Journal of Invertebrate Pathology 100 (2009) 189–192

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Journal of Invertebrate Pathology

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Short Communication

Effect of zinc on lysozyme-like activity of the seastar Marthasterias glacialis(Echinodermata, Asteroidea) mucus

L. Stabili a,b,*, P. Pagliara a

a Department of Biological and Environmental Sciences and Technology, University of Salento, via Prov.le Lecce-Monteroni, 73100 Lecce, Italyb Istituto per l’Ambiente Marino Costiero – CNR, via Roma 3, 74100 Taranto, Italy

a r t i c l e i n f o

Article history:Received 9 July 2008Accepted 29 January 2009Available online 6 February 2009

Keywords:LysozymeSeastarMarthasterias glacialisMucus

0022-2011/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.jip.2009.01.005

* Corresponding author. Address: Department of BSciences and Technology, University of Salento, via ProLecce, Italy. Fax: +39 0832 298626.

E-mail address: loredana.stabili@iamc.cnr.it (L. Sta

a b s t r a c t

Lysozyme represents the best characterized enzyme involved in the self-defense from bacteria. In thisstudy we analysed the effects of zinc on the lysozyme-like activity of the seastar Marthasterias glacialismucus. This activity, detected by measuring the cleared lysis area of dried Micrococcus lysodeikticus cellwalls on Petri dishes, was significantly reduced in presence of zinc. The results are discussed in the lightof elucidating the possible relationship between environmental contaminants and increased disease sus-ceptibility in seastars due to the decrease of antibacterial protection. The benefits of using the test of lyso-zyme activity to monitoring environmental pollution are highlighted.

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1. Introduction

Environmental pollution by metals, especially cadmium, mer-cury, lead, copper and zinc, has become one of the most importantproblems in marine coastal areas as a consequence of anthropo-genic inputs, via industrial run-off, river outflows, and domesticsewage (UNEP, 1996). Zinc, is an essential trace element poten-tially harmful at elevated levels. The concentrations of this metal,in pristine water systems, range from 2 � 10�6 to 1 � 10�4 mg/L,but in contaminated areas concentrations up to 1.5 � 10�2 mg/Lcan be found (Van den Berg et al., 1987). Zinc is used as fertiliser,furthermore its release in the Mediterranean Sea, including Italiancoasts, is due to industrial pollution from the mines, refineries andother industrial activities (Çelo et al., 1999).

In marine invertebrates heavy metals affect survival, growth,reproduction, metabolism and immunity (Matozzo et al., 2001;Mydlarz et al., 2006). A number of studies on the impact of heavymetals on the immune system have been carried out in molluscs,crustaceans and oligochaetes (Mydlarz et al., 2006) whilst, littleinformation is known about echinoderms. Canicattì and Grasso(1988) observed that high zinc ion concentrations produce aninhibitory effect on the hemolytic activity of Holothuria polii.

Lysozyme, identified in a wide range of organisms, representsthe best characterized enzyme involved in self-defense from bacte-ria (Jolles and Jolles, 1984). This enzyme cleaves the b1–4 bonds

ll rights reserved.

iological and Environmentalv.le Lecce- Monteroni, 73100

bili).

