Riboflavin content of coelomocytes in earthworm (Dendrodrilus rubidus) field populations as a molecular biomarker of soil metal pollution

Environmental Pollution 157 (2009) 3042–3050

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Riboflavin content of coelomocytes in earthworm (Dendrodrilus rubidus) field populations as a molecular biomarker of soil metal pollution Barbara Plytycz a, *, Urszula Lis-Molenda a, Malgorzata Cygal a, Edyta Kielbasa a, Anna Grebosz a, Micha1 Duchnowski b, Jane Andre c, A. John Morgan c a b c

Institute of Zoology, Jagiellonian University, Ingardena 6, PL 30-060 Krakow, Poland Institute of Environmental Sciences, Jagiellonian University, Krakow, Poland Cardiff School of Biosciences, Main Building, Cardiff University, Cardiff CF10 3TL, Wales, UK

Soil metal pollution reduces riboflavin content of earthworm eleocytes.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 November 2008 Received in revised form 3 March 2009 Accepted 18 May 2009

The effect of Pb þ Zn on coelomocyte riboflavin content in the epigeic earthworm Dendrodrilus rubidus inhabiting three metalliferous soils and one reference soil was measured by flow cytometry and spectrofluorimetry. A reciprocal polluted4unpolluted worm transfer experiment (4-week exposure) was also performed. High proportions of autofluorescent eleocytes were counted in worms from all localities, but intense riboflavin-derived autofluorescence was detectable only in reference worm eleocytes. Other findings were: (i) fluorophore(s) other than riboflavin is/are responsible for eleocyte autofluorescence in residents of metalliferous soils; (ii) riboflavin content was reduced in the eleocytes of worms transferred from unpolluted to metal-polluted soil; (iii) the riboflavin content of D. rubidus eleocytes is a promising biomarker of exposure; (iv) COII mitochondrial genotyping revealed that the reference population is genetically distinct from the three mine populations; (v) metal exposure rather than genotype is probably the main determinant of inter-population differences in eleocyte riboflavin status. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Earthworms Coelomocytes Riboflavin Biomonitoring Pb Zn Genotyping

1. Introduction Innate immunity in earthworms is mediated by coelomocytes (Cooper, 1996), circulating fluid-suspended cells of two main types: amoebocytes originating from the mesothelial lining of the coelom (Hamed et al., 2002), and eleocytes, the latter being differentiated chloragocytes sloughed from chloragogenous tissue (Affar et al., 1998). The number and composition of coelomocytes in adult earthworms are species-specific, especially in regard to autofluorescent eleocytes, which are seldom present in representative species of certain genera (e.g. Lumbricus spp., Aporrectodea spp.) but are common in others (e.g. Eisenia fetida, Dendrobaena veneta, Allolobophora chlorotica, Dendrodrilus rubidus, Octolasion spp.) (Cholewa et al., 2006). Moreover, eleocytes, when present, contain species-specific amounts of autofluorescent riboflavin, as detected and measured by spectrofluorimetry (Koziol et al., 2006; Plytycz et al., 2006). Riboflavin possesses antioxidant properties (Iwanaga et al., 2007) and a recognised ability to stimulate components of the

* Corresponding author. Tel.: þ48 12 663 24 28; fax: þ48 12 634 37 16. E-mail address: [email protected] (B. Plytycz). 0269-7491/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2009.05.046

innate and acquired immune responses in mammals (Verdrengh and Tarkowski, 2005). Post-industrial sites are often characterised by shallow soils contaminated with mixtures of chemicals reflecting local anthropogenic history. Abandoned metal mine-associated soils are frequently inhabited by a limited number of earthworm species typically members of the litter-dwelling ‘epigeic’ ecophysiological category. In the UK these include D. rubidus, whose actively mineralizing calciferous glands and capacity to contain metal toxicosis by macro-accumulative intracellular immobilization enables it to establish sustainable populations on extremely polluted metalliferous soils with pHs ranging over several orders of magnitude (Morgan and Morgan, 1991). Excessive essential or nonessential metal exposures interfere with earthworm performance at all levels of biological organisation, from demographic parameters (Spurgeon et al., 1994) and cellular integrity (Plytycz et al., 2007b), to metabolome (Bundy et al., 2008) and transcriptome (Owen et al., 2008) profiles. Unsurprisingly, therefore, coelomocyte community structure, the immunocompetence of individual coelomocyte types, and molecular activities expressed within coelomocytes have been shown to be significantly altered by metal exposures under laboratory conditions (Fuge´re et al., 1996; Goven

B. Plytycz et al. / Environmental Pollution 157 (2009) 3042–3050

et al., 2005; Wieczorek-Olchawa et al., 2003; Homa et al., 2003, 2005, 2007; Brulle et al., 2008). Comparable observations have not been made on the coelomocytes of populations of earthworms with protracted, multi-generational, histories of exposure to heavily contaminated metalliferous field soils. The main aim of the present study was to measure and compare the riboflavin content in autofluorescent eleocytes of the small ‘gilttailed’ earthworm species D. rubidus sampled directly from a series of contrasting Pb/Zn mine-associated soils and an uncontaminated reference soil. There were two reasons for pursuing this aim. First, given that riboflavin has been functionally implicated in the vertebrate immune system, it would be revealing to know whether the riboflavin content of individual earthworm coelomocytes correlates with ‘real world’ metal exposures and, therefore, whether its assay is a potential easily measured molecular biomarker for contaminated land ecotoxicology applications. A second reason was to determine whether eleocyte riboflavin status provides any indication that certain mine-site earthworm populations have evolved mechanisms to alleviate immunotoxicity. To further ascertain the influence of soil metal content, rather than a population’s exposure history, on riboflavin content a short (4 weeks) reciprocal transfer experiment was performed using worms from one mine site and worms from the reference site, respectively. The experiment entailed maintaining replicated pairs of worms on their ‘own’ field soil and, in parallel, on the ‘other’ field soil for the designated period under standard laboratory conditions. A further, but subsidiary, aim of the study was to gain some provisional insight into a largely ignored, but potentially important, biotic confounding factor in field-based ecotoxicology: the unrecognised presence of cryptic or sibling species that may or may not differ in their responsiveness to chemical contaminants (LinkeGamenick et al., 2000a,b; Rocha-Olivares et al., 2004). It is generally acknowledged by classical taxonomists that D. rubidus is polymorphic, comprising four recognised morphs (Sims and Gerard, 1999). We, therefore, applied our recently designed mitochondrial genotyping markers to a small representative number of individual worms from our sampling sites to estimate their degrees of genetic relatedness.

