SOURCES, EMISSIONS AND CONTROL OF HEAVY METALS Heavy Metals and Metallothionein Concentration in Spiders from Variously Polluted Areas A. Babczyńska1, G. Wilczek1, E. Szulińska1, I. Franiel2 1Department of Animal Physiology and Ecotoxicology, University of Silesia, Bankowa 9, 40-007 Katowice POLAND, [email protected] 2Department of Ecology, University of Silesia, Bankowa 9, 40-007 Katowice POLAND, [email protected] Abstract Spiders inhabiting post industrial environments, such as waste heaps or ore-bearing areas, are exposed to high concentrations of metals, accumulated in the body of their prey. The survival and reproductive success of the predators depend on the strategy of metal neu- tralization. Although they are generally recognized as macroconcentrators of metals, what is especially true for actively hunting spiders, some groups of spiders, mainly web-building species, eliminate the excess of the xenobiotics. Both strategies require the synthesis of metal-binding molecules such as metallothioneins (Mts). The low-molecular, multifunc- tion protective proteins, often postulated as biomarkers of metal exposure are still not well recognized in spiders yet. The aim of this study was to asses quantitatively and qualitative- ly, by immunodetection method, Elisa, the concentration of metallothioneins in various spider species (web building Araneus diadematus (Araneidae), Agelena labyrinthica (Age- lenidae), Linyphia triangularis (Linyphidae) and actively hunting Xerolycosa nemoralis (Lycosidae) differing in hunting activity, diet composition and life activity. Adult males and females were collected from three variously polluted areas in southern Poland: Olkusz, ore- bearing post industrial site; Katowice-Wełnowiec: post metallurgic waste heap, Pilica: the reference, rural, area. The concentration of metallothioneins has been analyzed in relation to the metal concentration in spiders body. The analysis of results obtained shows a strong species-dependence of the Mts level, with the highest concentration in L. triangularis and stage-dependence with the lowest Mts concentration in eggs. The correlations of Mts con- centration and metals level in studied spider species has been also discussed. Key words: Elisa, heavy metals, metallothioneins, spiders Introduction the environment. The mechanism efficient- Metallothioneins (Mts) belong to the ly protects organisms against metal pollu- group of low molecular (6-7 kDa) cytosolic tion but it is also useful as a biomarker of termostable proteins of high cysteine con- exposure to heavy metals (Dabrio et al., tents (30%) and contain no aromatic amino- 2002; Yudkovski et al., 2008). acids (Dabrio et al., 2002, Amiard et al., Spiders inhabit such environments as 2006; Wilczek et al., 2008). The protein forests, meadows, human settlements, have been found in vertebrates, invertebrates sandy and rocky places as well as water and plants. They are constantly present in ecosystems. They control the quantity of organisms living in optimal conditions, but insect populations due to their low food under increased metal concentration in the specificity and hunting intensity (Nentwig, environment their synthesis increases (Ami- 1987). As secondary consumers they are ard et al., 2006). The presence of cysteine - especially exposed to metals in their food. SH groups causes their high affinity to metal High concentration of metals in their body ions. The proteins bind both biogenic (Cu, places them among co-called macroconcen- Zn) and toxic (Cd, Pb, Hg) metals, what is trators of metals. The elements enter spi- connected with their biological role. This ders' body through the alimentary tract and includes the regulation of homeostasis of they are accumulated mainly in midgut biogenic elements, necessary in metaloen- glands as mineral granules (Hopkin, 1986). zymes synthesis, and elimination of the The time of storage depends on various fac- excess of the elements. They take part in the tors such as spider gender and species, its detoxication of xenobiotic metals. The nutritional status and kind of metal (Wil- induction of metallothionein is a biochemi- czek, 2008). The granules can be either cal response to high metal concentration in stored lifelong or removed holocrynically 994 15th ICHMET SOURCES, EMISSIONS AND CONTROL OF HEAVY METALS into gut lumen and excreted (Hopkin, 1986). with BRC-185 bovine liver as reference Both strategies: storage or elimination require material (IRMM, Geel, Belgium). The per- the synthesis of metal binding molecules, centage recovery of spiked samples was high: such as metallothioneins. Therefore the aim 93-96% for the measured concentrations of of this work was to asses the role of metalloth- Pb, Cd, Cu, Zn. ioneins in metal neutralization in various