between N-acetylglucosamine and N-acetylmuramic acid of bacte-rial cell walls. A lysozyme-like activity was evidenced in the eggsand mucus of the seastar Marthasterias glacialis (Canicattì andD’Ancona, 1990; Stabili and Pagliara, 1994). Mucus typically formsa slippery coating and contains antimicrobial agents (Suzuki et al.,2003) that prevents bacteria and debris from accumulating on thebody surface. The basal levels of lysozyme in the mucus protect theorganism from bacteria living in the same environment. Conse-quently, a reduction of mucus antibacterial activity, due to highconcentrations of contaminants, could make the seastars more sus-ceptible to bacterial infection. A variety of studies have demon-strated that in invertebrates environmental perturbations,affecting immunological competence, can increase susceptibilityto infectious disease (Galloway and Depledge, 2001; Rivera et al.,2003). In this framework we analysed the effects of a high zinc con-centration on the lysozyme-like activity in the mucus of the seastarM. glacialis. We evaluated the effects of zinc on lysozyme-likeactivity by comparing control and stressed seastars. In addition,we measured the capacity of recovery of M. glacialis lysozyme-likeactivity after the zinc removal. As already observed by Goven et al.(1994) for the earthworm Lumbricus terrestris and Marcano et al.(1997) for the polychaete Eurythoe complanata, the evaluation oflysozyme-like activity reduction in seastars may give an early indi-cation of disease susceptibility and, ultimately, survival.

2. Materials and methods

After the reproduction season (March) 80 adult specimens of M.glacialis (Echinodermata, Asteroidea) were collected, by using SCU-BA equipment, in a coastal area of the Northern Ionian Sea (S. Cate-

Fig. 1. Marthasterias glacialis mucus lysozyme-like activity in control and zinctreated seastars at T0, T48 and in control and post-zinc treated seastars at T96.CT48 = control at T48; TT48 = treated seastars at T48; CT96 = control at T96;TT96 = treated seastars at T96. Each column represents the mean value ±SD (n = 40for CT0, TT0, CT48, TT48 CT96; n = 24 for TT96).

190 L. Stabili, P. Pagliara / Journal of Invertebrate Pathology 100 (2009) 189–192

rina, Lecce–Italy), where Zn concentration was 1 � 10�6 mg/L (Buc-colieri et al., 2004). In the laboratory, each animal was placed on alarge Petri dish, left in this position for 15 min and then the undi-luted mucus was collected (T0) by a sterile plastic pipette. The mu-cus was immediately centrifuged at 400g for 15 min at 4 �C and thesupernatant was stored at �80 �C and successively employed forlysozyme activity assay. The mucus protein concentration wasdetermined using the protein–dye binding kit Biorad (Bradford,1976). Individual mucus was diluted to obtain an equal proteinconcentration for the assays (0.29 ± 0.01 mg/mL).

After the mucus collection the seastars were divided into twosets of 40 individuals each. Individuals of each set were separatedinto five groups (8 individuals each one). The first set (control) wasplaced in five aquaria filled with filtered seawater (0.2 lm) and thesecond set (treatment) in five aquaria with filtered seawater andZnCl2 (final concentration of 5 mg/L). Aquaria were maintained at20 �C and 37‰ salinity. After 48 h (T48) individuals were removedfrom the aquaria and the mucus was collected. Control specimenswere then replaced in fresh filtered seawater. Instead, zinc treatedanimals were washed gently in seawater (15 min) to remove resid-ual of zinc and placed into fresh filtered seawater (post-zinc treat-ment). After 48 h of this treatment (T96) all the seastars wereremoved from aquaria and mucus was collected again. Along allthe experimental period the survival rates of both control and trea-ted seastars were evaluated.

Lysozyme-like activity was assayed at T0, T48 and T96 by usingthe agar diffusion lysozyme test as described by van Bijsterveld(1974). Briefly 700 lL of 5 mg/mL of dried Micrococcus lysodeikticuscells (Sigma) were diluted in 7 mL of 0.05 M phosphate buffer (PB)agarose, pH 5.2 then spread on a Petri dish. Five replicate wells(6.3 mm diameters) per individual organism were sunk and filledwith 30 lL of mucus. The diameter of the cleared zone of the fivereplicates was recorded after overnight incubation at 37 �C, aver-aged for each individual, and compared with those of referencesamples represented by hen-egg-white lysozyme (Merck), care-fully measured as concentration and volume. Zinc concentrationsranging from 1 to 10 mg/L were employed as negative control.