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2.2. Soil and tissue metal analyses Metal (Pb, Zn, Cu, Cd, and Ni) accumulation was measured in soil samples (S) and in whole-worm tissues (B) by atomic absorption spectrophotometer (Aanalyst 800, Perkin-Elmer), as described in detail elsewhere (Homa et al., 2003; Bednarska and Laskowski, 2008). Metal bioaccumulation factors (BAF) were estimated according to the formula: whole body earthworm metal concentration O soil metal concentration (i.e. BAF ¼ B O S). 2.3. Soil pH Soil pH was measured by a glass–calomel electrode in equilibrated suspensions of 16 g dry soil with 40 ml distilled H2O. 2.4. Harvesting of coelomocytes Earthworms were stimulated for 1 min (in 2007) or 30 s (in 2008) with a 4.5 V electric current to expel coelomic fluid with coelomocytes through the dorsal pores according to a procedure modified after Roch (1979). Briefly, after weighing, washing and dry-blotting, the earthworms were placed individually in Petri dishes containing 1–1.5 ml of extrusion fluid (PBS supplemented with 2.5 g/L EDTA) to prevent cell aggregation (Plytycz et al., 2006). Extruded coelomocytes were counted in a haemocytometer, and 1 ml suspensions used for spectrofluorimetry, and the remaining sample from each worm was fixed in 2% formalin for flow cytometry. 2.5. Flow cytometric measurement and analysis Samples of coelomocytes were analysed with a FACScalibur flow cytometer (BD Biosciences). During analytical experiments, 10,000 thresholded events per worm sample were collected and analysed on the basis of their forward scatter (FS) (for cell size) and sideward scatter (SS) (cell complexity) properties. Fluorescence FL1 (emission 530 nm; excitation 488 nm) was recorded. The resulting files were analysed using WinMDI 2.8 software (Joe Trotter, http://facs.scripps.edu), by producing dot plots and histograms of FL1 autofluorescence. 2.6. Spectrofluorimetric measurements and analysis The spectrofluorimetric measurements were performed on coelomocyte suspension lysates with 2% Triton (Sigma) using a Varian Cary Eclipse Spectrofluorimeter. Excitation spectra were recorded between 300 and 520 nm (excitation at 525 nm), while emission spectra were recorded between 380 and 700 nm using excitation at 370 nm. Presence of unbound riboflavin is expected when monitoring of fluorescence at 525 nm provides the excitation spectra with the two maxima, lower at 370 nm and higher at 450 nm, while excitation of sample at 370 nm results in emission spectrum with a maximum at 525 nm. Arbitrary units (AU) of fluorescence were read using Microsoft Excel v. 97.

2. Materials and methods 2.1. Earthworms and soil samples (Experiment 1) Soil samples, and between 12 and 26 (see Results section for ‘n’ values) adult (clitellate) specimens of D. rubidus, were collected in November 2007 from three subsites at the large Cwmystwyth Pb/Zn mine site in Wales (UK), where work was discontinued in about 1921: Cwmystwyth Cottage (CC; N52:21:31, W3:45:22), Cwmystwyth Stream (CS; N52:21:31, W3:45:25), and Cwmystwyth Cottage East (CE; N52:21:35, W3:44:59). Soil and worms were also collected from an unpolluted reference site, Pontcanna (P; N51:29:30, W3:12:02). Worms were transferred to the laboratory in their soils of origin contained in well ventilated plastic boxes, kept in darkness at a constant temperature room at 16  C, and used experimentally within 5 days. Some worms were subjected to coelomocyte retrieval, others were depurated for 2 days on moist filter paper prior to processing for tissue metal analyses. Posterior segments were amputated from a few worms, immediately snap-frozen in liquid N2, and stored at 80  C for subsequent genotyping. 2.1.1. Laboratory exposures (Experiment 2) Soil samples and clitellate specimens of D. rubidus were collected in November 2008 from the Pontcanna reference site (‘P’ worms, ‘p’ soil), and from the Cwmystwyth Cottage Pb/Zn mine site (‘C’ worms, ‘c’ soil) (see Section 2.1 for site locations). Field soils were thoroughly mixed and sieved to minimise compositional heterogeneity. The worms were acclimated to laboratory conditions (16  1  C; 24 h darkness) for 1 week. Then 8 pairs of worms per treatment were maintained on 200 g dry mass equivalent per pair for 4 weeks either their native field soils (i.e. groups ‘Pp’ and ‘Cc’), or transferred to the non-native soils (i.e. groups ‘Pc’ and ‘Cp’). The experimental worms were kept in plastic boxes with perforated lids; no food was added to the field soils, and moisture content (field capacity) was checked weekly. After exposure the worms were subjected to the same analyses as were performed on the worms examined immediately after field sampling (see Section 2.1).

