time. Lysosomal membrane stability was investigated by means of two tests: neutral red retention assay (NRRA) for evaluating haemocyte lysosomes and lysosomal latency test (LLT) for digestive cell lysosomes. Results indicate that the lysosomal response measured in haemocytes according to NRRA is a more valuable biomarker of anthropogenic stress in the framework both of passive and active biomonitoring in marine coastal environments. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Mytilus galloprovincialis; Lysosomes; Biomonitoring; Lagoon of Venice
1. Introduction The lysosomal system, which is remarkably well-developed in both digestive cells and haemocytes of mussels, is well-known as a target site for toxic metals and organic chemicals, due to its ability to accumulate them (Moore, 1985). As a consequence, cell health deteriorates after lysosomal damage induced by contaminants. Damage is * Corresponding author. Tel.: +39-041-5204-126; fax: +39-041-5207-622. E-mail address: [email protected] (L. Da Ros). 0141-1136/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(02)00123-X
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mainly due to rapid weakening of the lysosomal membranes, which may release hydrolitic enzymes into the cytoplasm (Moore, 1985), with subsequent enhanced protein catabolism up to the autophagic conditions indicating a stress syndrome (Hole, Moore, & Bellamy, 1995). Biomarkers are primarily used in monitoring coastal marine environments in order to detect signs of impaired health in marine organisms, thus providing measurable advance warning of changes in the environment. With this in mind, in the last few years growing efforts in research work have focused on developing and/or validating more reliable, cost-effective and sufficiently simple tests for measuring the lysosomal membrane stability in molluscs in the field (Lowe, Soverchia, & Moore, 1995; Ringwood, Conners, & Hoguet, 1998). This approach may ultimately supply reliable biomarkers for use as powerful screening tools when looking for biological effects of non-specific origin in coastal marine environments. The mussel Mytilus galloprovincialis is an indigenous species in the Lagoon of Venice, naturally widespread throughout the area, where it lives mainly subtidally, attached to submerged substrates. Besides being of commercial interest and extensively cultivated as a edible species, it has also become a scientifically interesting subject within the framework of local ‘‘mussel watch’’ investigations since the 1970s (Fossato, 1982). Only more recently has it started being used as a sentinel organism to monitor the biological effects of pollution in the lagoon waters (Livingstone et al., 1995; Lowe, Fossato, & Depledge, 1995). The purpose of this study was to apply two methods of evaluating lysosomal fragility: the well-established lysosomal latency test (LLT) applied to digestive cells (Moore, 1976), and the more recently proposed neutral red retention assay (NRRA) for haemocytes (Lowe et al., 1995). Two different biomonitoring methods were also tested: passive biomonitoring and the transplantation approach.
2. Materials and methods 2.1. Biomonitoring Specimens of M. galloprovincialis were collected in April 1999 (for LLT) and April–June 2000 (for NRRA) from five sites in the lagoon showing different types and degrees of pollution (Fossato, Campesan, Craboledda, Dolci, & Stocco, 2000): Chioggia, Treporti, Salute, Canale Vittorio Emanuele (CVE), Marghera (i.e., SM15, the largest discharge point, which receives industrial waste from the surrounding industrial area). In addition a clean farming area, Pellestrina, was used as control site. 2.2. Transplantation For the LLT, in October 1999, specimens of M. galloprovincialis from the clean area, Pellestrina, were placed in the polluted sites and collected after 6 weeks. For NRRA, in April 2000, mussels from Pellestrina were transplanted into the most
L. Da Ros et al. / Marine Environmental Research 54 (2002) 817–822
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polluted site, SM15. Samples of both transplanted specimens and controls were collected simultaneously at days 1, 2, 7, 15 and 30 after transplantation. Indigenous mussels from SM15 were collected at days 15 and 30. 2.3. NRRA The ability to retain the neutral red dye by haemocytic lysosomes was evaluated adopting well-established protocols (Lowe et al., 1995; Ringwood et al., 1998). 2.4. LLT The latency of the enzyme N-acetyl-b-hexosaminidase (NAH) was evaluated by assessing the fragility of lysosomal membranes (Moore, 1976). The amount of reaction product for NAH was determined on an image analysis system (Image-Pro Plus rel. 4.0.0.9). 2.5. Statistical analyses Data were compared using the Mann–Whitney U-test (NRRA) and one-way analysis of variance (ANOVA; LLT). 3. Results and discussion 3.1. Neutral Red Retention Assay Biomonitoring revealed a higher percentage of destabilised haemocytes (P < 0.001) in indigenous mussels sampled inside the Lagoon (except at Treporti), compared with controls (Fig. 1a). The most important findings of the transplantation approach were the significant, persistent increase in destabilisation (P < 0.001) in transplanted specimens after 15 days, and the lack of significant differences between indigenous and transplanted specimens at days 15 and 30 (Fig. 1b). These results indicate preliminarily that lysosomal destabilisation in mussel haemocytes is quite a stable parameter which may be used in the Lagoon of Venice as a biomarker of effect more than of exposure. Although this is consistent with no evidence of earlier modifications—from 24 h to 7 days after transplantation, no differences were detected between transplanted mussels and controls—it is partially in contrast with the results of other authors (Ringwood et al., 1998) who found oysters to show a rapid increase in destabilization (less than 24 h) after treatment with copper. However, the lack of similar data from field transplantations, either for M. galloprovincialis or for other mollusc species, does not allow definitive conclusions to be made regarding this behaviour. 3.2. NAH Lysosomal Latency Test Comparisons among all samples, controls and indigenous, showed no significant differences (Table 1), although it must be noted that latency values in controls from
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Fig. 1. (a) Neutral Red Retention Assay as a lysosome destabilisation index of Mytilus galloprovincialis from study sites (n=10, means SE). ns=P <00.5, *** P<0.001 (Mann–Whitney U-test). Neutral Red Retention Assay as a lysosome destabilisation index of Mytilus galloprovincialis between controls and Marghera (SM15) specimens over 30-day period. (n=10, means SE) ns=P >0.05, **=P <0.01, ***=P <0.001 (Mann–Whitney U-test)).
