© 2005 International Council for the Exploration of the Sea
Acetylcholinesterase activity in hosts (herring Clupea harengus) and parasites (Anisakis simplex larvae) from the southern Baltic
Sea Fisheries Institute ul. Kollataja 1, 81-332 Gdynia, Poland
*Correspondence to M. Podolska: tel: +48 58 620 17 28; fax: +48 58 620 28 31. e-mail: bilbo{at}mir.gdynia.pl.
This study compares the acetylcholinesterase (AChE) activity of herring Clupea harengus infected with Anisakis simplex larvae and non-infected individuals caught in coastal waters of the southern Baltic. Acetylcholinesterase activity was measured spectrophotometrically. Generalized linear models were applied to analyse the dependence of AChE activity on the area of sampling and the biological parameters of fish and their parasites. The AChE activity of herring was higher in samples from the western and central coasts (regarded as "clean" waters) than in fish caught in the semi-enclosed areas of the Gulf of Gda
sk and Vistula Lagoon (regarded as "polluted" sites). The opposite relationship was noted in the activity of AChE extracted from A. simplex larvae. In male hosts, the parasitic AChE activity was markedly higher than in the females in all examined areas.
Keywords: AChE, Anisakis simplex, Baltic Sea, Clupea harengus, herring
Received 30 November 2004; accepted 1 August 2005.
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Introduction |
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The Baltic Sea is a shallow sea with a large catchment area and limited exchange with the North Sea, which can effectively trap pollutants from point and diffuse sources. Furthermore, low water temperatures and ice cover cause slow biodegradation of chemicals. These conditions make the Baltic Sea a special ecosystem which is highly sensitive to pollutants.
Pollutant exposure may lead to severe consequences for fish populations inhabiting these waters. Biomarkers, which include a number of molecular, cellular, and physiological parameters, have been identified as a powerful and cost-effective approach to obtaining information on the state of the environment and the effects of pollution on living biological resources. Owing to the inherent attributes of fish biology, the overwhelming numbers of confounding factors in field studies, and the fact that environmental exposure involves chemical mixtures, the specificity of effect of any one compound is difficult to identify. However, it is possible to assess effects between populations with high and low levels of exposure. Since parasites are affected directly by pollution or indirectly by their effects on the intermediate and definitive hosts (MÖller, 1987), indices such as metabolic or parasitic markers might be useful, economical, and reliable indicators for determining the effects of pollutants on ecosystems (Marcogliese and Cone, 1997; Overstreet, 1997; Broeg et al., 1999).
Lewis and Hoole (2003) summarized how aquatic hosts and their parasites are likely to indicate changes in water quality and thus play a significant role in developing understanding of natural aquatic ecosystems. Pollutants might promote increased parasitism in aquatic animals, especially fish, by impairing the immune response of the hosts or favouring the survival and reproduction of intermediate hosts. Alternatively, decreased parasitism might ensue through the toxicity of the pollutant to free-living stages and intermediate hosts or by the alteration of the host's physiology (Khan and Thulin, 1991).
Studies on the occurrence of diseases and parasites of marine fish also constitute an important component of the monitoring programmes implemented in ICES Member Countries in order to assess the biological effects of anthropogenic activities in the marine environment (Lang and Mellergaard, 1999). The rationale behind the use of herring (Clupea harengus) in this study was to investigate biomarker response in more and less contaminated areas and to target a pelagic, migratory species. The biological effects in the pelagic systems of the Baltic Sea have not been thoroughly investigated to date, with most studies usually having been restricted to bottom-dwelling, non-migratory fish species inhabiting coastal regions. Additionally, herring is a fish species commonly infected with Anisakis simplex – a nematode parasite of marine organisms (Grabda, 1974; KÜhlmorgen-Hille, 1979, 1983; Lang et al., 1990). The presence of these parasites indicates that the herring must have migrated to feed outside the Baltic Sea.
The results of the authors' previous field study on biomarkers in flounder (Platichthys flesus) from the southern Baltic Sea indicated that there were statistically significant differences in the acetylcholinesterase (AChE) activity of the measured biomarkers between reference and contaminated sites (Napierska and Podolska, 2005). Acetylcholinesterase is a very important enzyme in nerve impulse transmission in both vertebrates and invertebrates. Acetylcholinesterase terminates the transmission of neural impulses by the rapid hydrolysis of acetylcholine (ACh) into the inactive products of choline and acetic acid (Barnard, 1974). Acetylcholine is the primary neurotransmitter in the sensory and neuromuscular systems of fish. The levels of acetylcholine at the cholinergic synapse and the neuromuscular junction must be regulated, and the role of AChE is to prevent the accumulation of ACh. Acetylcholinesterase inhibition results in an increase in the ACh level that causes continuous and excessive stimulation of the neural system. This can lead to tetany, paralysis, and even death (Kirby et al., 2000). Many chemicals can affect AChE activity. Strong inhibitory effects have been identified in carbamates, organophosphates, and organochlorine pesticides. However, a number of other important contaminants have also been shown to have anti-AChE properties (Olson and Christensen, 1980) including heavy metals (Zinkl et al., 1991), hydrocarbons, and detergents (Payne et al., 1996). The monitoring of AChE activity in fish has become a technique that is applied commonly to diagnose environmental exposure to cholinergic poisons.