2.7. Earthworm genotyping Genomic DNA was extracted from CC (n ¼ 5), CS (n ¼ 4), CE (n ¼ 5) and P (n ¼ 5) earthworms using the QIAamp DNA Micro Kit (Qiagen Ltd, UK) according to the manufacturer’s instructions. For each genotyping PCR reaction w100 ng DNA template was amplified using 10 pmol/ml forward and reverse primer (50 -TAGCTCACTTAGATGCCA and 50 -GTATGCGGATTTCTAATTGT respectively), 10 mM dNTP mix and 5 U/ml Taq DNA polymerase buffered with 5 Mg-free Taq PCR amplification buffer and supplemented with MgCl2 (1.5 mM). The reaction was denatured at 95  C for 10 min and then cycled 35 times at 95  C for 30 s, 30 s at 48  C and 72 for 1 min. This was followed by a 10-min final extension at 72  C. Products (469 bp) were resolved by electrophoresis in 1 TAE buffer at 120 V for approximately 30 min in a Pharmacia GNA-100 tank. Nucleic acid bands were visualised on a UV gel documentation system. Prior to sequencing PCR clean-ups were performed using ExoSAP-IT (Amersham Pharmacia, UK) reagents. Exonuclease 1 (0.25 ml) and Shrimp Alkaline Phosphatase (0.5 ml) were mixed with PCR product (10 ml) and incubated at 37  C for 45 min followed by 80  C for 15 min. DNA was sequenced using ABI PRISMÒ BigDye v3.1 Terminator Sequencing technology (Applied Biosystems, Foster City, USA) on the ABI PRISMÒ 3100 DNA Sequencer run by the Cardiff University Molecular Biology Support Unit. Raw sequence traces were confirmed using Finch TV before being imported into Mega v3.1 for alignment and tree construction. The distance-based neighbour joining (NJ) algorithm, using p-distances, was used to estimate tree topology and calculate branch lengths. 2.8. Statistical analysis Results were expressed as means þ standard errors. Differences between means were determined by non-parametric Mann–Whitney ‘U’ test (Microsoft Excel v. 97), with the level of significance established at p < 0.05.

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Table 1 Soil pH and heavy metal contents from the reference (R) and post-industrial (M) sites from Wales, UK. Means are from 3 samples. Most samples were collected in 2007; *samples collected in 2008.

3. Results

Sites

Soil pH and soil metal (Cd, Cu, Ni, Pb, Zn) concentrations are presented in Table 1. The data confirm that the circumneutral (pH ¼ 6.6) Pontcanna (P) reference site is unpolluted. Soil at the acidic (pH ¼ 4.5) CS is contaminated only with Pb. Concentrations of Cd, Cu, Pb, and (especially) Zn are high in the alkaline (pH ¼ 8.0) soil enclosed within the roofless abandoned cottage designated CC; CE cottage-associated soil, in comparison with CC, has a circumneutral pH of 6.8, and is predominantly contaminated with Pb and Zn.

Soil pH

Metal Concentration (mg/kg dry mass) Pb

Zn

Cd

Cu

Ni

R

Pontcanna Pontcanna*

P p

6.6 6.4

84 91

199 120

0.7 0.3

26 33

14 14

M

Cwmystwyth Cottage Cwmystwyth Stream Cwmystwyth Cottage-East Cwmystwyth*

CC CS CE c

8 4.5 6.8 7.4

9141 941 6043 5833

15448 179 7556 7594

49 0.2 18 22

120 40 264 90

31 20 30 30

3.1. Soil characteristics

3.2. Metal accumulation in whole earthworm bodies (Experiment 1) Metal concentrations and bioaccumulation factors (BAF) of D. rubidus analysed soon after field collection are summarized in Fig. 1. Reference-site worms contained very low concentrations of essential (Cu, Ni, Zn) and non-essential (Cd, Pb) metals.

BAF

mg/kg dry weight Pb

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ab

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CS

CE

Fig. 1. Heavy metal content in whole bodies (left panel) and body accumulation factors (BAF – see text for definition; right panel,) in D. rubidus collected directly from field sites in 2007: Pontcanna reference site P (open bars), and metal-polluted subsites Cwmystwyth Cottage (CC), Cwmystwyth Stream (CS), and Cwmystwyth Cottage East (CE) (solid bars). Plotted data are means þ SE, and the numbers of individual worms analysed (‘n’ values) were 11, 16, 5, and 5, respectively. Means not sharing lower-case letters are significantly different (p < 0.05) according to the Mann–Whitney ‘U’ test.

B. Plytycz et al. / Environmental Pollution 157 (2009) 3042–3050

Earthworms inhabiting the three metalliferous sites (CC, CS, and CE) contained relatively high concentrations of Pb and Zn, with CC worms also contained high Cd content. Whole-worm metal concentrations at all four sites can be arranged in the following ascending series: Cd (P ¼ CS < CE << CC); Cu (P < CS < CE,CC); Ni (P < CS,CC < CE); Pb (P << CE < CS < CC); Zn (P < CS < CE << CC). BAF values for Cu and Ni were relatively low, being consistently below unity across all sites. BAF values indicate that Pb, Zn and especially Cd are rendered more available to the earthworms inhabiting the acidic CS soil (Fig. 1).

populations, but not significantly higher than that recorded in the reference (P) population (Fig. 2). 3.4. Autofluorescent eleocytes in field sampled D. rubidus (Experiment 1) Flow cytometry of D. rubidus coelomocytes confirmed the presence both of agranular amoebocytes and granular eleocytes (E), the latter exhibiting strong autofluorescence 1 (FL1) (Fig. 3A), similar to that described previously in this species sampled from an unpolluted habitat (Cholewa et al., 2006; Plytycz et al., 2006).