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Table 1 Lysosomal Latency Test (mins) in indigenous and transplanted samples of Mytilus galloprovincialis (6 weeks of transplantation) from study sites (n=10, means SE)a Stations Pellestrina
Treporti
Salute
CVE
SM15
Chioggia
Minutes Indigenous 9.00 1.55 10.001.29 ns 7.750.79 ns 9.25 1.18 ns 10.751.75 ns 12.50 2.24ns mussels Transplanted 12.752.34 8.750.77* 8.251.12* 7.50 0.84* 8.75 1.72 6.00 0.67* mussels (from control area) a
ns=P > 0.05, *=P <0.05 (ANOVA, transplanted vs controls).
Pellestrina were lower than those indicated as ‘‘normal’’ for M. edulis (Lowe et al., 1995; Moore, 1976). The apparent lack of response may be due to the fact that our control samples were not suitable, since they came from an area free of contaminants but continually exposed to sudden hydrological modifications (F. Bianchi, personal communication). Instead, the transplanted organisms always showed significantly lower values (P < 0.05) in comparison with control samples, except in site SM15 (Table 1). Although here too control values were quite low, transplanted specimens were generally in a worse condition than indigenous ones, suggesting that latency detected by the histochemistry of NAH in digestive cell lysosomes is an early sign of exposure to adverse conditions. This study demonstrates the usefulness of the lysosomal destabilization approach in environmental monitoring, the need to apply a number of biomarker methods in order to consider the multiple aspects of the biological response, and the importance of the transplantation approach in marine coastal monitoring.
Acknowledgements This work was partly supported by the Consorzio per la Gestione del Centro di Coordinamento delle Attivita` di Ricerca inerenti il Sistema lagunare di Venezia (CO.RI.LA). References Fossato, V. U. (1982). Vies journees etud. pollutions. Cannes: CIESM. Fossato, V. U., Campesan, G., Craboledda, L., Dolci, F. & Stocco, G. (2000). In: The Venice Lagoon ecosystem, man and the biosphere series, vol. 25, Pearl River, NY: Parthenon Publishing, 97–126. Hole, M. H., Moore, M. N., & Bellamy, D. (1995). Mar. Ecol. Prog. Ser., 122, 173–178. Livingstone, D. R., Lemaire, P., Matthews, A., Peters, L. D., Porte, C., Fitzpatrick, P. J., Forlin, L., Nasci, C., Fossato, V. U., Wotton, N., & Goldfarb, P. (1995). Marine Environmental Research, 39, 235– 240.
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Lowe, D. M., Fossato, V. U., & Depledge, M. H. (1995). Mar. Ecol. Prog. Ser., 129, 189–196. Lowe, D. M., Soverchia, C., & Moore, M. N. (1995). Aquatic Toxicology, 33, 105–112. Moore, M. N. (1976). Cell Tissue Research, 175, 279–287. Moore, M. N. (1985). MarinePollution Bulletin, 16, 134–139. Ringwood, A. H., Conners, D. E., & Hoguet, J. (1998). Mar. Ecol. Prog. Ser., 166, 163–171.
