Department of Evolutionary Immunology, Institute Of

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ACTA BIOLOGICA CRACOVIENSIA Series Zoologia 54: 11–17, 2012

Re s t o r a t i o n o f Co e l o m o c y t e s i n t h e Ea r t h w o r m De n d r o b a e n a Ve n e t a

Ma ł g o r z a t a Kl i m e k *, Je r z y Kr u k ** a n d Ba r b a r a Pł y t y c z *1

* Department of Evolutionary Immunology, Institute of Zoology, Jagiellonian University, Gronostajowa 9, 30-387 Cracow, Poland ** Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Cracow, Poland

Accepted September 28, 2012

We have previously shown that Dendrobaena veneta belongs to the earthworm species which possesses two main cohorts of coelomocytes, namely amoebocytes and autofluorescent eleocytes, the latter storing moderate amounts of riboflavin. The aim of the present experiments performed at 17oC was to follow the restoration of amoebocytes and eleocytes, as well as riboflavin stores in eleocytes after experimental extrusion of coelomocyte-containing coelomic fluid through the dorsal pores induced by electrostimulation (4.5V, 1 min). The analyses were conducted using a combination of cell counts, flow cytometric detection of eleocytes, and spectrofluorimetric measurements of riboflavin in coelomocyte lysates. It has been found that the depleted coelomocyte system recovers slowly. The number of amoebocytes reaches the level characteristic of that in undisturbed worms in 4 weeks, while the number of elecytes is still below the control level 6 weeks after extrusion. The amount of riboflavin stored in recovering chloragocyte- derived eleocytes is higher than that in mature eleocytes for at least 4 weeks.

Key words: Earthworms, coelomocytes, amoebocytes, eleocytes, riboflavin

Introduction wide range of organic wastes are often exposed to high diversity of microorganisms; thus, their im- In natural and semi-natural conditions Dendro- mune system is strongly challenged by microbial baena veneta inhabits dung and compost heaps. antigens and various sorts of pollutants and has It is also cultured on municipal sludge (Lo e h r et to be very efficient to cope with them successfully. al., 1985; Lo f t s -Ho l m i n , 1986) or paper sludge Therefore it is worthwhile to explore the mecha- (Fa y o l l e et al., 1997) for the fishing bait market nisms of immunity of earthworm species adapted and/or vermicomposting (for a review see Mo r g a n , to such extreme antigenic challenges. 2010; Ya d a v and Ga r g , 2011). Composting spe- Earthworm immunity comprises cellular and cies, like D. veneta and Eisenia sp., consuming a humoral defense mechanisms, recently reviewed

1 e-mail: [email protected]

PL ISSN 0001-530X © Polish Academy of Sciences, Cracow 2012 12 Klimek et al. by Bi l e j et al. (2011). Like other annelid inverte- Materials and Methods brates, earthworms possess the metameric coelomic cavity filled with coelomic fluid containing Earthworms various antimicrobial factors like lysozyme and antimicrobial peptides and free wandering cells Adult Dendrobaena veneta (Oligochaeta; Lumbri- named coelomocytes. The earthworm coelomo- cidae), purchased from the commercial supplier cytes consist of amoebocytes, being classical im- (EKARGO, Słupsk), were reared in commercial soil munocytes (according to Ot t a v i a n i ’s nomenclature, (PPUH BIOVITA, Tenczynek) under controlled la- 2011), plus a species-specific portion of eleocytes, boratory conditions (17oC; 12:12 LD). The worms being detached chloragocytes derived from the were kept in plastic boxes with perforated lids chloragogen tissue that surrounds the intestine. and the moisture level was checked weekly. The The chloragocytes/eleocytes, but not amoebo- worms were fed ad libitum a mixed diet comprised cytes, exhibit autofluorescence, which predispo- of dried/boiled nettle (Urtica dioica) and dandelion ses them to flow cytometric analysis (Ch o l e w a et (Taraxacum officinale) leaves, boiled/dried tea lea- al., 2006). This autofluorescence is restricted to ves, and powdered commercial mouse pellets. The chloragosomal vesticles (Pl y t y c z et al., 2007) and experiments were performed in May-June. comes from – among other sources – riboflavin (Ko z i o l et al., 2006; Cy g a l et al., 2007). Riboflavin (vitamin B2) plays an important role in immunity Restoration of the coelomocyte system of animals (e.g. Po w e r s , 2003; Ve r d r e n g h and Ta r - k o w s k i , 2005) and plants (Do n g and Be e r , 2000; At the start of the six-week experiments, 30 in- As a i et al., 2010), and it is responsible for quorum dividuals with similar body weight (1.27+0.23 g) sensing in bacteria (Ra j a m a n i et al., 2008; At k i n s o n were put into 5 separate boxes with fresh soil co- and Wi l l i a m s , 2009). The amount of riboflavin sto- vered with perforated lids, forming 5 experimen- red in eleocytes is species-specific (Pl y t y c z et al. tal groups, 6 worms per group. The worms were 2006) and changes in a species-specific manner in subjected to electrostimulation for coelomocyte response to various edaphic factors (Pl y t y c z et al., extrusion, at various intervals of time before the 2011; Pl y t y c z & Mo r g a n , 2011). end of the experiments. Thus, they had various Coelomocyte-containing coelomic fluid can be time periods for recovery of their coelomocyte sy- expelled through dorsal pores during convulsive stems: 6 weeks (6W), 4 weeks (4W), 2 weeks (2W), body movements of worms stressed by various and 1 day (1D). After 6 weeks from the start of the mechanical stimuli (e.g. predators) or chemical experiments all these animals, their body weight factors. The presence of dorsal pores is commonly reaching 1.66+0.33 g, were once again subjected exploited for the non-invasive quantitative retri- to electrostimulation to assess the recovery of the eval of coelomocyte-containing coelomic fluid of coelomocyte system, while the intact animals from worms stimulated by alcohol (Co o p e r et al., 1995), the last box were electrostimulated for the first ultrasounds (He n d a w i et al., 2004) or mild electric time and served as the control group with no time current (Ro c h et al., 1979), the latter procedure to recover (0D) (Fig. 1). being applied also in the present studies. Dendroabena veneta belongs to the species with a high number of eleocytes storing moderate Coelomocyte extrusion amounts of riboflavin which is also accumulated in chloragocytes of the chloragogen tissue (Ma z u r The earthworms were stimulated for 1 minute with et al., 2011; Pl y t y c z and Mo r g a n 2011). We have an electrical current (4.5V) to expel coelomic flu- previously shown that restoration of the coelomo- id with suspended coelomocytes through the dor- cyte number of D. veneta after experimental ex- sal pores. Briefly, the weighed earthworms were trusion is a long-lasting temperature-dependent individually placed in Petri dishes containing 3 process (Ol c h a w a et al. 2003). The aim of the pre- mL of extrusion fluid (phosphate-buffered saline, sent research is to continue these investigations PBS, supplemented with 2.5 g/L ethylenediami- focusing on restoration of riboflavin-storing eleo- ne tetra-acetic acid, EDTA; Sigma-Aldrich); 2mL cytes. samples of the extruded coelomocyte suspensions Restoration of Earthworm Coelomocytes 13