A

3.3. Earthworm body weight and coelomocyte numbers (Experiment 1)

P17

FL-1

B a

60

ab

b

c

AFC %

BW [g]

0,2

CC11

SSC

The fresh body weights of D. rubidus were fairly similar (ranging from about 0.2 to 0.3 g) at the reference and two of the metalliferous sites, CC and CS; CE worms, despite being fully clitellated, in comparison had a significantly lower mean body weight (w0.1 g) (Fig. 2). The total number of coelomocytes counted in the extruded coelomic fluids was similar in the reference worms (P) and the contaminated CC and CS sites, but was significantly lower in the worms from the third polluted site, CE. When coelomocyte number was expressed as a ratio of fresh body weight, CS worms possessed a significantly higher value than did the other two polluted-site

b

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0.5

0.3 0.2 0.1 P

P

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12 b

10 ab a

6

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Fig. 2. Fresh body weight (BW; g), total coelomocyte numbers per animal (TC), and coelomocyte numbers per fresh body weight (TC/BW) of D. rubidus collected directly from field sites: reference site P (open bars) and metal-polluted subsites CC, CS, CE (solid bars). Plotted data are means þ SE, and the ‘n’ values were 11, 16, 5, and 5, respectively. Means not sharing lower-case letters are significantly different (p < 0.05) according to the Mann–Whitney ‘U’ test.

TAFC/BW [106/g]

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Fig. 3. Flow cytometric analysis of coelomocytes of D. rubidus collected directly from field sites. (A) Representative examples of density plots of coelomocytes from the individual earthworms P17 (from unpolluted P site) and CC11 (from the polluted CC site). Cell fluorescence (FL1-axis) versus cell granularity/complexity (SSC axis). Groups of dots to the right of FL1-axis are granular autofluorescent cells, i.e. eleocytes (E); (B) percentages of eleocytes (i.e. autofluorescent coelomocytes (AFC)); (C) total AFC numbers; (D) AFC per body weights of D. rubidus from the reference site P (open bar) and metal-polluted sites CC, CS, CE (solid bars). Data presented as means þ SE. Representative experiment on 5 individuals per group. Samples from the same individuals were assayed for riboflavin content (see Fig. 4). Means not sharing lower-case letters are significantly different (p < 0.05) according to the Mann–Whitney ‘U’ test.

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The percentage of autofluorescent coelomocytes, i.e. eleocytes (% AFC, Fig. 3B) in the four field populations ranged from about 25 to 40%. Earthworms from the most metal-polluted site (CC) possessed a significantly higher % AFC value than their counterparts from the other two metalliferous sites (CS and CE); reference-site (P) worms had an intermediate value, but not significantly different from any of the mine-site worms (Fig. 4B). Total number of AFC (TAFC, Fig. 3C), and TAFC normalized to fresh body weight (TAFC/BW; Fig. 3D) was lowest in CE-site worms, but these values were not significantly different from those in CC-site worms. 3.5. Spectrofluorimetry in field sampled D. rubidus (Experiment 1) Fig. 4A and B show typical fluorescence excitation (left) and emission (right) spectra of coelomocyte lysates prepared from representative individual D. rubidus inhabiting the reference site (P17) and the heavily polluted CC site (CC11), respectively. A typical riboflavin bimodal excitation spectrum was apparent in the case of sample P17; but such a signature was clearly absent from sample CC11 (Fig. 4A). Moreover, a characteristic riboflavin emission peak at 525 nm (lex ¼ 370 nm) was observed in the P17 sample but not in CC11 (Fig. 4B). These samples confirm the strong accumulation of riboflavin in the eleocytes of D. rubidus inhabiting unpolluted soil. The second fluorescence peak (at 450 nm) is much higher than the first peak (at 370 nm) riboflavin-derived excitation spectra (Fig. 4A) (Koziol et al., 2006; Plytycz et al., 2006). The theoretical 450/370 riboflavin signature ratio of w1.5 is similar to the mean value observed in samples from reference (P)-site worms, whilst the 450/370 ratio value is close to unity for the flat excitation spectra from samples of worms from each of the metal-polluted sites (Fig. 4C). The amount of riboflavin is proportional to fluorescence emission intensity measured at 525 nm (expressed as arbitrary units, AU) (Plytycz et al., 2006). In Fig. 4D the mean AU value is significant only in the coelomocyte lysates of D. rubidus from the unpolluted

reference site (P); its value is negligible in samples from mine-site worms (CC, CS, CE). 3.6. Laboratory-based reciprocal transfer exposures (Experiment 2) Whole-worm tissue metal contents at the end of the reciprocal transfer exposure period of 4 weeks are presented in Fig. 5A and B. The Pb concentration was negligible in reference worms maintained on their own clean soil (Pp worms), but was significantly increased after exposure to Cwmystwyth Cottage soil (Pc worms) although significantly lower than in the mine-site worms maintained on their native metalliferous soil (Cc worms). The Pb content of worms transferred from metal polluted to the reference soil (Cp worms) was not reduced (Fig. 5A). Tissue Zn contents were relatively low in both the Pp and Pc worms; Cc and Cp worm treatment groups both had higher Zn concentrations than Pp and Pc worms. Worms transferred from their native polluted soil to clean soil (Cp) had lost some of their Zn burden but the mean value after 4 weeks was not significantly different from that of their counterparts maintained on their ‘own’ soil (Fig. 5B). The short exposure period indicates that non-essential Pb accumulates more rapidly than essential Zn in the tissues of earthworms transferred from native clean soil to a metalliferous soil. Moreover, the high issue burdens of Pb and Zn measured in the mine-site worms (Cc) were not significantly diminished by a 4-week ‘depuration period’ on clean reference soil (Cp). Whilst the accumulated or retained tissue metal concentrations are not necessarily linked to organism health, it is germane that Sheppard et al. (1998) estimated the depuration half-life of Zn in earthworms at 69 days. Body weights (Fig. 5C) and coelomocyte numbers (Fig. 5D) were unaffected in earthworms transferred from their native soils to a contrasting field soil. The percentage of autofluorescent coelomocytes was highest in worms from the metalliferous soil (Cc) (Fig. 5E), whilst the amount of riboflavin was highest in worms from the unpolluted reference soil (Pp) (Fig. 5F). As predicted by the

