Riboflavin content in the coelomocytes of contrasting earthworm species is differentially affected by edaphic variables including organic matter and metal content

Pedobiologia 54S (2011) S43–S48

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Pedobiologia - International Journal of Soil Biology journal homepage: www.elsevier.de/pedobi

9th International Symposium on Earthworm Ecology

Riboflavin content in the coelomocytes of contrasting earthworm species is differentially affected by edaphic variables including organic matter and metal content Barbara Plytycz a,∗ , Malgorzata Klimek a , Beata Anna Klimek b , Wojciech Szymanski c , Jerzy Kruk d , A. John Morgan e a

Institute of Zoology, Jagiellonian University, Krakow, Poland Institute of Environmental Sciences, Jagiellonian University, Krakow, Poland c Institute of Geography and Spatial Management, Jagiellonian University, Krakow, Poland d Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Krakow, Poland e Cardiff School of Biosciences, Main Building, Cardiff University, Cardiff CF10 3US, Wales, UK b

a r t i c l e

i n f o

Article history: Received 13 October 2010 Received in revised form 10 July 2011 Accepted 19 July 2011 Key words: Eleocytes Riboflavin Eisenia andrei Dendrobaena veneta Allolobophora chlorotica Metals, Spectrofluorometery

a b s t r a c t The riboflavin content in extruded coelomocyte lysates derived from Dendrodrilus rubidus may serve as a sensitive bioindicator of soil metal pollution: the vitamin (B2) content has previously been found to be high in worms from unpolluted soil but low in worms inhabiting Zn/Pb mine soils, aerially deposited Ni-contaminated soil, and in worms experimentally transferred from clean soil to the metalliferous field soils. The aim of the present work was to extend these observations by comparing the number and riboflavin composition of coelomocytes retrieved from three lumbricid species (Allolobophora chlorotica, Dendrobaena veneta, Eisenia andrei) after 4-week exposures to an unpolluted commercial soil, two geochemically contrasting unpolluted field soils, and two different Zn/Pb/Cd-polluted soils from the Bukowno district in South Poland. Whilst eco-physiologically contrasting, these three earthworm species share the trait of possessing relatively high numbers of eleocytes, a category of immune-competent coelomocyte rich in autofluorescent riboflavin. Spectroflurometric analysis indicated that coelomocyte riboflavin content in worms maintained in strongly metalliferous soils or in unpolluted sandy-clay and loamy-sand soils was increased in coleomocytes from epigeic D. veneta and E. andrei species, whilst was decreased in endogeic A. chlorotica. In conclusion, the riboflavin content of earthworm coelomocytes is affected in species-specific ways by edaphic variables, including organic matter and metal pollution. © 2011 Elsevier GmbH. All rights reserved.

Introduction In some lumbricid earthworm species (Aporrectodea spp., Lumbricus spp.) the immune-competent coelomocytes freely floating in the coelomic fluid are comprised almost exclusively of one morphologically distinguishable cell type, the amoebocytes. In other lumbricids (Allolobophora chlorotica, Dendrobaena veneta, Dendrodrilus rubidus, Eisenia spp., Octolasion spp.) the free coelomocyte community is more diverse, comprising of amoebocytes and significant percentages of granular chloragocyte-derived eleocytes (Cholewa et al. 2006; Olchawa et al. 2006). Coelomocyte composition and functional characteristics vary in response to several factors, including intrinsic seasonal cyclic changes (Kurek and

∗ Corresponding author at: Institute of Zoology, Jagiellonian University, Ingardena 6, PL 30-060 Krakow, Poland. Tel.: +48 12 663 24 28; fax: +48 12 634 37 16. E-mail address: [email protected] (B. Plytycz). 0031-4056/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.pedobi.2011.07.003