Analysis of variance (ANOVA) was used to test for differences inthe lysozyme-like activity between control and treatment seastars.Prior to analysis the assumption variance homogeneity was testedby Levene test. Given the unbalanced design and the skewed nat-ure of the data, p-values were obtained by a permutational proce-dure. Therefore, tests for the significance of all terms involved inthe full ANOVA model were made by using a distance-based per-mutational multivariate analysis of variance (PERMANOVA; Ander-son, 2001) based on Euclidean distances on untransformed data.Each term in the analysis was tested using the highest number ofpossible random permutations of the appropriate units (Andersonand ter Braak, 2003). The analyses were performed using the add-on package PERMANOVA + (Anderson et al., 2008) in the PRIMERv6 computer program (Clarke and Gorley, 2006). Analysis of co-variance (ANCOVA) among diameter of lysis and seastars sizewas also performed.

3. Results

Lysozyme-like activity of M. glacialis mucus, evaluated at thedifferent times, and expressed as mean values of lysis diameter,is shown in Fig. 1. Before zinc treatment (T0), the mean diameterof lysis was 6.5 ± 0.3 mm corresponding to 0.39 mg/mL of hen-egg-white lysozyme. After 48 h (T48), in control conditions themean diameter of lysis was 6.47 ± 0.34 mm and in treated organ-isms was 1.9 ± 0.4 mm (corresponding to 0.11 mg/mL of hen-egg-white lysozyme). The difference in lysozyme-like activity betweencontrols and treatments was significant (P < 0.05). After 48 h ofzinc removal (T96), we evidenced the absence of significant differ-

ences in lysozyme–like activity between control and post-zinc sea-stars (mean diameter 6.48 ± 0.33 mm and 6.23 ± 0.35 mmrespectively). Thus the treated specimens showed the capacity ofrecovery from the chemical stress. In Table 1 we summarized dataon the body sizes as well as the mucus lysozyme activity of bothcontrol and post-zinc treated animals.

After 48 h of zinc treatment, the mortality rate was approxi-mately 40% with no differences among the seastars with differentbody sizes (Table 1). The control groups of M. glacialis insteadshowed no mortality.

ANCOVA showed that diameter of lysis and seastars size signif-icantly covariate both at T0 (MScov 7.1439; MSdenom 0.01756,P < 0.01) and T96 (MScov 6.1036; MSdenom 0.018544; P < 0.01). ANO-VA highlighted the significance of the interaction Treat-ment � Time (Tr � Ti) suggesting that differences betweencontrol vs zinc treatment varied with times. Post-hoc comparisonsof treatment within each of the three levels of the factor time iden-tified these differences as occurring in T48, where the mean diam-eter of lysis in control was by far higher than that observed in zinctreatment (Fig. 1). No significant differences between treatmentswere observed in T96 and, as expected, in T0. ANOVA also indicatedthe lack of a tankness effect that is differences between treatmentsin time did not vary among tanks, as indicated by the lack of signif-icance of the Ta (Tr) � Ti (Table 2).

4. Discussion

Available literature on zinc impact on marine invertebrates sug-gests that concentrations ranging from 0.097 to 11.3 mg/L, arehighly toxic (Martin et al., 1989). In this study we used a high zincion concentration taking into account of both its high concentra-tion in some marine invertebrates eaten by seastar (Viviani,1992) and the accumulation of this heavy metal by metallothio-nein-like proteins in seastars (den Besten et al., 1990). Our resultsshow that:

— lysozyme-like activity of M. glacialis mucus was inhibited inshort-term by the presence of high zinc ion concentrations.

Zinc might act directly on the enzyme or indirectly through theorganism itself. Anyway the suppression of lysozyme-like activity,by increasing the susceptibility of seastars to bacteria, may portenda decrease in immunocompetence and pathological manifesta-

Table 1Body size dimension (cm) and mucus lysozyme-like activity expressed as diameter of lysis (mm) in both control and post-zinc treated seastars (T96). Each value represents themean of five replicates ± SD.