B

A P17

AU

AU

P17

CC11

CC11 300 325 350 375 400 425 450 475 500

470 485 500 515 530 545 560 575 590 605

wavelength (nm)

C

2.0

D

a 1.5

b b

b

1.0 0.5

20

a

16

AU

450 / 370

wavelength (nm)

12 8

b

b

CC

CS

b

4

0.0 P

CC

CS

CE

0

P

CE

Fig. 4. Spectrofluorimetric analysis of coelomocyte lysates of D. rubidus collected directly from field sites. AU ¼ arbitrary units of fluorescence intensity. (A) Representative examples of excitation spectra (at 525 nm, top-left). (B) Emission spectra (lex ¼ 370 nm, top-right) of the individual earthworms P17 (from unpolluted P site; the number is the number assigned to the individual worm during sampling) and CC11 (from the metalliferous CC site). (C) Ratios of AU values measured at 450 nm and 370 nm (compare with panel ‘A’). (D). AU emission values measured at 525 nm (proportional to riboflavin content) (compare with panel ‘B’) in samples from the reference site (P) and the three metal-polluted sites CC, CS, and CE. Data presented as means þ SE. Representative experiment on 5 individuals per group. Samples from the same individuals were assayed for autofluorecent coelomocyte AFC content (see Fig. 3). Means not sharing lower-case letters are statistically significantly different (p < 0.05) according to the Mann–Whitney ‘U’ test.

B. Plytycz et al. / Environmental Pollution 157 (2009) 3042–3050

A 20000

C

0,3 a

Pb (mg/kg)

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BW (g)

c 15000

0,2

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Zn (mg/kg)

TCN (106)

6000

1000

a

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a

a

0

5000

0

3047

30

ab

a a

20 10

F

15

RF (AU)

0

10

a

5

ab b

ab

0 Fig. 5. Effects of experimental transference (4 weeks) of D. rubidus worms (reference worms ¼ ‘P’; polluted-site worms ¼ ‘C’) from their native unpolluted soil (‘p’) to a metalliferous soil (‘c’), or vice versa – Experiment 2. Pp ¼ worms from the unpolluted site in their ‘own’ reference soil (open bars of left panel); Pc ¼ worms from the unpolluted site transferred to polluted soil (grey bars of left panel); Cc ¼ worms from the polluted site in their ‘own’ polluted soil (black bars of right panel); Pc ¼ worms from the polluted site transferred to the unpolluted reference soil (grey bars of right panel). (A) Pb content (mg/kg dry mass) in whole-worm tissues; (B) Zn content (mg/kg dry mass) in whole worm tisues; (C) worm fresh body weight, BW (g); (D) total number of coelomocytes, TC; (E) percentage of autofluorescent coelomocytes, AFC; (F) riboflavin (RF) content (AU - arbitrary units). The data are presented as means þ SE, and the ‘n’ values (i.e. number of individual worms measured) in each panel are: A & B ¼ 5; C–G ¼ 14, 7, 8, and 7, respectively. Means not sharing lowercase letters are significantly different (p < 0.05) according to the Mann–Whitney ‘U’ test.

observations in Experiment 1 (see Fig. 4), the amount of riboflavin was negligible or absent in worms maintained constantly on their native metal-polluted soil (Cc group), and it was not significantly increased during 4-week maintenance on unpolluted soil (Cp group). In contrast, the amount of riboflavin in reference-site worms maintained for 4 weeks in the metal-polluted soil was appreciably lowered, albeit not quite to the extent of being statistically significant (cf. Pp and Pc groups). 3.7. Earthworm genotyping (Experiment 1) Fig. 6 shows a phylogenetic tree constructed using the distancebased neighbour joining (NJ) algorithm, based upon p-distance, of the COII mitochondrial gene of D. rubidus individuals from the

reference P site and from metal-polluted CC, CS, CE sites. The individuals from the unpolluted site are grouped in a clade (open squares) separate from the individuals from metalliferous CC, CS, CE sites (solid squares). Tree topology is well supported by statistical bootstrap analysis (based upon 3000 replicates). 4. Discussion It has long been known that the organelles known as ‘chloragosomes’ found exclusively in oligochaete annelids not only have a complex matrix with the capacity to sequester high concentrations of O-seeking cations, but that they also possess strong redox properties conferred by their metalloporphyrin, thiamine, carotenoid, and riboflavin contents (Fischer, 1975). More recently,

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0.02 Fig. 6. Phylogenetic tree based on p-distance of the COII mitochondrial gene of D. rubidus individuals from the reference P soil (open squares) and from metal-polluted CC, CS, and CE sites (solid squares).