Plytycz 2003) and developmental age (Sauvé and Fournier 2005), as well as external mediators such as thermal (Cygal et al. 2007) and heavy metal stress (Kwadrans et al. 2008; Dutkiewicz et al. 2009; Homa et al. 2010; Plytycz et al. 2009, 2010). For these reasons there is an ongoing interest in measuring disturbances in the cytology (Gastaldi et al. 2007; Plytycz et al. 2007; Fuchs et al. 2010) and transcriptome of the coelomocytes (Brulle et al. 2006, 2008; Homa et al. 2010) of ecologically relevant earthworms (Turbé et al. 2010) as tools for biomonitoring soil contamination. Flow cytometry is a particularly effective high-throughput method for determining the percentage of eleocytes in coelomocyte suspensions derived from earthworm species where this cell type is prevalent. Eleocytes are conveniently characterised due to their complexity and autofluorescence, the latter primarily deriving from riboflavin (Koziol et al. 2006; Plytycz et al. 2006) with secondary contributions from other fluorophores, including lipofuscins (Valembois et al. 1994; Cygal et al. 2007). Moreover, the riboflavin content of coelomocyte lysates from eleocyte-rich

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species can also be rapidly and objectively measured by spectrofluorometry. The effectiveness of the two assay techniques was demonstrated in two recent comparative studies on the coelomocytes of field populations of the epigeic gilt-tail species D. rubidus inhabiting, or experimentally exposed to, unpolluted and metalliferous (Pb/Zn-contaminated abandoned mine-associated, or aerially deposited Ni/Cu-contaminated) soils. It was found (Plytycz et al. 2009, 2010) that the percentage of autofluorescent riboflavinstoring eleocytes in D. rubidus did not consistently correspond to the riboflavin content in the corresponding lysates, probably because eleocytes were equally numerous in worms from both unpolluted and metalliferous sites but the riboflavin content of individual eleocytes was depleted in metal-exposed worms. This notion was confirmed by laboratory reciprocal transfer experiments where, for example, the riboflavin content in D. rubidus originating from an unpolluted soil was almost extinguished by worms’ maintenance for several weeks in Zn/Pb or Ni/Cu field soils. Thus, it was concluded that coelomocyte-lysate riboflavin content at least in D. rubidus is potentially a sensitive biomarker of soil metal pollution (Plytycz et al. 2009, 2010). At the present time it is not known whether cytometric variables, including the eleocyte-associated riboflavin content, of the coelomocytes of other lumbricids with a high eleocyte presence are also responsive to elevated soil metal levels in much the same way as D. rubidus coelomocytes evidently are. The main aim of the present study was to determine whether the coelomocytes of the two ‘manure worms’ Eisenia andrei and D. veneta that are especially suitable for laboratory toxicity tests, as well as the coelomocytes of the widely distributed endogeic species A. chlorotica, are affected by experimental exposure to four contrasting soils: two contrasting uncontaminated ‘reference’ soils, and two metalliferous soils differing mainly in the intensity of Zn contamination. Materials and methods Soil samples and characteristics Reference (R) soil was purchased from a commercial supplier (PPUH BIOVITA, Tenczynek, Poland). Field soil samples were collected from four areas in Southern Poland: unpolluted Sierbowice (forest; SF) and Zlozeniec (meadow; ZM); and two polluted localities at Bukowno (forest; BF), and Bukowno (meadow; BM), respectively. Individual soils were thoroughly mixed and sieved (2 mm stainless steel) to minimise compositional heterogeneity. Soil pH, organic matter, and granulometry (texture) were assessed as described previously (Homa et al. 2003).