Control animals

i ii iii iv v

Body-size (cm) Lysis (mm) Body-size(cm) Lysis (mm) Body-size (cm) Lysis (mm) Body-size (cm) Lysis (mm) Body-size (cm) Lysis (mm)

21.5 5.98 ± 0.12 22.5 5.95 ± 0.15 21.0 5.98 ± 0.11 22.5 5.92 ± 0.14 20.5 5.96 ± 0.1122.0 6.21 ± 0.13 24.5 6.06 ± 0.11 23.5 6.38 ± 0.14 23.0 6.27 ± 0.16 24.5 6.15 ± 0.1125.0 6.25 ± 0.12 24.5 6.2 ± 0.11 24.0 6.38 ± 0.15 23.5 6.53 ± 0.13 24.0 6.23 ± 0.1326.5 6.35 ± 0.11 25.0 6.37 ± 0.15 26.5 6.39 ± 0.14 25.5 6.37 ± 0.11 25.5 6.28 ± 0.1130.0 6.45 ± 0.15 26.0 6.62 ± 0.11 27.5 6.47 ± 0.11 28.5 6.52 ± 0.15 27.0 6.47 ± 0.1529.0 6.90 ± 0.13 31.0 6.57 ± 0.13 30.0 6.88 ± 0.14 30.5 6.78 ± 0.16 30.5 6.65 ± 0.1332.5 6.94 ± 0.14 32.0 6.92 ± 0.14 33.5 6.94 ± 0.15 33.5 6.91 ± 0.17 33.5 6.81 ± 0.1533.5 6.85 ± 0.16 34.5 6.83 ± 0.13 34.0 6.94 ± 0.15 33.5 6.82 ± 0.15 34.5 6.92 ± 0.15

Treated animals

vi vii viii ix x

Body-size (cm) Lysis (mm) Body-size (cm) Lysis (mm) Body-size (cm) Lysis (mm) Body-size (cm) Lysis (mm) Body-size (cm) Lysis (mm)

21.0 5.62 ± 0.14 21.0 Dead 20.5 5.62 ± 0.11 20.0 5.65 ± 0.12 21.5 5.66 ± 0.1423.5 Dead 22.0 5.98 ± 0.11 22.5 Dead 24.0 Dead 22.5 6.12 ± 0.1525.0 6.1 ± 0.13 26.0 6.12 ± 0.13 24.0 6.35 ± 0.12 24.5 5.99 ± 0.12 23.0 Dead27.5 6.12 ± 0.11 27.5 6.14 ± 0.13 26.0 Dead 27.0 Dead 28.0 6.19 ± 0.1627.5 6.27 ± 0.13 28.0 Dead 28.5 6.49 ± 0.13 29.5 6.31 ± 0.14 28.5 Dead29.0 Dead 30.0 6.48 ± 0.11 30.0 6.53 ± 0.14 30.0 Dead 31.0 6.66 ± 0.1432.0 Dead 30.5 Dead 34.0 6.63 ± 0.15 32.0 Dead 32.5 6.67 ± 0.1534.5 6.63 ± 0.12 35.0 6.64 ± 0.11 34.5 Dead 33.0 6.67 ± 0.15 33.5 Dead

The small Roman numerals indicate the seastar groups: five groups (eight individuals each one) for control and five groups (eight individuals each one) for treatment.

Table 2Results of ANOVA testing for lysozyme-like activity differences in control vs zinc treatment across the three times.