confocal microscopy has facilitated the detection and localisation of riboflavin in the chloragosomes of the eleocytes of certain earthworm species (Plytycz et al., 2007a), including D. rubidus (in preparation). In the present study, spectrofluorimetry detected significant riboflavin content only in the coelomocytes of D. rubidus from an unpolluted reference site. Earthworms inhabiting three contrasting metal-polluted soils did not contain detectable riboflavin signatures in their eleocytes despite the fact that these cells were consistently autofluorescent. These observations indicate that other fluorophores are putatively responsible for eleocyte autofluorescence in the metal-stressed earthworm populations. This notion is supported by the laboratory-based reciprocal transfer experiment, where it was found that earthworms from unpolluted reference soil kept for 4 weeks in the metal-polluted soil (Pc) had diminished riboflavin content whilst their eleocytes autofluorescence was unchanged. This pattern corresponded with avid Pb accumulation in the tissues of Pc treatment group worms. In contrast, the reverse transfer of worms inhabiting metal-polluted soil to unpolluted reference soil (Cp worms) did not experience measurable changes in their coelomocyte characteristics. This corresponds with the limited clearance of accumulated Pb, and Zn from the tissues during the relatively short transference period. It may be germane to note that the acknowledged source of systemic eleocytes is the chloragogenous tissue that is primarily attached to the coelomic surfaces of the alimentary canal (Affar et al., 1998),

a tissue that also serves as the primary site for Pb and Zn sequestration in earthworms (Cotter-Howells et al., 2005). Whilst the earthworm biomarker approach to contaminated land assessment has been widely promulgated (e.g. Spurgeon et al., 2000), the critical review by Forbes et al. (2006) raises a number of important challenges that warrant being addressed. Of particular concern are the issues of sensitivity and potential confounding factors. It would appear that riboflavin autofluorescence is a good marker of soil metal pollution because the signal was extinguished in the coelomocytes of field worms inhabiting all three metalliferous soils. However, the biochemical trait assayed in the present study was very sensitive and not sufficiently discriminating to provide any evidence of differential, site-specific, metal tolerance in any of the worm populations inhabiting mine-associated soils despite the fact that metal body-burdens and bio-concentration factors showed appreciable inter-site differences. The riboflavin content of earthworm eleocytes might, therefore, be a cytological biomarker whose effective deployment range is restricted either to short-duration exposures in the laboratory or to field populations inhabiting metalliferous soils that fall into the categories of ‘‘slight contamination’’ or ‘‘contaminated’’ (HMSO, 1991). The interpretation of our findings has hitherto assumed that the metals accumulated by the field worms interfere with riboflavin metabolism or consumption within the worms. An alternative possibility is that the diet of mine-site worms is impoverished in riboflavin due to metal-evoked inhibition of microbes and/or plants capable of synthesising the vitamin. It is imperative to establish whether the reduced riboflavin content of earthworm coelomocytes is a direct metal-mediated endogenous or an indirect exogenous event, or a combination of both. Forbes et al. (2006) and Morgan et al. (2007) have drawn attention to the ecotoxicological consequences of ignoring deep genetic differences amongst field populations. Our genotyping findings on relatively small numbers of individual D. rubidus indicate that the individual worms comprising the three metalliferous-site populations cluster within one genetic lineage, whilst the individuals from the reference population cluster to form a different lineage. This very limited dataset implies that there could be a correlation between genotype and riboflavin content. Specifically, it is possible that the D. rubidus lineage inhabiting the mine sites is a previously unrecognised cryptic species with low intrinsic riboflavin content in its coelomocytes. Alternatively, it is plausible that the non-detectable riboflavin in mine-site worms is a genuine environmental (e.g. metal toxicity) rather than a genetic effect because the phenotypic outlier population in terms of body weight and total coelomocyte content was one of the mine site populations, Cwmystwyth East (CE), not the genetically distinct reference population (P). To resolve these issues it would be necessary to examine a large number of genotyped populations, including several different reference populations belonging to the same genetic lineage as some of the mine site populations. Moreover, it might be advisable to expose D. rubidus of a defined genotype derived from an unpolluted site for protracted periods to a range of metalliferous field soils under laboratory conditions to determine the temporal effects on riboflavin content and its contribution to the autofluorescence of eleocytes. A complimentary approach would entail producing F1 and F2 progeny of mine-site F0 adults in an unpolluted standard soil under laboratory conditions. Riboflavin deficiency in the eleocytes of these laboratory-bred earthworms with no individual history of metal exposure would support the notion that the disturbance in riboflavin uptake/synthesis is a constitutive, genetically determined, phenomenon. Conversely, the presence of riboflavin would confirm that environmental factors are the predominant causative agents of the defect in earthworms resident on metalliferous field soils.