Field application of lysosomal destabilisation indices in the mussel Mytilus galloprovincialis: biomonitoring and transplantation in the Lagoon of Venice (north-east Italy) L. Da Ros*, F. Meneghetti, C. Nasci Istituto di Biologia del Mare, Consiglio Nazionale delle Ricerche, Castello 1364/A, I-30122 Venezia, Italy
Abstract A field study was carried out in the Lagoon of Venice (north-east Italy) with the aim of evaluating the potential use of lysosomal destabilisation as a biomarker of anthropogenic stress in the autochthonous mussel Mytilus galloprovincialis. Two different approaches were adopted in biomonitoring six sites in the Lagoon, evaluating indigenous populations of mussels and organisms transplanted from a reference site and checked at several points in time. Lysosomal membrane stability was investigated by means of two tests: neutral red retention assay (NRRA) for evaluating haemocyte lysosomes and lysosomal latency test (LLT) for digestive cell lysosomes. Results indicate that the lysosomal response measured in haemocytes according to NRRA is a more valuable biomarker of anthropogenic stress in the framework both of passive and active biomonitoring in marine coastal environments. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Mytilus galloprovincialis; Lysosomes; Biomonitoring; Lagoon of Venice
1. Introduction The lysosomal system, which is remarkably well-developed in both digestive cells and haemocytes of mussels, is well-known as a target site for toxic metals and organic chemicals, due to its ability to accumulate them (Moore, 1985). As a consequence, cell health deteriorates after lysosomal damage induced by contaminants. Damage is * Corresponding author. Tel.: +39-041-5204-126; fax: +39-041-5207-622. E-mail address: [email protected] (L. Da Ros). 0141-1136/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(02)00123-X
818
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mainly due to rapid weakening of the lysosomal membranes, which may release hydrolitic enzymes into the cytoplasm (Moore, 1985), with subsequent enhanced protein catabolism up to the autophagic conditions indicating a stress syndrome (Hole, Moore, & Bellamy, 1995). Biomarkers are primarily used in monitoring coastal marine environments in order to detect signs of impaired health in marine organisms, thus providing measurable advance warning of changes in the environment. With this in mind, in the last few years growing efforts in research work have focused on developing and/or validating more reliable, cost-effective and sufficiently simple tests for measuring the lysosomal membrane stability in molluscs in the field (Lowe, Soverchia, & Moore, 1995; Ringwood, Conners, & Hoguet, 1998). This approach may ultimately supply reliable biomarkers for use as powerful screening tools when looking for biological effects of non-specific origin in coastal marine environments. The mussel Mytilus galloprovincialis is an indigenous species in the Lagoon of Venice, naturally widespread throughout the area, where it lives mainly subtidally, attached to submerged substrates. Besides being of commercial interest and extensively cultivated as a edible species, it has also become a scientifically interesting subject within the framework of local ‘‘mussel watch’’ investigations since the 1970s (Fossato, 1982). Only more recently has it started being used as a sentinel organism to monitor the biological effects of pollution in the lagoon waters (Livingstone et al., 1995; Lowe, Fossato, & Depledge, 1995). The purpose of this study was to apply two methods of evaluating lysosomal fragility: the well-established lysosomal latency test (LLT) applied to digestive cells (Moore, 1976), and the more recently proposed neutral red retention assay (NRRA) for haemocytes (Lowe et al., 1995). Two different biomonitoring methods were also tested: passive biomonitoring and the transplantation approach.
2. Materials and methods 2.1. Biomonitoring Specimens of M. galloprovincialis were collected in April 1999 (for LLT) and April–June 2000 (for NRRA) from five sites in the lagoon showing different types and degrees of pollution (Fossato, Campesan, Craboledda, Dolci, & Stocco, 2000): Chioggia, Treporti, Salute, Canale Vittorio Emanuele (CVE), Marghera (i.e., SM15, the largest discharge point, which receives industrial waste from the surrounding industrial area). In addition a clean farming area, Pellestrina, was used as control site. 2.2. Transplantation For the LLT, in October 1999, specimens of M. galloprovincialis from the clean area, Pellestrina, were placed in the polluted sites and collected after 6 weeks. For NRRA, in April 2000, mussels from Pellestrina were transplanted into the most
L. Da Ros et al. / Marine Environmental Research 54 (2002) 817–822