Soil and tissue metal analyses Metal accumulation was measured in soils and whole-worm tissues by atomic absorption spectrophotometer (Aanalyst 800, Perkin-Elmer) as described previously (Homa et al. 2003). Metal body accumulation factors (BAF) were calculated as ratios of wholebody metal concentration to ‘total’ soil metal concentration. Harvesting coelomocytes Earthworms were stimulated for 30 s with a 4.5 V electric current to expel coelomic fluid with suspended coelomocytes through the dorsal pores. Briefly, after weighing, washing and dry-blotting, the earthworms were placed individually in Petri dishes containing 3 mL of extrusion fluid (phosphate-buffered saline, PBS, supplemented with 2.5 g/L ethylenediamine tetraacetic acid, EDTA) (Plytycz et al. 2006). Freshly prepared 2 mL suspensions were used for spectrofluorometry, and the remaining sample from each worm was fixed in 2% formalin for coelomocyte counting in haemocytometer and for flow cytometry. Each worm was used for coelomocyte harvesting only once. 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-H (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. Spectrofluorometric measurements and analysis The spectrofluorometric measurements were performed on coelomocyte suspension lysates (2% Triton; Sigma–Aldrich) using Perkin-Elmer Spectrofluorometer LS50B. Excitation spectra were recorded between 300 and 520 nm (emission at 525 nm), whilst emission spectra were recorded between 380 and 700 nm using excitation at 370 nm. The spectrofluorometric signatures of unbound riboflavin are two excitation maxima (370 nm and 450 nm) and one emission maximum (525 nm). Arbitrary units (AU) of fluorescence were recorded using Microsoft Excel v. 2007. Statistical analysis

Earthworms Adult E. andrei were collected in September 2007 and 2008 from an unpolluted area near Muszyna in the Sadecki Mountains, Poland; A. chlorotica was collected from a relatively metal-free site in Krakow; D. veneta was purchased from a commercial supplier (EKARGO Slupsk, Poland). The worms were maintained under standard conditions in the soil (R) from the commercial supplier until used for laboratory exposures. For experiments, groups of worms (6 per group) belonging to each of the three species were maintained for 4 weeks each of field soils (SF, ZM, BF, BM) and the commercial soil (R). Experiments were conducted under controlled conditions (16 ± 1 ◦ C; 24 h darkness). Exposed worms were kept in plastic boxes with perforated lids and the moisture content was checked weekly. Worms were fed weekly (1 g/200 g dry soil equivalent) on a mixed diet comprised of flour, boiled/dried/powdered tea leaves, and powdered mouse feed.

Within each species, worm body weights, percentages of autofluorescent eleocytes, and riboflavin content of worms maintained in the commercial reference soil (R groups) were combined, and the respective values from other exposure groups (ZM, SF, BF, and BM) were expressed as fold-differences in relation to the combined ‘R’group value. Results were expressed as means ± standard errors. Differences between means were determined by non-parametric Mann–Whitney ‘U’ test (Statgraphic PLUS 5.0), with the level of significance established at P < 0.05. Results Soil characteristics Soil pH, granulometry, % organic matter (OM) content, and metal (Ni, Cu, Zn, Cd, Pb) concentrations are presented in Table 1. The

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Table 1 Characteristics of subject soils obtained from a commercial supplier (R) and from four field sites (ZM, SF, BF, BM) in Southern Poland (means of 3 samples ± SE). The New Dutch List of appropriate soil action values (*www.ContaminatedLAND.co.uk) are also presented as benchmarks. Sites

Commercial supplier Zlozeniec (meadow) Sierbowice (forest) Bukowno (forest) Bukowno (meadow) Action values*

R ZM SF BF BM

Soil type

Organic matter (%)

pH

Organic Sandy-clay Loamy-sand Organic Organic

51.7 2.1 3.5 28.0 23.8

6.1 6.2 5.1 5.6 7.1 6–7

data confirmed the unpolluted states of the commercial reference R soil. All investigated field soils are free of Cu pollution whereas Ni content in the BM soil (38 mg kg−1 ) is in the action value rage for this metal (35–75 mg kg−1 ). The two soils from unpolluted areas of southern Poland are slightly contaminated with Cd (mainly sandyclay soil from the Zlozeniec meadow, ZM) and Pb (both loamy-sand soil from Sierbowice forest and sandy-clay Zlozeniec meadow). In contrast, the organic soil from Bukowno forest (BF) and Bukowno meadow (BF) are predominantly contaminated with Zn, Cd, and Pb, which exceed by wide margins the New Dutch action concentrations (www.ContaminatedLAND.co.uk). The two polluted soils have much higher OM values (24%, 28%) than both unpolluted soils (2%, 3.5%), but the reference soil (R) has an exceptionally high OM of 52%.