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In conclusion, a simple hypothetical scheme may be tentatively proposed to interpret our observations on the effects of soil metal contaminants on the composition and autofluorescent properties of the eleocytes of D. rubidus. Chronic exposure to metalliferous soils manifestly increases the metal body burden, the large proportion of which is sequestered within the chloragogenous tissue (Morgan and Morgan, 1998), the likely source of eleocytes. In any event, the induction of metallothionein indicates that coelomocytes are metal targets (Homa et al., 2005). Metal stress initiates a cascade of molecular and cellular events. Metals compromise earthworm immunocompetence (Goven et al., 2005), thus increasing the likelihood of systemic invasion by soil-borne pathogens (Wieczorek-Olchawa et al., 2003; Olchawa et al., 2006). Eleocytes become depleted of their endogenous riboflavin as the molecule is released to potentiate the cellular immune response. The accumulating metal ions, and possibly riboflavin itself, cause peroxidative damage leading to accelerated lipofuscin deposition. The ‘model’ predicts, therefore, that eleocyte autofluorescence in D. rubidus is normally attributable largely if not exclusively in the unstressed state to the presence of riboflavin, and that metal intrusion triggers the functional release of riboflavin and its replacement by an autofluorescent product of toxicosis, probably lipofuscin. It is important to recognise that a number of other metabolically active molecules are autofluorescent, including nicotinamide adenine dinucleotide (NADH) (Pitts et al., 2001), but the proposed model may have merit because recent findings indicate that the riboflavin/lipofuscin balance may be a sensitive indicator of soil pollution intensity (Cygal et al., 2007). Acknowledgements We thank to Prof. Ryszard Laskowski for fruitful discussions, and Beata Klimek and Anna Stefanowicz for metal analyses. This work was supported by a grant (No PB3502/PO1/32) from the Ministry of Science and Education. JA was funded via a Ph.D. Studentship awarded by the UK Natural Environment Research Council. References Affar, E.B., Dufour, M., Poirier, G.G., Nadeau, D., 1998. Isolation, purification and partial characterization of chloragocytes from the earthworm species Lumbricus terrestris. Molecular and Cellular Biochemistry 185, 123–133. Bednarska, A., Laskowski, R., 2008. Effects of nickel and temperature on the ground beetle Pterostichus oblongopunctatus (Coleoptera: Carabidae). Ecotoxicology 17, 189–198. Brulle, F., Cocquerelle, C., Mitta, G., Castric, V., Douay, F., Lepreˆtre, A., Vandenbulcke, F., 2008. Identification and expression profile of gene transcripts differentially expressed during metallic exposure in Eisenia fetida coelomocytes. Developmental & Comparative Immunology 32, 1441–1453. Bundy, J.G., Sidhu, J.K., Rana, F., Spurgeon, D.J., Svendsen, C., Wren, J.F., Stu¨rzenbaum, S.R., Morgan, A.J., Kille, P., 2008. Systems toxicology’ approach identifies coordinated metabolic responses to copper in a terrestrial non-model invertebrate, the earthworm Lumbricus rubellus. BMC Biology 6, 25. Cholewa, J., Feeney, G.P., O’Reilly, M., Stu¨rzenbaum, S.R., Morgan, A.J., Plytycz, B., 2006. Autofluorescence in eleocytes of some earthworm species. Folia Histochemica et Cytobiologica 44, 65–71. Cooper, E.L., 1996. Earthworm immunity. In: Rinkevich, B., Mu¨ller, W.E.G. (Eds.), Invertebrate Immunology. Springer – Verlag Berlin, Thomson Press (India), New Delphi, pp. 10–35. Cotter-Howells, J., Kille, P., Charnock, J.M., Winters, C., Fry, J.C., Morgan, A.J., 2005. Metal coordination in earthworm cells and tissues: correlative electron probe X-ray mapping and EXAFS analyses. Environmental Science and Technology 39, 7731–7740. Cygal, M., Lis, U., Kruk, J., Plytycz, B., 2007. Coelomocytes and fluorophores of the earthworm Dendrobaena veneta raised at different ambient temperatures. Acta Biologica Cracoviensia, Series Zoologia 49, 5–11. Fischer, E., 1975. Structural basis of cation exchange, complex formation and redox properties in chloragosomes. Acta Biologica Academiae Scientiarum Hungaricae 26, 75–84. Forbes, V.E., Palmqvist, A., Bach, L., 2006. The use and misuse of biomarkers in ecotoxicology. Ecotoxicology Environmental Chemistry 25, 272–280. Fuge´re, N., Brousseau, P., Krzystniak, K., Coderre, D., Fournier, M., 1996. Heavy metal-specific inhibition of phagocytosis and different in vitro sensivity of