spi- For statistical analyses all assays were der species differing in hunting strategy and based on six samples, performed in duplicate. intensity, diet composition and life activity in The results were reported as mean values ± relation to metal pollution of their habitats. SD. Normality was checked by the Kol- mogorov-Smirnov test. The data were tested Materials and Methods. for homogeneity of variance using Levene's Five spider species, abundantly inhab- test of equality of error variances. Whenever a iting polluted environments, were chosen for significant effect was observed, Least Signifi- this study. Web building Araneus diadema- cant Difference (LSD) test was used for post tus, (Araneidae) build large orb webs high hoc one-way analysis of variance (ANOVA). above the ground and feed mainly on flying Results with p < 0.05 were considered signif- insects such as Diptera and Lepidoptera. icant. The data were analyzed using Agelena labyrinthica (Agelenidae) spreads STATISTICA 8.0 package (StatSoft, Inc. its webs close to the ground and hunts main- (2007). STATISTICA (data analysis software ly for Coleoptera, Hymenoptera, Brachycera system), version 8.0. www.statsoft.com). (Wilczek, 2008). Linyphia triangularis, (Linyphiidae) constructs its horizontal webs Results among branches of bushes and pine trees The highest metallothionein concen- and eats mainly Nematocera and tration for all studied spider species was Homoptera. Xerolycosa nemoralis (Lycosi- found in individuals collected from the heav- dae) is an active hunter that moves on the ily polluted area of Olkusz (Table 1, 2) and it surface of soil, and its diet consists mainly of was always was significantly, up to 30 times, larval stages of Diptera, Collembola, higher than in spiders from the reference Coleoptera and Hymenoptera. Adult males site. The animals from the waste heap in and females were collected from three vari- Wełnowiec had lower Mts level than the spi- ously polluted areas in southern Poland: ders from Olkusz, but it was still more than Olkusz, ore-bearing post industrial site; 4.5 times higher than in the control, irre- Katowice-Wełnowiec: post metallurgic waste spectively of the species. The highest Mts heap, Pilica: the reference, rural, area. concentration from among various spiders Metallothioneins were measured by species was found in web building L. triangu- enzyme-linked immunosorbent assay follow- laris from all study sites (Table 1, 3). ing the protocol optimized for spiders. Mouse From metals studied, cadmium accu- Metallothionein monoclonal antibody mulated mainly in the mid gut glands of A. (Stressgen), goat anti-mouse IgG Polyclonal labyrinthica and X. nemoralis. In these spi- Antibody, AP Conjugate (Stressgen) and p- ders concentration of the element was about nitrophenylephosphate (Sigma) as a reaction 6 times higher than in the remaining spider substrate. The color intensity, reflecting the species from all study sites (Table 4). Among concentration of metallothionein calculated all studied species the accumulation of cad- according to metallothioneine standard mium was higher in the individuals collect- curve, was measured using Tecan Infinite ed in heavily polluted habitats. The level of M200 microplate reader at 405 nm. Metal- cadmium in spiders from Olkusz and lothionein concentration was expressed as % Wełnowiec was 2-3 times higher than in the of total protein. Protein concentration was specimen from Pilica in each spider species assayed according to Bradford (1976). (Table 4). Metal concentration was measured The highest Cu concentration was with a Solaar Unicam 939 atomic absorption found in female A. labyrinthica from spectrophotometer in an air- acetylene flame Wełnowiec, significantly exceeding the val- for Zn and Cu and in a PU-93 090X graphite ues noted In individuals of the same species furnace for Cd and Pb. Metal levels were cal- from the remaining sites. In actively hunting culated on the basis of similarly prepared X. nemoralis the highest copper concentra- Merck standards. The accuracy of determi- tion was found in individuals from the refer- nation was controlled using spiked samples ence site (Table 5). 15th ICHMET 995 SOURCES, EMISSIONS AND CONTROL OF HEAVY METALS Discussion cules with sulphydryl groups (Brown 1982, Results described above suggest that Hopkin, 1989). Unlike actively hunting X. metallothionein concentration depends on nemoralis and tunnel web building A. the metal level in spiders' body, related to the labyrinthica, two other spider species, L, tri- level of the elements in their habitats. The angularis and A. diadematus, collected in the concentration of the proteins was higher in reference site accumulated lower amount of spiders that accumulated higher amounts of cadmium