µg g-1 b.w.

Ni

Metal concentrations; X ± SE (mg kg−1 ) Ni

Cu

Zn

Cd

Pb

7 ± 0.5 10 ± 0.7 5 ± 0.9 20 ± 6.2 38 ± 9.2 35–210

8 ± 0.9 6 ± 0.8 6 ± 0.4 33 ± 6.5 31 ± 6.1 36–190

19 ± 3.3 120 ± 10.7 90 ± 10.9 5772 ± 1045 27,790 ± 2796 140–720

0.5 ± 0.3 1.6 ± 0.18 0.8 ± 0.1 65.1 ± 11 144.8 ± 4.8 0.8–12

11 ± 2.9 59 ± 8.3 114 ± 4.8 1717 ± 272 1941 ± 215 85–530

Metal concentration in worm bodies and body accumulation factors (BAFs) The concentrations of Ni and Cu in whole-worm tissues, as well as the body accumulation factors (BAFs) of these metals, were low in the three investigated worm species maintained on all the tested soils. In contrast, Zn, Cd, and Pb concentrations were significantly increased in worms maintained for 4 weeks in metalcontaminated metals BF, BM), being especially high in the case of Zn in worms exposed to heavily Zn-contaminated BM soil despite the soil’s circumneutral pH (7.1). Some inter-species differences were recorded: metal concentrations were consistently lower in E. andrei than in D. veneta and A. chlorotica exposed to BF or BM soils (Fig. 1). BAFPb values were below unity in all treatment groups; BAFZn and

BAF

Dv

6

Ea

Ach

0 R

ZM

SF

BF

30

3

20

2

10

1 R

ZM

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15

4000

10

2000

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100

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0

6000

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SF

BF

BM

R

ZM

SF

BF

BM

R

ZM

SF

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BM

R

ZM

SF

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0 R

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BF

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30 20

50

10 0

0 R

Pb

R

BM

0

Cd

Ach

0.2

0

Zn

Ea

0.4

4 2

Cu

Dv

0.6

ZM

SF

BF

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600

0.6

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0.4

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0

0 R

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Fig. 1. Effects of soil quality on metal content in whole bodies (␮g g−1 dry body weights) and body accumulation factors (BAF; i.e. ratio of body to soil metal concentration) in adult worms D. veneta (Dv), E. andrei (Ea), and A. chlorotica (Ach) after 4-week exposures to various soil samples: reference soil from a commercial supplier (R), and geochemically contrasting field soils from southern Poland: sandy clay from Zlozeniec meadow (ZM), loamy sand from Bukowno forest (BF), and metalliferous organic-rich soils from Bukowno forest (BF) and Bukowno meadow (BM). Means + SE; n = 3.

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Riboflavin content in coelomocyte lysates Appreciable between-treatment and inter-species differences were observed in the riboflavin contents of coelomocyte lysates. In the case of D. veneta upward trends are obvious in Pb-polluted loamy-sand SF soil, and metalliferous organic BF and BM soils, albeit not statistically significant due to the large standard deviations, as only 6 earthworms were used for each conditions. The riboflavin content of E. andrei coelomocyte lysates was significantly higher after exposure to Cd-polluted sandy-clay soils (ZM) and to both heavily polluted organic soils (BF and BM) compared with worms maintained on the reference (R) soil. In contrast, A. chlorotica exposed to ZM, SF, and BF soils had significantly lower riboflavin in coelomocyte lysates compared with the reference treatment, and downward trend was also recorded in BM soil (Fig. 2c).