3049

heterogenous coelomocytes from Lumbricus terrestris (Oligochaeta). Toxicology 109, 157–166. Goven, A.J., Venables, B.J., Fitzpatrick, L.C., 2005. Earthworms as ecosentinels for chemical-induced immunotoxicity. In: Tryphonas, H., Fournier, M., Blakeley, B.R., Smits, J.E.G., Brousseau, P. (Eds.), Investigative Immunotoxicology. CRC Press, pp. 91–106. HMSO (Her Majesty’s Stationery Office), 1991. Protection of Workers and the General Public During the Development of Contaminated Land. Health Safety Executive, London, p. 20. Hamed, S.S., Kauschke, E., Cooper, E.L., 2002. Cytochemical properties of earthworm coelomocytes enriched by Percoll. In: Beschin, A., Bilej, M., Cooper, E.L. (Eds.), A New Model for Analyzing Antimicrobal Peptides with Biomedical Applications. IOS Press, Ohmsha, pp. 29–37. Homa, J., Niklinska, M., Plytycz, B., 2003. Effect of heavy metals on coelomocytes of the earthworm Allolobophora chlorotica. Pedobiologia 47, 640–645. Homa, J., Olchawa, E., Stu¨rzenbaum, S.R., Morgan, A.J., Plytycz, B., 2005. Early-phase immunodetection of metallothionein and heat shock proteins in extruded earthworm coelomocytes after dermal exposure to metal ions. Environmental Pollution 135, 275–280. Homa, J., Stu¨rzenbaum, S.R., Morgan, A.J., Plytycz, B., 2007. Disrupted homeostasis in coelomocytes of Eisenia fetida and Allolobophora chlorotica exposed dermally to heavy metals. European Journal of Soil Biology 43, 273–280. Iwanaga, K., Hasegawa, T., Hultquist, D.E., Harada, H., Yoshikawa, Y., Yanamadala, S., Liao, H., Visovatti, S.H., Pinsky, D.J., 2007. Riboflavin-mediated reduction of oxidant injury, rejection, and vasculopathy after cardiac allotransplantation. Transplantation 83, 747–753. Koziol, B., Markowicz, M., Kruk, J., Plytycz, B., 2006. Riboflavin as a source of autofluorescence in Eisenia fetida coelomocytes. Photochemistry and Photobiology 82, 570–573. Linke-Gamenick, I., Forbes, V.E., Me´ndez, N., 2000a. Effects of chronic fluoranthene exposure on sibling species of Capitella with different development modes. Marine Ecology Progress Series 203, 191–203. Linke-Gamenick, I., Vismann, B., Forbes, V.E., 2000b. Effects of fluoranthene and ambient oxygen levels on survival and metabolism of three sibling species of Capitella (Polychaeta). Marine Ecology Progress Series 203, 191–203. Morgan, J.E., Morgan, A.J., 1991. Differences in the accumulated metal concentrations in two epigeic earthworm species (Lumbricus rubellus and Dendrodrilus rubidus) living in contaminated soils. Bulletin of Environmental Contamination and Toxicology 47, 296–301. Morgan, J.E., Morgan, A.J., 1998. The distribution and intracellular compartmentation of metals in the endogeic earthworm Aporrectodea caliginosa sampled from an unpolluted and a metal-contaminated site. Environmental Pollution 99, 167–175. Morgan, A.J., Kille, P., Stu¨rzenbaum, S., 2007. Microevolution and ecotoxicology of metals in invertebrates. Environmental Science and Technology 41, 1085–1096. Olchawa, E., Bzowska, M., Stu¨rzenbaum, S.R., Morgan, A.J., Plytycz, B., 2006. Heavy metal exposure affects the coelomocyte-bacteria balance in earthworms: environmental interactions between abiotic and biotic stressors. Environmental Pollution 142, 373–381. Owen, J., Hedley, B.A., Svendsen, C., Wren, J., Jonker, M.J., Hankard, P.K., Lister, L.J., Stu¨rzenbaum, S.R., Morgan, A.J., Spurgeon, D.J., Blaxter, M.L., Kille, P., 2008. Transcriptome profiling of developmental and xenobiotic responses in a keystone soil animal, the oligochaete annelid Lumbricus rubellus. BMC Genomics 9, 266 (open access). Pitts, J.D., Sloboda, R.D., Dragnev, K.H., Dmitrovsky, E., Mycek, M.A., 2001. Autofluorescence characteristics of immortalized and carcinogen-transformed human bronchial epithelial cells. Journal Biomedical Optometry 6, 31–40. Plytycz, B., Homa, J., Koziol, B., Rozanowska, M., Morgan, J.M., 2006. Riboflavin content in autofluorescent earthworm coelomocytes is species-specific. Folia Histochemica et Cytobiologica 44, 275–280. Plytycz, B., Klimek, M., Homa, J., Tylko, G., Kolaczkowska, E., 2007a. Flow cytometric measurement of neutral red accumulation in earthworm coelomocytes: novel assay for studies on heavy metal exposure. European Journal of Soil Biology 43, 116–120. Plytycz, B., Homa, J., Aziz, A., Molna´r, L., Kille, P., Morgan, A.J., 2007b. Earthworm for monitoring metal contamination: from cells to molecules. In: Plattenberg, R.H. (Ed.), Environmental Pollution: New Research. Nova Science Publishers, New York, pp. 47–98. Roch, P., 1979. Protein analysis of earthworm coelomic fluid: (1) polymorphic system of natural hemolysin of Eisenia fetida andrei. Developmental & Comparative Immunology 4, 599–608. Rocha-Olivares, A., Fleeger, J.W., Foltz, D.W., 2004. Differential tolerance among cryptic species: a potential cause of pollutant-related reduction in genetic diversity. Environmental Toxicology and Chemistry 23, 2132–2137. Sheppard, S.C., Evenden, W.G., Cornwell, T.C., 1998. Depuration and uptake kinetics of I, Cs, Mn, Zn, and Cd by the earthworm (Lumbricus terrestris) in radio-spiked litter. In: Sheppard, S.C., Bembridge, J.D., Holmstrup, M., Posthuma, L. (Eds.), Advances in Earthworm Ecotoxicology, Society of Environmental Toxicology and Chemistry (SETAC), Pensacola, FL, USA, pp. 151–164. Sims, R.W., Gerard, B.M., 1999. Earthworms. Notes for the identification of British species. In: Barnes, R.S.K., Crothers, J.H. (Eds.), Synopses of the British Fauna (New Series). Published for the Linnean Society of London and the Estuarine and Coastal Sciences Association by Field Studies Council, Shrewsbury, UK. Spurgeon, D.J., Hopkin, S.P., Jones, D.T., 1994. Effects of cadmium, copper, lead and zinc on growth, reproduction and survival of the earthworm Eisenia fetida

3050

B. Plytycz et al. / Environmental Pollution 157 (2009) 3042–3050

(Savigny): assessing the environmental impact of point-source metal contamination in terrestrial ecosystems. Environmental Pollution 84, 123–130. Spurgeon, D.J., Svendsen, C., Rimmer, R.V., Hopkin, S.P., Weeks, A.M., 2000. Relative sensitivity of life-cycle and biomarker responses in four earthworm species exposed to Zn. Environmental Toxicology and Chemistry 19, 1800–1808.

Verdrengh, M., Tarkowski, A., 2005. Riboflavin in innate and acquired immune responses. Inflammation Research 54, 390–393. Wieczorek-Olchawa, E., Niklinska, M., Miedzobrodzki, J., Plytycz, B., 2003. Effects of temperature and soil pollution on the presence of bacteria, coelomocytes and brown bodies in coelomic fluid of Dendrobaena veneta. Pedobiologia 47, 702–709.