The role of AChE in many parasite species differs from that in free-living organisms and might be significant in host–parasite interactions. Edwards et al. (1971) described parasitic AChE as a "biochemical holdfast" that permits them to exist in a preferred habitat. This enzyme is produced by the excretory/secretory (E/S) system of nematodes (Blackburn and Selkirk, 1992; Griffiths and Pritchard, 1994). Some parasite species secrete AChE to inhibit the intestinal peristalsis of the host, which might prevent parasite expulsion by host muscular contraction, while other species can increase peristaltic movement to move more quickly in the host alimentary tract (Lee, 1969, 1970, 1972; Lee and Foster, 1995). According to Selkirk et al. (2001), acetylcholinesterases secreted by nematodes might act on alternative substrates to acetylcholine.
The current investigation focused on the effects of neurotoxic pollutants present in the Baltic ecosystem on pelagic fish (herring) and its parasites (A. simplex larvae) with respect to the host–parasite relationship in AChE activity.
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Material and methods |
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Sample collection and handling
Samples of Baltic herring were collected during the spring spawning period (from April to May) in the coastal waters of ICES Subdivisions 24–26 in four locations: 1, western coast (Pomeranian Bay); 2, central coast (Rowy); 3, eastern coast (Gulf of Gda
sk); and 4, Vistula Lagoon (Figure 1). Immediately after capture, the fish were stored in polystyrene boxes with ice (to avoid the postmortem migration of parasites into the flesh of the fish). Total body length (cm), weight (g), as well as sex and gonad developmental stage according to Maier's scale (FAO, 1965) were determined for each fish. Otoliths were extracted from each individual to estimate age and the stock component of herring according to the Kompowski (1969) classification. Age was determined as the number of hyaline (winter) rings on the otolith. The presence of A. simplex larvae in the body cavity of each fish was recorded by macroscopic examination. The length of most of the fish (84%) was in the range 23–26 cm. Only fish belonging to the southern coast stock component (n = 225) were chosen for further analyses. These individuals were placed with their anterior part facing leftwards, and a sample of muscle tissue was dissected from the posterior part of the fish, close to the tail fin. The same location was used for both infected and non-infected animals. Samples of herring muscle and A. simplex larvae from the fish were taken and frozen immediately at –80°C for biochemical analysis.
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owo); 4, Vistula Lagoon.Preparation of tissue homogenates
AChE extraction was performed on 400–500 mg of muscle tissue using a 0.02 M phosphate buffer (pH 7.0) containing 0.1% Triton X 100. The tissue was homogenized in four volumes of buffer (4 ml buffer per g tissue wet weight) and centrifuged at 10 000 x g for 20 min at 4°C. An aliquot of the supernatant (the "S9" fraction) was stored at –80°C and used in the assay. All A. simplex larvae found in one host individual (5–53 individuals of parasites) were pooled as one sample and treated exactly like the muscle samples.
Enzyme activity determination
AChE activity determinations were performed using a method described by Ellman et al. (1961), adapted for use with a microplate reader (Bocquené and Galgani, 1998). The enzyme kinetic was monitored at 412 nm. The standard reaction mixture, final volume 0.380 ml, contained 0.02 M phosphate buffer, pH 7.0, 0.5 mM DTNB [5,5'-dithiobis(2-nitrobenzoic acid)], and 2.6 mM ACTC (acetylthiocholine chloride). Protein concentration was determined as described by Bradford (1976), using Protein Kit II from Bio-Rad laboratories and a bovine serum albumin as the protein standard.