Discussion

Fig. 2. Effects of soil quality on body weights and coelomocytes non-invasively retrieved from adult worms D. veneta (Dv), E. andrei (Ea), and A. chlorotica (Ach) after 4-week exposure to various soil samples: reference soil from a commercial supplier (R), and geochemically contrasting field soils from southern Poland: sandy clay from Zlozeniec meadow (ZM), loamy sand from Bukowno forest (BF), and metalliferous organic-rich soils from Bukowno forest (BF) and Bukowno meadow (BM). (a) Worm body weights (BW); (b) Percentages of autofluorescent eleocytes in extruded coelomocytes (E); (c) Riboflavin content in coelomocyte lysates (RF). All values recalculated as a ratio to the mean value in the reference group (R) for each species scaled to 1. Means + SE; n = 6. Asterisks – means significantly different from R group of the species according to Mann–Whitney ‘U’ test (P < 0.05).

BAFCd values in worms exposed to polluted BF and BM soils were also below unity, and significantly lower than corresponding BAF values in worms exposed to unpolluted soils (Fig. 1). Worm survival and body weights All worms survived 4-week laboratory exposures to the five test soils differing to different degrees in terms of background geochemistry and metal pollution intensity. At the end of experimental period, body weights were diminished only in worms maintained on the more acidic of the two Bukowno metalliferous soils (BF), with the change being statistically significant in D. veneta and A. chlorotica (Fig. 2a). Cell counts – autofluorescent eleocytes The percentages of autofluorescent eleocytes were especially stable across the treatments in D. veneta; more variability was observed in this cytometric parameter in E. andrei and A. chlorotica, but there were no statistically significant differences in any exposure group compared with worms maintained on the unpolluted reference (R) soil (Fig. 2b).

Given that animals are incapable of synthesizing riboflavin and thus derive the water-soluble vitamin (B2) from dietary sources (Fischer and Bacher 2006), and given that riboflavin has indirect and direct roles in potentiating innate immune responses in vertebrates (Verdrengh and Tarkowski 2005), the present study had two subsidiary aims: (i) to determine whether the riboflavin status of the test species differed when maintained on unpolluted soils containing very different organic matter contents; (ii) to discuss aspects of the metabolism of riboflavin in order to promote hypothesis-driven future research on the micronutrient in the context of earthworm immunity. In the present studies the riboflavin content of non-intrusively extruded coelomocytes was affected in different ways in the three lumbricids D. veneta, E. andrei and A. chlorotica, when they were maintained under normalized laboratory conditions in an unpolluted organic-rich commercial soil and four field soils, two relatively unpolluted and two metal-polluted. The two earthworm species D. veneta and E. andrei that are normally found inhabiting organic-rich substrates such as manure and compost heaps experienced either an obvious upward trend (D. veneta) or significantly elevated (E. andrei) riboflavin status after 4-weeks on metalliferous, relatively organic, soils or on relatively metal-free sandy-clay or loamy-sand soils (Table 2). This was a puzzling finding in the case of the polluted-soil treatment because previous observations on D. rubidus indicate that the riboflavin status of the riboflavin-storing eleocytes tends to be diminished by metal exposures (Plytycz et al. 2009, 2010). It is possible that, despite being transferred from the organic-rich ‘holding’ commercial soil of presumably high micronutrient, including riboflavin, status to soils of significantly to extremely lower organic matter content, the regular supplementation of the substrates with surface-deposited food counteracts the negative influences of low organic matter and/or metals on eleocyte riboflavin. However, this notion appears not to apply to the ecophysiologically contrasting endogeic species A. chlorotica. This species, a relatively pH intolerant dweller of mineralized soil, experienced reduction of coelomocyte riboflavin contents relative to reference-soil worms when maintained on metalliferous and relatively metal-free field soils despite being fed a nutrient-rich supplement just like the two other subject lumbricids (Table 2). It is possible that the endogeic worm either did not encounter food deposited on the surface, or did not consume it because it was not in an appropriate state of mineralization. Whilst it is possible that A. chlorotica was restricted through a combination of behaviour and physiology traits to nutrients intrinsic to the exposure soils, the maintenance of its body mass during the period of experimental exposures indicated that the nutrient availabilities in the finite soil volumes (i.e. the net amounts of nutrients)

