ICES Journal of Marine Science, 63: 161e168 (2006) doi:10.1016/j.icesjms.2005.08.

001

Acetylcholinesterase activity in hosts (herring Clupea harengus) and parasites (Anisakis simplex larvae) from the southern Baltic
Magdalena Podolska and Dorota Napierska
Podolska, M., and Napierska, D. 2006. Acetylcholinesterase activity in hosts (herring Clupea harengus) and parasites (Anisakis simplex larvae) from the southern Baltic. e ICES Journal of Marine Science, 63: 161e168. 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 sh and their parasites. The AChE activity of herring was higher in samples from the western and central coasts (regarded as sk clean waters) than in sh caught in the semi-enclosed areas of the Gulf of Gdan 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.
2005 International Council for the Exploration of the Sea. Published by Elsevier Ltd. All rights reserved.

Keywords: AChE, Anisakis simplex, Baltic Sea, Clupea harengus, herring. Received 30 November 2004; accepted 1 August 2005. M. Podolska and D. Napierska: 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@mir.gdynia.pl.

Introduction
The Baltic Sea is a shallow sea with a large catchment area and limited exchange with the North Sea, which can eectively trap pollutants from point and diuse 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 sh populations inhabiting these waters. Biomarkers, which include a number of molecular, cellular, and physiological parameters, have been identied as a powerful and cost-effective approach to obtaining information on the state of the environment and the eects of pollution on living biological resources. Owing to the inherent attributes of sh biology, the overwhelming numbers of confounding factors in eld studies, and the fact that environmental exposure involves chemical mixtures, the specicity of eect of any one compound is dicult to identify. However, it is possible to assess eects between populations with high and low levels of exposure. Since parasites are aected directly by pollution or indirectly by their eects on the intermediate and den ller, 1987), indices such as metabolic or paritive hosts (Mo asitic markers might be useful, economical, and reliable
1054-3139/$32.00

indicators for determining the eects 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 signicant role in developing understanding of natural aquatic ecosystems. Pollutants might promote increased parasitism in aquatic animals, especially sh, 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 hosts physiology (Khan and Thulin, 1991). Studies on the occurrence of diseases and parasites of marine sh also constitute an important component of the monitoring programmes implemented in ICES Member Countries in order to assess the biological eects 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 eects in the pelagic systems of the Baltic Sea have not been

2005 International Council for the Exploration of the Sea. Published by Elsevier Ltd. All rights reserved.

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M. Podolska and D. Napierska

thoroughly investigated to date, with most studies usually having been restricted to bottom-dwelling, non-migratory sh species inhabiting coastal regions. Additionally, herring is a sh species commonly infected with Anisakis simplex e a nematode parasite of marine organisms (Grabda, 1974; Ku hlmorgen-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 eld study on biomarkers in ounder (Platichthys esus) from the southern Baltic Sea indicated that there were statistically signicant dierences 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 sh. 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 aect AChE activity. Strong inhibitory eects have been identied 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 sh has become a technique that is applied commonly to diagnose environmental exposure to cholinergic poisons. The role of AChE in many parasite species diers from that in free-living organisms and might be signicant in hosteparasite 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; Griths 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 eects of neurotoxic pollutants present in the Baltic ecosystem on pelagic sh (herring) and its parasites (A. simplex larvae) with respect to the hosteparasite relationship in AChE activity.

Material and methods

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 24e26 in four locations: 1, western coast (Pomeranian Bay); 2, central coast (Rowy); 3, eastern sk); and 4, Vistula Lagoon (Figure 1). coast (Gulf of Gdan Immediately after capture, the sh were stored in polystyrene boxes with ice (to avoid the postmortem migration of parasites into the esh of the sh). Total body length (cm), weight (g), as well as sex and gonad developmental stage according to Maiers scale (FAO, 1965) were determined for each sh. Otoliths were extracted from each individual to estimate age and the stock component of herring according to the Kompowski (1969) classication. 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 sh was recorded by macroscopic examination. The length of most of the sh (84%) was in the range 23e26 cm. Only sh belonging to the southern coast stock component (n Z 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 sh, close to the tail n. The same location was used for both infected and non-infected animals. Samples of herring muscle and A. simplex larvae from the sh were taken and frozen immediately at 80(C for biochemical analysis.

Preparation of tissue homogenates

AChE extraction was performed on 400e500 mg of muscle tissue using a 0.02 M phosphate buer (pH 7.0) containing 0.1% Triton X 100. The tissue was homogenized in four volumes of buer (4 ml buer per g tissue wet weight) and centrifuged at 10 000 ! 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 (5e53 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 and Galgani, 1998). The ena microplate reader (Bocquene zyme kinetic was monitored at 412 nm. The standard reaction mixture, nal volume 0.380 ml, contained 0.02 M phosphate buer, 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.