Source Degrees of freedom Sum of squares Mean squares F-ratio P (perm) Unique perms

Tr 1 25.25 25.25 294.04 0.0002 248Ti 2 211.96 105.98 2614.7 0.0002 4981Ta(Tr) 8 0.25639 3.20E+02 0.3352 0.9518 4985Tr � Ti 2 211.96 105.98 2614.7 0.0002 4988Ta(Tr) � Tia 12 4.99E+02 4.16E+01 4.35E+02 1 4982Residual 175 16.732 9.56E + 02Total 200 689.38

Tr = Treatment; Ti = Time; Ta = Tank.The significance of the main effects of Treatment (Tr) and Time (Ti) indicated in italic are not discussed in the text due to their involvement in significant higher Tr � Tiinteraction (indicated in bold). P-values associated to F-tests were based on a permutational procedure (see text for details).

a Term has one or more empty cells.

L. Stabili, P. Pagliara / Journal of Invertebrate Pathology 100 (2009) 189–192 191

tions. Immunosuppressive effects induced by several pollutantshave been already observed in other marine invertebrates and in-sects (Chu and Hale, 1994; Sorvari et al., 2007).

- The seastars have the capacity of recovery from the chemicalstress as evidenced by the absence of differences in lysozymeactivity between control and post-zinc treated animals.

- The significant covariance between body size and lysozymeactivity observed represents an interesting issue that could beconsidered in future studies.

Taking into account of both the susceptibility of M. glacialislysozyme-like activity to zinc and the simpleness, rapidity and sen-sitivity of the lysozyme test, we suggest its employment in assess-ing risks of animal exposure to chemical contamination. This couldgive an additional support to the conventional immunological testsemployed i.e. direct coelomocyte counts and cell-mediated immu-nity. In order to evaluate if the variations of lysozyme-like activitydo not highlight a single stress effector, but rather a general stresscondition, further studies will be undertaken. In particular eitherdifferent zinc concentrations, inducing lysozyme-like activityreduction but no mortality, or other xenobiotics will be employedto generalize our results. Thence, a lysozyme-like activity variationcould be thought of as taking a patient’s temperature with a ther-

mometer. Unusual temperatures do not inform about the disease:they inform only about the variation from the normal.

Acknowledgments

Many thanks to Dr. Antonio Terlizzi and Dr. Luigi Musco for theprecious suggestions in statistical analysis.

References

Anderson, M.J., 2001. A new method for non-parametric multivariate analysis ofvariance. Aus. J. Ecolo. 26, 32–46.

Anderson, M.J., ter Braak, C.J.F., 2003. Permutation tests for multi-factorial analysisof variance. J. Stat. Comput. Simulat. 73, 85–113.

Anderson, M.J., Gorley, R.N., Clarke, K.R., 2008. PERMANOVA+ for PRIMER: Guide toSoftware and Statistical Methods. PRIMER-E, Plymouth, UK.

Bradford, M.M., 1976. A rapid and sensitive method for the quantification ofmicrogram quantities of proteins using the principle of protein–dye binding.Anal. Biochem. 72, 248–254.

Buccolieri, A., Buccolieri, G., Cardellicchio, N., Dell’Atti, A., Di Leo, A., Maci, A.,Petronio, B.M., 2004. Distribution and speciation of metals in surface sedimentsof the Taranto Gulf (Ionian Sea, Southern Italy). Annu. Chim. 94, 469–478.

Canicattì, C., D’Ancona, G., 1990. Biological protective substances in Marthasteriasglacialis (Asteroidea) epidermal secretion. J. Zool. Lond. 222, 445–454.

Canicattì, C., Grasso, M., 1988. Biodepressive effect of zinc on humural effector ofthe Holothuria polii immune response. Mar. Biol. 99, 393–396.

Çelo, V., Babi, D., Baraj, B., Çullaj, A., 1999. An assessment of heavy metal pollution inthe sediments along the Albanian coast. Water Air Soil Pollut. 111, 235–250.

192 L. Stabili, P. Pagliara / Journal of Invertebrate Pathology 100 (2009) 189–192

Chu, L.E., Hale, R.C., 1994. Relationship between pollution and susceptibility toinfectious disease in the eastern oyster, Crassostrea virginica. Mar. Environ. Res.38, 243–256.