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Table 2 Opposite effects of organic matter and metal pollution in natural soil samples on riboflavin content in coelomocytes retrieved from epigeic and endogeic earthworm species after 4-week exposure. Riboflavin content similar to that in the control worms (∼); increased (>); significantly increased (); decreased (<); significantly decreased (). Sites

Soil type

Metal soil pollution: moderate, very strong

Riboflavin content in coelomocyte samples from earthworm species Epigeic

Commercial supplier Zlozeniec (meadow) Sierbowice (forest) Bukowno (forest) Bukowno (meadow)

R ZM SF BF BM

Organic Sandy-clay Loamy-sand Organic Organic

None Cd Pb Zn, Cd, Pb Ni, Zn, Cd, Pb

were not growth-limiting even in the sandy organic-poor field soils. That the riboflavin stores in earthworm species containing riboflavin-laden eleocytes display differential species-related labilities when faced with ostensibly similar edaphic variables, including metal pollutants, seems to be evident from the present study. Interpreting such inter-species differences is hampered by limited knowledge of the vitamin’s availability, distribution, and fate in natural terrestrial environments. This is compounded by the rather imprecise knowledge of the specific dietary preferences and microbial trophic interrelationships of different earthworm species (Curry and Schmidt 2007). There is some indication that different plant species have different riboflavin contents (Brown et al. 1997), and that the riboflavin content of plant tissues and riboflavin release from plant roots can be increased, for example, under conditions of iron deficiency (Welkie and Miller 1989). Whether such ecological factors contribute to differences in the differential riboflavin responses of contrasting earthworm species occupying different niches within terrestrial ecosystems remains to be seen. Even less well known is the physiology and biochemistry of riboflavin in earthworms. It is plausible to hypothesise that when dietary riboflavin supply exceeds the earthworm demand it is stored in the chloragosome granules of chloragocytes/eleocytes, but if supply becomes limited or if demand is ramped upwards then cellular riboflavin status diminishes. In principle, riboflavin depletion can arise in at least four ways: (i) the functional inaccessibility of stabilized riboflavin; (ii) the exhaustion of reserves through upregulated flavin-dependent immune events; (iii) compensatory reactions of coelomocytes whose immune-competence has been compromised by environmental contaminants; and, (iv) the indiscriminate stimulation of enzymes such as flavokinase by elevated cellular Zn availability and leading to the consumption of precursor molecules. Overall, our observations indicated that the quantity of dietary riboflavin is a likely determinant of the flavin status of earthworm cells and tissues. In addition, inter-species differences in the susceptibility to metal exposure of riboflavin storage and/or metabolism revealed under laboratory conditions are difficult to explain given the paucity of current knowledge. We conclude, therefore, that studies on the interactions of metal contaminants with flavins designed to identify modes of metallo-toxicity in sentinel organisms such as earthworms are desirable because of the wide and intimate involvement of flavins in fundamentally important metabolic pathways. For example, nitric oxide synthase (NOS) multienzyme systems contain one FAD, FMN and protoporphyrin IX heme iron per subunit (Mittal et al. 1995). NOS catalyses the production of nitric oxide (NO) whose functions range from signalling to participation in responses to infection (Perry and Marletta 1998). Interestingly, both sets of authors presented data showing conclusively that NOS not only binds metals but is inhibited to different extents by, for example, Ni and Zn. It is becoming evident, therefore, that the impacts of different metal contaminants in ‘real’ field

