Cholinesterase Activities in the Adductor Muscle of The

Antarctic Science 18 (1), 15–22 (2006) © Antarctic Science Ltd Printed in the UK DOI: 10.1017/S0954102006000022 Cholinesterase activities in the adductor muscle of the Antarctic scallop Adamussium colbecki STEFANO BONACCI*, ILARIA CORSI and SILVANO FOCARDI Department of Environmental Sciences “G. Sarfatti”, University of Siena, Via P.A. Mattioli 4, I-53100 Siena, Italy *[email protected] Abstract: Antarctica is regarded as one of the most pristine parts of the Earth but even this remote ecosystem is affected by contamination and high levels of certain heavy metals, such as cadmium, which may occur naturally in Antarctic waters. The bivalve scallop Adamussium colbecki is considered a key species of Antarctic benthic ecosystems and a sensitive target for bioaccumulation of xenobiotics and metals. Since cholinesterases (ChEs) in the adductor muscle of A. colbecki presumably play a prominent physiological role through regulation of swimming movements, the main aims of this study was to characterize ChE activities in adductor muscle of A. colbecki and to investigate their sensitivity to organophosphate pesticides and heavy metals. The results suggest that an acetylcholinesterase-like enzyme in the adductor muscle of the scallop has low sensitivity to organophosphates but was significantly inhibited by exposure to cadmium. Received 1 December 2004, accepted 27 June 2005 Key words: aquatic pollutants, benthos, bivalve, Cd, ChE, DFP Introduction Antarctic scallop that is considered a key species of the The remote continent of Antarctica is regarded as one of the Antarctic benthic marine ecosystem (Mauri et al. 1990, most pristine areas of the Earth. However, several studies Berkman & Nigro 1992, Chiantore et al. 1999, Cerrano have reported organic xenobiotics and heavy metals in et al. 2001). Swimming movements are performed by shell- biotic and abiotic matrices of the Antarctic marine clapping which seems to be significant for escape from environment and their bioaccumulation in the marine food predators and locally unfavourable conditions (Ansell et al. chain (Fuoco et al. 1991, 1996, Bargagli 2000). It is 1998), enabling at least some of the population to avoid therefore now widely recognized that Antarctica is affected mortality, as in other scallop species (Peterson et al. 1982, by man-made contaminants and naturally occurring heavy Vacchi et al. 2000). Swimming is achieved by contraction metals. Most trace elements have both anthropogenic and of the adductor muscle, which forces water out of the shell natural origins (Bargagli 2000). Sanchez-Hernandez (2000) cavity through a pair of small jets located dorsally on either reported that metal contents in nearshore environments may side of the hinge. This method of locomotion seems very be predominantly controlled by phenomena not directly effective in spite of the low temperatures of Antarctic waters linked to anthropogenic contamination and that the high (Stocchino 1992, Ansell et al. 1998). levels in biota do not necessarily indicate anthropogenic Research has been done into bioaccumulation of impact. In particular, the Southern Ocean exhibits very high xenobiotics and metals by A. colbecki. Although concentrations of cadmium, regarded as a major biotoxic bioaccumulation is mainly influenced by the level a species element and pollutant (Bordin et al. 1987, Nolting et al. occupy in the food chain, Focardi et al. (1993) found higher 1991, Bargagli et al. 1996). In most Antarctic offshore levels of organochlorine contaminants in whole body of environments the input of metals due to geochemical and A. colbecki than in any other invertebrate or fish species anthropogenic sources and from long-range transport is hierarchically located at higher levels. Strong accumulation negligible, but concentrations of Cd in seawater and biota of Cd (a metal usually considered an environmental may be higher than in water and organisms from polluted contaminant) has been reported (Mauri et al. 1990, Bargagli coastal areas (Berkman & Nigro 1992, Bargagli et al. 1996, et al. 1996). Scallops exposed in vivo to Cd and Cu for Bargagli 2000, Sanchez-Hernandez 2000). High seven days showed high levels of both metals in gills which concentrations of Cd were measured by Rainbow (1989) in for Cu decreased sharply during 14 days of Antarctic crustaceans. It has been inferred that Cd decontamination, whereas Cd levels did not vary (Berkman enrichment in seawater and biota may be due to upwelling & Nigro 1992). The higher cadmium levels in tissues of of deep nutrient-rich water, as the Southern Ocean is A. colbecki than in those of other bivalve species, including isolated and there is no evidence of wind-borne or Mediterranean