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polluted site, SM15. Samples of both transplanted specimens and controls were collected simultaneously at days 1, 2, 7, 15 and 30 after transplantation. Indigenous mussels from SM15 were collected at days 15 and 30. 2.3. NRRA The ability to retain the neutral red dye by haemocytic lysosomes was evaluated adopting well-established protocols (Lowe et al., 1995; Ringwood et al., 1998). 2.4. LLT The latency of the enzyme N-acetyl-b-hexosaminidase (NAH) was evaluated by assessing the fragility of lysosomal membranes (Moore, 1976). The amount of reaction product for NAH was determined on an image analysis system (Image-Pro Plus rel. 4.0.0.9). 2.5. Statistical analyses Data were compared using the Mann–Whitney U-test (NRRA) and one-way analysis of variance (ANOVA; LLT). 3. Results and discussion 3.1. Neutral Red Retention Assay Biomonitoring revealed a higher percentage of destabilised haemocytes (P < 0.001) in indigenous mussels sampled inside the Lagoon (except at Treporti), compared with controls (Fig. 1a). The most important findings of the transplantation approach were the significant, persistent increase in destabilisation (P < 0.001) in transplanted specimens after 15 days, and the lack of significant differences between indigenous and transplanted specimens at days 15 and 30 (Fig. 1b). These results indicate preliminarily that lysosomal destabilisation in mussel haemocytes is quite a stable parameter which may be used in the Lagoon of Venice as a biomarker of effect more than of exposure. Although this is consistent with no evidence of earlier modifications—from 24 h to 7 days after transplantation, no differences were detected between transplanted mussels and controls—it is partially in contrast with the results of other authors (Ringwood et al., 1998) who found oysters to show a rapid increase in destabilization (less than 24 h) after treatment with copper. However, the lack of similar data from field transplantations, either for M. galloprovincialis or for other mollusc species, does not allow definitive conclusions to be made regarding this behaviour. 3.2. NAH Lysosomal Latency Test Comparisons among all samples, controls and indigenous, showed no significant differences (Table 1), although it must be noted that latency values in controls from
820
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Fig. 1. (a) Neutral Red Retention Assay as a lysosome destabilisation index of Mytilus galloprovincialis from study sites (n=10, means SE). ns=P <00.5, *** P<0.001 (Mann–Whitney U-test). Neutral Red Retention Assay as a lysosome destabilisation index of Mytilus galloprovincialis between controls and Marghera (SM15) specimens over 30-day period. (n=10, means SE) ns=P >0.05, **=P <0.01, ***=P <0.001 (Mann–Whitney U-test)).
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Table 1 Lysosomal Latency Test (mins) in indigenous and transplanted samples of Mytilus galloprovincialis (6 weeks of transplantation) from study sites (n=10, means SE)a Stations Pellestrina
Treporti
Salute
CVE
SM15
Chioggia
Minutes Indigenous 9.00 1.55 10.001.29 ns 7.750.79 ns 9.25 1.18 ns 10.751.75 ns 12.50 2.24ns mussels Transplanted 12.752.34 8.750.77* 8.251.12* 7.50 0.84* 8.75 1.72 6.00 0.67* mussels (from control area) a
ns=P > 0.05, *=P <0.05 (ANOVA, transplanted vs controls).
Pellestrina were lower than those indicated as ‘‘normal’’ for M. edulis (Lowe et al., 1995; Moore, 1976). The apparent lack of response may be due to the fact that our control samples were not suitable, since they came from an area free of contaminants but continually exposed to sudden hydrological modifications (F. Bianchi, personal communication). Instead, the transplanted organisms always showed significantly lower values (P < 0.05) in comparison with control samples, except in site SM15 (Table 1). Although here too control values were quite low, transplanted specimens were generally in a worse condition than indigenous ones, suggesting that latency detected by the histochemistry of NAH in digestive cell lysosomes is an early sign of exposure to adverse conditions. This study demonstrates the usefulness of the lysosomal destabilization approach in environmental monitoring, the need to apply a number of biomarker methods in order to consider the multiple aspects of the biological response, and the importance of the transplantation approach in marine coastal monitoring.
Acknowledgements This work was partly supported by the Consorzio per la Gestione del Centro di Coordinamento delle Attivita` di Ricerca inerenti il Sistema lagunare di Venezia (CO.RI.LA). References Fossato, V. U. (1982). Vies journees etud. pollutions. Cannes: CIESM. Fossato, V. U., Campesan, G., Craboledda, L., Dolci, F. & Stocco, G. (2000). In: The Venice Lagoon ecosystem, man and the biosphere series, vol. 25, Pearl River, NY: Parthenon Publishing, 97–126. Hole, M. H., Moore, M. N., & Bellamy, D. (1995). Mar. Ecol. Prog. Ser., 122, 173–178. Livingstone, D. R., Lemaire, P., Matthews, A., Peters, L. D., Porte, C., Fitzpatrick, P. J., Forlin, L., Nasci, C., Fossato, V. U., Wotton, N., & Goldfarb, P. (1995). Marine Environmental Research, 39, 235– 240.
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Lowe, D. M., Fossato, V. U., & Depledge, M. H. (1995). Mar. Ecol. Prog. Ser., 129, 189–196. Lowe, D. M., Soverchia, C., & Moore, M. N. (1995). Aquatic Toxicology, 33, 105–112. Moore, M. N. (1976). Cell Tissue Research, 175, 279–287. Moore, M. N. (1985). MarinePollution Bulletin, 16, 134–139. Ringwood, A. H., Conners, D. E., & Hoguet, J. (1998). Mar. Ecol. Prog. Ser., 166, 163–171.