Endogeic

D. veneta

E. andrei

A. chlorotica

Control ∼ > > >

Control  ∼  

Control    <

soils upon flavins, and flavin-mediated pathways in earthworm immune-competent coelomocytes can be better understood by selectively assaying the relative fluorescent signals emanating from riboflavin and its functional metabolic derivatives, FAD and FMN. This enterprise should gain impetus from the recently described (Rhee et al. 2009) bifunctional chemosensor for flavins (referred to as PTZ-DPA) that facilitates the real-time monitoring under physiological conditions of riboflavin kinase (RF to FMN), FAD synthetase (FMN to FAD), and alkaline phosphatase (FMN to riboflavin). Finally, research efforts to detect, localize, and determine the functionality of riboflavin binding protein in earthworms are urgently required, especially since recent observations indicate that the protein binds and possibly chaperones Cu (Smith et al. 2008). Acknowledgments We thank the students from Professor Ryszard Laskowski group for field collection of soil samples. This work was supported by a grant No. PB3502/PO1/32 from the Ministry of Science and Education in Poland. References Brown, M.R., Jeffrey, S.W., Volkman, J.K., Dunstan, G.A., 1997. Nutritional properties of microalgae for mariculture. Aquaculture 151, 315–331. Brulle, F., Mitta, G., Cocquerelle, C., Vieau, D., Lemière, S., Leprêtre, A., Vandenbulcke, F., 2006. Cloning and real-time PCR testing of 14 potential biomarkers in Eisenia fetida following cadmium exposure. Environ. Sci. Technol. 40, 2844–2850. Brulle, F., Cocquerelle, C., Mitta, G., Castric, V., Donay, F., Leprêtre, A., Vandenbulcke, F., 2008. Identification and expression profile of gene profiles differentially expressed during metallic exposure in Eisenia fetida coelomocytes. Dev. Comp. Immunol. 32, 1441–1453. Cholewa, J., Feeney, G.P., O’Reilly, M., Stürzenbaum, S.R., Morgan, A.J., Plytycz, B., 2006. Autofluorescence in eleocytes of some earthworm species. Folia Histochem. Cytobiol. 44, 65–71. Curry, J.P., Schmidt, O., 2007. The feeding ecology of earthworms – a review. Pedobiologia 50, 463–477. Cygal, M., Lis, U., Kruk, J., Plytycz, B., 2007. Coelomocytes and fluorophores of the earthworm Dendrobaena veneta raised at different ambient temperatures. Acta Biol. Cracov. Zool. 49, 5–11. Dutkiewicz, R., Klimek, M., Klimek, B., Stefanowicz, A.M., Płytycz, B., 2009. Effects of cadmium, copper, lead or nickel-contaminated soil on amoebocytes of the earthworm, Aporrectodea caliginosa. Acta Biol. Cracov. Zool. 51, 73–79. Fischer, M., Bacher, A., 2006. Biosynthesis of vitamin B2 in plants. Physiology 126, 304–318. Fuchs, J., Piola, L., González, E.P., Oneto, M.L., Basack, S., Kesten, E., Casabé, N., 2010. Coelomocyte biomarkers in the earthworm Eisenia fetida exposed to 2,4,6trinitrotoluene (TNT). Exp. Monit. Assess., doi:10.1007/s10661-010-1499-z. Gastaldi, L., Ranzato, E., Capri, F., Hankard, P., Pérès, G., Canesi, L., Viarengo, A., Pons, G., 2007. Application of a biomarker battery for the evaluation of the sublethal effects of pollutants in the earthworm Eisenia andrei. Comp. Biochem. Phys. C 146, 398–405. Homa, J., Niklinska, M., Plytycz, B., 2003. Effect of heavy metals on coelomocytes of the earthworm Allolobophora chlorotica. Pedobiologia 47, 640–645. Homa, J., Klimek, M., Kruk, J., Cocquerelle, C., Vandenbulcke, F., Plytycz, B., 2010. Metal-specific effects of metallothionein gene induction and riboflavin content in coelomocytes of Allolobophora chlorotica. Ecotox. Environ. Saf., doi:10.1016/j.ecoenv.2010.07.01. Koziol, B., Markowicz, M., Kruk, J., Plytycz, B., 2006. Riboflavin as a source of autofluorescence in Eisenia fetida coelomocytes. Photochem. Photobiol. 82, 570–573.

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