ones (Brooks & Rumsby 1965, Viarengo anthropogenic input of this metal (Bargagli et al. 1996). et al. 1993), may be related to the intense feeding rate of the Adamussium colbecki (Smith, 1902) is an endemic Antarctic scallop (Palmer & Rand 1977, Chiantore et al. 15 Downloaded from https://www.cambridge.org/core. Open University Library, on 20 Jan 2020 at 17:06:27, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0954102006000022 16 STEFANO BONACCI et al. 1998, 1999). These results suggest that A. colbecki may be a and less specific butyrylcholinesterase, also known as suitable species for studying the effects of Cd pseudocholinesterase or non-specific esterase (BChE, EC contamination in Antarctic marine biota. In light of this, and 3.1.1.8) (Silver 1974, Massoulié & Toutant 1988). AChE under the consideration of its circum-Antarctic distribution hydrolyses the neurotransmitter acetylcholine (ACh) at (Dell 1972, Berkman & Nigro 1992) the scallop was cholinergic synapses in all living organisms and occurs in recently proposed for monitoring anthropogenic impact many molecular forms (Talesa et al. 2002). AChE is mainly, near Antarctic scientific bases (SCAR 1994, 1996). In but not exclusively, present in the synaptic cleft (Massoulié addition, Viarengo et al. (1997) suggested the possible et al. 1993) and is assumed to be a target enzyme of utilization of metallothioneins as a biomarker of Antarctic organophosphates (OPs) and carbamates (CBs) in most marine pollution utilizing the Antarctic scallop A. colbecki species (Boone & Chambers 1996). Recent studies provide as a bioindicator. Moreover, it has been inferred that evidence that ChE activities may also be affected by a wide populations of A. colbecki could collapse under the effect of range of contaminants, including heavy metals (Bocquené limited anthropogenic pressure (Berkman 1990). Since the et al. 1990, Najimi et al. 1997, Guilhermino et al. 1998). scallop is considered a key species for pelagic-benthic Inhibition of AChE results in accumulation of the coupling in the littoral system (Chiantore et al. 1998) and is neurotransmitter ACh in neuromuscular junctions, causing an important secondary producer in the Antarctic benthic hyperpolarization of the post-synaptic membrane and system (Ansell et al. 1998), insult or chronic stress overstimulation of cholinergic receptors, both signs of disrupting the population structure of A. colbecki may also cholinergic toxicity (Carlock et al. 1999, Pope 1999). While significantly affect higher ecological levels of the Antarctic ChEs have been extensively studied in vertebrates, less nearshore marine ecosystem (Fossi 2000). research has been done on other species, particularly Cholinesterases (ChEs) are a class of serine hydrolases molluscs, where the classification is much more ambiguous. ubiquitous in the animal kingdom and are presumed to have Recent attempts have been made to classify ChE types in evolved through mutation of an ancestral esterase form, marine invertebrata by substrate preference and sensitivity achieving higher specificity for hydrolyzing choline esters. to specific inhibitors, as is done for vertebrate They play a prominent role in nerve impulse transmission cholinesterases (Sturm et al. 1999, Chuiko 2000). The same (Silver 1974, Massoulié et al. 1993). Vertebrate ChEs are methodology has been applied in studies involving marine now classified as acetylcholinesterase (AChE, EC 3.1.1.7) bivalve species (Basack et al. 1998, Talesa et al. 2001, Fig. 1. Map of the Terra Nova Bay Italian scientific station in Antarctica, Ross Sea, showing location of the sampling site for A. colbecki. Downloaded from https://www.cambridge.org/core. Open University Library, on 20 Jan 2020 at 17:06:27, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0954102006000022 CHOLINESTERASES IN THE ANTARCTIC SCALLOP ADAMUSSIUM COLBECKI 17 2002, Galloway et al. 2002). addition of substrate. Three thiocholine esters (ASCh, In view of the prominent ecological role played by ChE BSCh and PrSCh) were used as substrates. Initial assay activities in the adductor muscle of A. colbecki conditions in the reaction mixture (final volume 300 μl) (transmission of nerve impulses and regulation of were as follows: 25 mM Tris-HCl buffer (pH 7.6, 1 mM swimming movements) the main aims of the present CaCl2), 40μl DTNB (0.333 mM, final concentration) and 40 research were: μl of sample. The reaction was started by adding 40 μl of substrate (2 mM, final concentration). Spontaneous 1) to characterize ChE activities in adductor muscle of hydrolysis was determined in the absence of supernatant. A. colbecki, and ChE activities were expressed as enzyme units x mg total 2) to investigate

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