Acetylcholinesterase activity in herring and parasites

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163
S T U W
13 12 11 10 9

55 2

8 7 6 3 4 5 4 3

54

1
14 15 16 17 18 19

2 1

20

sk (Or1owo); 4, Figure 1. Location of herring sampling sites: 1, western coast (Pomeranian Bay); 2, middle coast (Rowy); 3, Gulf of Gdan Vistula Lagoon.

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 sh and its parasites. The following models were tted:

Model 1 AChE activity in herring including infected

and non-infected individuals (n Z 225) lnAChEZLTCCFCageCsexCgonCareaCinf Cerror;

Model 2 AChE activity in herring with parasitic AChE

activity as the covariate (n Z 98) lnAChEZLTCCFCnCACTCageCsexCgonCarea Cerror;

Model 3 AChE activity in parasites (n Z 98)

lnAChEZLTCCFCageCsexCgonCareaCerror; where LT is the total length of sh, CF the sh body condition factor, n the number of parasites, ACT the parasitic AChE activity, age the age group of sh, sex, the sex of sh, gon the gonad maturity stage, area the sampling area, and inf is the presence of A. simplex larvae. First, the full model (all considered variables and factors included) was tted. Corner point parameterization

was used, i.e. the factor eects for level one were assumed to be zero for all factors. Thus, the factor eects for other levels can be regarded as the dierences between the eect at a given level and the eect at level one. Body length and condition factor of sh, 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 signicance of factors and the covariates was tested and only signicant terms were left in the nal model. Similarly, factor levels that did not produce a signicantly dierent 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 signicant increase in deviance (the GLM measure of discrepancy between modelled and observed values) were left in the model. The signicance of the eects and factor levels was tested with an F-test. The distributions of the model residuals were analysed to test the model assumptions and performance.

Results
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 sh. 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

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M. Podolska and D. Napierska hosts (Figure 4). The model explained 35.3% of the deviance.

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.

AChE activity models

Only the area eect was signicant in the model of AChE activity in herring (model 1). This eect was lowest in the sk and highest on the western coast (Figure 2). Gulf of Gdan The model explained only 11.9% of the deviance. Parasitic AChE activity and sampling area were signicant 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 sk (Figure 3a). The eseect was lowest in the Gulf of Gdan sk and Vistula timated AChE activity for the Gulf of Gdan Lagoon was not signicantly dierent (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. Sampling area and host sex were signicant in the model of AChE activity in A. simplex larvae (model 3) (Table 3). In contrast to the host AChE activity model, the area eect was highest in eastern areas (Figure 3b). The Gulf of sk and Vistula Lagoon eects were not signicantly Gdan dierent. AChE activity was higher in male than female

Discussion
Acetylcholinesterase activity is usually high in sh living in clean environments and decreases in polluted areas. The study by Kirby et al. (2000) indicated that there was cholinesterase inhibition in ounder 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 et al. (1995) indihigher in less polluted waters. Bocquene cated high inhibitory activities of several organophosphate pesticides (OP) and carbamate (CB) compounds on AChE extracts of marine organisms, including sh. It is reported that acetylcholinesterase activity was directly inhibited by organophosphates in European sea bass (Dicentrarchus lab` et al., 2003) and by carbamates in rainbow trout rax) (Varo (Oncorhynchus mykiss) (Zinkl et al., 1987). The relationship between OP and/or CB exposure and AChE inhibition was also studied in several other sh species e mosquitosh (Gambusia anis) (Boone and Chambers,

Table 1. Mean AChE activity (nM min1 mg1 protein) in herring muscles (a) and A. simplex parasites (b) from the studied areas. Males (a) Herring non-infected Area sk Gulf of Gdan Vistula Lagoon Middle coast Western coast n 11 15 12 15 53 sk Gulf of Gdan Vistula Lagoon Middle coast Western coast 15 4 15 15 49 102 AChE activity 19.88 22.85 20.02 27.91 23.02 17.88 26.37 26.90 28.56 24.60 23.78 Male hosts (b) A. simplex larvae Area sk Gulf of Gdan Vistula Lagoon Middle coast Western coast n 13 4 15 11 43 AChE activity 137.04 99.30 39.04 64.20 80.71 s.e. 22.90 6.82 4.56 7.94 9.57 n 13 16 11 15 55 s.e. 2.12 1.57 1.63 2.98 1.18 1.31 1.38 3.51 2.18 1.45 0.93 n 10 14 14 15 53 16 24 15 15 70 123 Females AChE activity 20.61 19.21 23.90 27.94 23.19 19.16 22.17 29.34 29.13 24.51 23.94 Female hosts AChE activity 82.17 86.26 35.10 51.20 65.50 s.e. 14.17 7.27 6.16 5.55 5.14 n 26 20 26 26 98 Average 109.60 88.87 37.37 56.70 72.17 s.e. 14.29 6.02 3.65 4.72 5.12 s.e. 1.92 1.43 2.66 2.33 1.17 1.38 1.20 2.97 2.92 1.13 0.82 n 21 29 26 30 106 31 28 30 30 119 225 Average 20.23 21.09 22.11 27.93 23.11 18.54 22.77 28.12 28.84 24.55 23.87 s.e. 1.41 1.10 1.64 1.86 0.83 0.94 1.08 2.27 1.79 0.89 0.61

Non-infected sum Herring infected

Infected sum Sum

Sum

Acetylcholinesterase activity in herring and parasites

% of variance accounted for = 11.9 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 Gulf of Gdask Vistula Lagoon

165

Middle coast

Western coast

Figure 2. Eects of area with standard errors estimated for the model of AChE activity of infected and non-infected herring.