Clarke, K.R., Gorley, R.N., 2006. PRIMER v6: User Manual/Tutorial. PRIMER-E,Plymouth.

den Besten, E.J., Herwig, H.J., Zandee, D.I., Voogt, P.A., 1990. Cadmium accumulationand metallothionein-like proteins in the sea star Asterias rubens. Arch. Environ.Contamin. Toxicol. 19, 858–862.

Galloway, T.S., Depledge, M.H., 2001. Immunotoxicity in Invertebrates:measurement and ecotoxicological relevance. Ecotoxicology 10, 5–23.

Goven, A.J., Chen, S.C., Fitzpatric, L.C., Venables, B.J., 1994. Lysozyme activity inearthworm (Lumbricus terrestris) coelomic fluid and coelomocytes: Enzymeassay for immunotoxicity of xenobiotics. Environ. Toxicol. Chem. 3, 607–613.

Jolles, P., Jolles, J., 1984. What’s new in lysozyme research? Med. Cell. Biochem. 63,165–189.

Marcano, L., Nusetti, O., Rodríguez-Grau, J., Briceño, J., Vilas, J., 1997. Coelomic fluidlysozyme activity induction in the polychaete Eurythoe complanata as abiomarker of heavy metal toxicity. Bull. Environ. Contamin. Toxicol. 59, 22–28.

Martin, M., Hunt, J.W., Anderson, B.S., Palmer, F.H., 1989. Experimental evaluation ofthe mysid Holmesimysis costata as a test organism for effluent toxicity testing.Environ. Toxicol. Chem. 8, 1003–1012.

Matozzo, V., Ballarin, L., Pampanin, D.M., Marin, M.G., 2001. Effects of copper andcadmium exposure on functional responses of hemocytes in the clam, Tapesphilippinarum. Arch. Environ. Contamin. Toxicol. 41 (2), 163–170.

Mydlarz, L.D., Jones, L.E., Drew Harvell, C.D., 2006. Innate immunity, environmentaldrivers, and disease ecology of marine and freshwater invertebrates. Annu. Rev.Ecol. Evol. Syst. 37, 251–288.

Rivera, M.T., De Souza, A.P., Araujo-Jorge, T.C., De Castro, S.L., Vanderpas, J., 2003.Trace elements, innate immune response and parasites. Clin. Chem. Lab. Med.41 (8), 1020–1025.

Sorvari, J., Rantala, L.M., Rantala, M.J., Hakkarainen, H., Eeva, T., 2007. Heavy metalpollution disturbs immune response in wild ant populations. Environ. Pollut.145, 324–328.

Stabili, L., Pagliara, P., 1994. Antibacterial protection in Marthasterias glacialis eggs:characterization of lysozyme-like activity. Comp. Biochem. Physiol. B. 109 (4), 709–713.

Suzuki, Y., Tasumi, S., Tsutsui, S., Okamoto, M., Suetake, H., 2003. Moleculardiversity of skin mucus lectins in fish. Comp. Biochem. Physiol. B 136, 723–730.

UNEP. 1996. State of the Marine and Coastal Environment in the MediterraneanRegion. MAP Technical Report Series No. 100. UNEP, Athens.

Van Bijsterveld, O.P., 1974. Standardization of a lysozyme test for a commerciallyavailable medium. Its use for the diagnosis of the sicca syndrome. Arch.Ophthalmol. 91, 432–434.

Van den Berg, C.M.G., Merks, A.G., Duursma, E.K., 1987. Organic complexation andits control of the dissolved concentrations of copper and zinc in the Scheldtestuary. Estuar. Coast. Shelf. Sci. 24, 785–797.

Viviani, R., 1992. Eutrophication, marine biotoxins, human health. In: Vollenweider,R.A., Marchetti, R., Viviani, R. (Eds.), Marine Coastal Eutrophication. Elsevier,Amsterdam, pp. 631–662.