Statistical analysis
Generalized linear models (GLM) (McCullagh and Nelder, 1989) were applied to analyse the dependence of the AChE activity on the sampling area and the biological parameters of the fish and its parasites. The following models were fitted:
- Model 1 AChE activity in herring including infected and non-infected individuals (n = 225)
- Model 2 AChE activity in herring with parasitic AChE activity as the covariate (n = 98)
- Model 3 AChE activity in parasites (n = 98)
- Model 2 AChE activity in herring with parasitic AChE activity as the covariate (n = 98)
First, the full model (all considered variables and factors included) was fitted. Corner point parameterization was used, i.e. the factor effects for level one were assumed to be zero for all factors. Thus, the factor effects for other levels can be regarded as the differences between the effect at a given level and the effect at level one. Body length and condition factor of fish, the number of parasites and their AChE activity were taken as covariates, while age, sex, gonad stage, and area were treated as factors in the analysis. The error was assumed to be normal and the identity link function was used. Next, the significance of factors and the covariates was tested and only significant terms were left in the final model. Similarly, factor levels that did not produce a significantly different response of enzymatic activity were grouped into new factor levels. The tests were performed by deletion, i.e. only those terms whose deletion did not result in a significant increase in deviance (the GLM measure of discrepancy between modelled and observed values) were left in the model. The significance of the effects and factor levels was tested with an F-test. The distributions of the model residuals were analysed to test the model assumptions and performance.
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Results |
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A. simplex larvae were found in herring in the body cavity, the mesentery, and on the gonads and pyloric processes. The number of parasites ranged from 1 to 71 per fish.
The mean activity levels of AChE were higher in herring samples taken from the western coast (Pomeranian Bay) where the contaminant input is assumed to be lower than in samples from eastern areas (Table 1), while the opposite relationship was noted in the activity of AChE extracted from A. simplex larvae. In all areas, parasitic AChE activity was markedly higher in male than in female hosts.
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AChE activity models
Only the area effect was significant in the model of AChE activity in herring (model 1). This effect was lowest in the Gulf of Gda
sk and highest on the western coast (Figure 2). The model explained only 11.9% of the deviance.
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Parasitic AChE activity and sampling area were significant in the model of host enzymatic activity with A. simplex AChE activity as a covariate and explained 23.4% of the variance (model 2). Similar to the previous model, the area effect was lowest in the Gulf of Gda
sk (Figure 3a). The estimated AChE activity for the Gulf of Gdask and Vistula Lagoon was not significantly different (Table 2). This also applied to enzyme activity in open sea areas (middle and western coast). AChE activity in the host is negatively correlated with the activity of this enzyme in parasites.
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Sampling area and host sex were significant in the model of AChE activity in A. simplex larvae (model 3) (Table 3). In contrast to the host AChE activity model, the area effect was highest in eastern areas (Figure 3b). The Gulf of Gda
sk and Vistula Lagoon effects were not significantly different. AChE activity was higher in male than female hosts (Figure 4). The model explained 35.3% of the deviance.
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Discussion |
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Acetylcholinesterase activity is usually high in fish living in "clean" environments and decreases in "polluted" areas. The study by Kirby et al. (2000) indicated that there was cholinesterase inhibition in flounder tissues in samples from a number of contaminated English estuaries that were studied as compared with a clean control estuary where it was not noted. Galgani et al. (1992) demonstrated that AChE activity in the muscles of North Sea dab (Limanda limanda) varied according to the contamination gradient and was higher in less polluted waters. Bocquené et al. (1995) indicated high inhibitory activities of several organophosphate pesticides (OP) and carbamate (CB) compounds on AChE extracts of marine organisms, including fish. It is reported that acetylcholinesterase activity was directly inhibited by organophosphates in European sea bass (Dicentrarchus labrax) (Varò et al., 2003) and by carbamates in rainbow trout (Oncorhynchus mykiss) (Zinkl et al., 1987). The relationship between OP and/or CB exposure and AChE inhibition was also studied in several other fish species – mosquitofish (Gambusia affinis) (Boone and Chambers, 1997), largemouth bass (Micropterus salmoides), bluegill sunfish (Lepomis macrochirus), golden shiners (Notemigonus crysoleucas) (Carr et al., 1997), mummichog (Fundulus heteroclitus) (Karen et al., 1998), and threespined stickleback (Gasterosteus aculeatus) (Sturm et al., 1999).