1997), largemouth bass (Micropterus salmoides), bluegill sunsh (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 inuence of contaminants. AChE inhibition in herring was observed in the semi-enclosed areas of the sk and Vistula Lagoon, in contrast to the signifGulf of Gdan icantly 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

ounder (P. esus) was signicantly lower at sampling sites sk (regarded as polluted) located within the Gulf of Gdan 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 ounder could be associated with the heavy anthropogenic pressure of industrial and agricultural wastes. The existence of extremely low thresholds for induction of inhibitory eects on AChE suggests that detection in sh is possible after exposure to insecticide concentrations of around 0.1e1 mg l1 (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),

a) host
0.60 0.40 0.20 0.00

% of variance accounted for = 23.4

factor effect

b) parasite
0.00 -0.20 -0.40 -0.60 -0.80 -1.00 -1.20 Gulf of Gdask Vistula Lagoon Middle coast

% of variance accounted for = 34.7

Western coast

Figure 3. Eects of area with standard errors estimated for the model of AChE activity in herring muscles (a) and A. simplex parasites (b).

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Table 3. Parameter estimates with standard error (s.e.) for the nal model of AChE activity in A. simplex. Percentage of variance accounted for 35.3%. Parameter Intercept sk C Vistula Lagoon Gulf of Gdan Middle coast Western coast Male hosts Female hosts Estimate 4.6 0 0.99 0.51 0 0.26 s.e. p

Table 2. Parameter estimates with standard error (s.e.) for the nal model of AChE activity in herring. Percentage of variance accounted for 22%. Parameter Intercept Parasitic AChE activity sk C Vistula Lagoon Gulf of Gdan Open sea Estimate 3.16 0.002 0 0.23 s.e. p

0.09 !0.001 0.001 0.022 Aliased 0.08 0.004

0.11 !0.001 Aliased 0.14 !0.001 0.13 !0.001 Aliased 0.11 0.026

hydrocarbons, and detergents (Payne et al., 1996). Contami sk and Vistula Lagoon is well docunation in the Gulf of Gdan mented. Higher levels of persistent organic pollutants (POPs) have been recorded in sediment samples collected from the sk than in those taken from open sea areas (PazGulf of Gdan dro, 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 sk. Sapota (2004) established the level of polychloriof Gdan nated 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 sk (with the highest pollution levels coming from the Gdan 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. sk as the site most contamThey identied the Gulf of Gdan inated by DDTs among the studied areas of the Bothnian sk. The contaminant Bay, Bothnian Sea, and the Gulf of Gdan input in Pomeranian Bay is assumed to be lower than that of sk (Konat and Kowalewska, 2001; Emeis the Gulf of Gdan 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 eect 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 signicant, negative correlation between host and parasite AChE activity was noted. The dierence 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 para sk. The results sites (highest level) from the Gulf of Gdan 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 ndings of Lee (1996) are related to the role of AChE secreted by parasites and its inuence on the inammatory response in host tissues. Lee also stated that ACh enhances the release of lysosomal enzymes which are mediators of acute inammation; thus, the AChE secreted by nematodes may interfere with this

0.00

-0.10

factor effect

-0.20

-0.30

-0.40

-0.50

male hosts

female hosts

Figure 4. Eect of host sex with standard errors estimated for the model of AChE activity in A. simplex parasites.

Acetylcholinesterase activity in herring and parasites process by hydrolyzing the ACh before it reaches its target cells, reducing inammation in the immediate vicinity of the nematodes. On the other hand, recent ndings indicate that ACh inhibits the release of pro-inammatory mediators in mammals by deactivating peripheral macrophages (Tracey, 2002; De Simone et al., 2005). It is possible that ACh could have both eects on the immune system: proinammatory when acting on muscarinic receptors (Sato et al., 1998), and anti-inammatory 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 inuence the inammatory response in the sh. 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 hosts acetylcholine. The ndings 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 sh. 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 inuence of dierent factors. Fish are usually exposed simultaneously to many dierent 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 sh is scant (Hoole et al., 2003). Multidisciplinary approaches, which include the elds of parasitology, ecotoxicology, ecology, and sh biology, could help ll the gaps in existing knowledge.

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