The results of the present study might suggest that herring populations which spawn in southern Baltic waters are also exposed to the influence of contaminants. AChE inhibition in herring was observed in the semi-enclosed areas of the Gulf of Gdask and Vistula Lagoon, in contrast to the significantly higher enzymatic activity noted in the coastal area of the open Southern Baltic. Napierska and Podolska (2005) demonstrated that muscle enzyme activity in southern Baltic flounder (P. flesus) was significantly lower at sampling sites located within the Gulf of Gdask (regarded as "polluted") than in the open sea waters of the central coast (regarded as a "reference" area). The authors argued that the inhibition of AChE activity noted in flounder could be associated with the heavy anthropogenic pressure of industrial and agricultural wastes. The existence of extremely low thresholds for induction of inhibitory effects on AChE suggests that detection in fish is possible after exposure to insecticide concentrations of around 0.1–1 µg l–1 (Klaverkamp and Hobden, 1980; Habig et al., 1986). However, a number of other important contaminants have been shown to have anti-AChE properties, including heavy metals (Zinkl et al., 1991), hydrocarbons, and detergents (Payne et al., 1996). Contamination in the Gulf of Gdask and Vistula Lagoon is well documented. Higher levels of persistent organic pollutants (POPs) have been recorded in sediment samples collected from the Gulf of Gdask than in those taken from open sea areas (Pazdro, 2004). Sediments from the Vistula Lagoon are polluted by chlorinated hydrocarbons (Sapota, 1997) and trace elements (Szefer et al., 1999). According to Glasby and Szefer (1998), sediments from the Vistula Lagoon are much less polluted with Zn, Pb, Cd, and Ag than those from the Gulf of Gdask. Sapota (2004) established the level of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) in seawater of the southern Baltic Sea and reported quite high concentrations of PCBs and DDTs in the Gulf of Gdask (with the highest pollution levels coming from the Vistula River). Strandberg et al. (1998) investigated the contamination status of a wide range of organochlorine compounds through herring body burden studies and found HCHs, DDTs, HCB, and PCBs in every sample investigated. They identified the Gulf of Gdask as the site most contaminated by DDTs among the studied areas of the Bothnian Bay, Bothnian Sea, and the Gulf of Gdask. The contaminant input in Pomeranian Bay is assumed to be lower than that of the Gulf of Gdask (Konat and Kowalewska, 2001; Emeis et al., 2002). It seems more likely that reactions in muscular AChE activity observed in the herring in this study, if caused by pollution, could be attributed to the integrated effect of several classes of contaminants.
There was no relationship between AChE activity in herring and the presence and/or number of A. simplex larvae. A significant, negative correlation between host and parasite AChE activity was noted. The difference between AChE activity in A. simplex larvae and herring was the highest in polluted areas. The most divergence was observed between the AChE activity in the male hosts (lowest level) and their parasites (highest level) from the Gulf of Gdask. The results could suggest that AChE levels in parasites may be inversely related to the enzyme level in their hosts. Anderson and May (1992) observed that the survival and fecundity of parasitic nematodes is limited by the immune response of the host. According to some authors (Brownlee and Fairweather, 1999; Lazari et al., 2004), the precise role of AChEs secreted by parasites is uncertain. One of the most important functions of AChE secreted by nematodes is the modulation of host immunity (Rhoads, 1984; Pritchard, 1993). The findings of Lee (1996) are related to the role of AChE secreted by parasites and its influence on the inflammatory response in host tissues. Lee also stated that ACh enhances the release of lysosomal enzymes which are mediators of acute inflammation; thus, the AChE secreted by nematodes may interfere with this process by hydrolyzing the ACh before it reaches its target cells, reducing inflammation in the immediate vicinity of the nematodes. On the other hand, recent findings indicate that ACh inhibits the release of pro-inflammatory mediators in mammals by deactivating peripheral macrophages (Tracey, 2002; De Simone et al., 2005). It is possible that ACh could have both effects on the immune system: pro-inflammatory when acting on muscarinic receptors (Sato et al., 1998), and anti-inflammatory when acting on nicotinic receptors (Borovikova et al., 2000; Wang et al., 2003).
In polluted environments the suppression of AChE activity in herring might lead to the accumulation of acetylcholine, which could influence the inflammatory response in the fish. This biochemical response by the host could be adverse for parasite survival, so nematodes produce a large amount of AChE in order to inactivate the host's acetylcholine. The findings of Fournier et al. (1993) suggest that AChE overproduction might protect organisms against organophosphates, but such a mechanism was reported in Drosophila melanogaster. There is no data on how pesticides and other chemicals can interfere with the AChE secreted by internal parasites of fish.
It also cannot be ruled out that both of the processes observed (increasing parasitic AChE activity and decreasing host AChE activity) changed independently under the influence of different factors. Fish are usually exposed simultaneously to many different stress factors, such as adverse environmental conditions, pollutants, or infectious agents (parasites, bacteria, etc.), and their combination can disturb physiological equilibrium. Existing knowledge of how all of these stressors interact in fish is scant (Hoole et al., 2003). Multidisciplinary approaches, which include the fields of parasitology, ecotoxicology, ecology, and fish biology, could help fill the gaps in existing knowledge.
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