6W by producing dot plots of cell size versus FL1 au- (1) G N I

T tofluorescence. S E T

(2) R O F

Spectrofluorimetry and analysis L S A T

2W V N E E (3) I R M I

T Spectrofluorometric measurements were perfor- R E E

1D R

med on 2 mL coelomocyte-suspension lysates (ly- X E E

(4) T

sed with 2% Triton; Sigma-Aldrich) using a Per- Y F C O

kin-Elmer LS50B spectrofluorimeter. Emission M T O R L A (5) spectra of riboflavin were recorded in the 380-680 E T S O

0D C nm range (lambda at 370 nm), while excitation spectra were recorded in the 300-500 nm range (lambda at 525 nm). The spectrofluorimetric si- -6w -4w -2w -1d 0 gnatures of unbound riboflavin were characteri- TIME BEFORE TESTING sed by two maxima (at 370 nm and 450 nm) in the excitation spectrum, and a maximum at 525 nm Fig. 1. Scheme for experiments on restoration of coelomo- in the emission spectrum. Arbitrary units (AU) of cytes of Dendrobaena veneta depleted by extrusion induced fluorescence were recorded using Microsoft Excel Fig. 1 by electrostimulation. At the start of the experiments 30 worms with similar body weight were divided into 5 experi- v. 97. The amount of riboflavin in the sample was mental groups (6 worms per group) and put into fresh soil proportional to the maximum at 525 nm in the samples. The worms from groups 1-4 were stimulated for co- emission spectrum. elomocyte extrusion at selected time points (grey marks), i.e. 6 weeks (-6w), 4 weeks (-4w), 2 weeks (-2w) or 1 day (-1d) before the end of the experiments at time 0, while group 5 Statistical analysis served as the control. At the end of the experiments (time 0) coelomocytes from all the five groups of worms were extruded by electrostimulation (black marks) and comparisons were Coelomocyte-connected parameters were calcula- made between the control worms stimulated for the first time ted using Microsoft Excel version 97. The results at time 0 (0D, control) and those restoring their immune sy- concerning restoration of coelomocyte systems stems for 6 weeks (6W), 4 weeks (4W), 2 weeks (2W), or 1 day were expressed as means +/- standard errors. Dif- (1D) (thick lines). ferences between the means were determined by ANOVA (STATGRAPHICS Plus 5.0), with the level were used for spectrofluorimetric analysis, while 1 of significance established at P<0.05. mL was fixed in 2% formalin (Sigma) and used for cell counts in haemocytometer and for flow cytometry. Results

As illustrated in Fig. 2, full restoration of the num- Flow cytometric measurement and analysis ber of D. veneta coelomocytes after experimental depletion by extrelectrostimulation is a long-la- Samples of coelomocytes were analysed with a sting process (Fig. 2a,b,c). Under the conditions of FACScalibur flow cytometer (BD Biosciences). Du- the present experiments, the number of coelomo- ring analytical experiments, 10000 thresholded cytes (CN), including both amoebocytes (AN) and events per worm sample were collected and analy- eleocytes (EN), is statistically significantly lower sed on the basis of their forward scatter (FS) (for compared with the respective control (black bars), cell size) and sideward scatter (SS) (cell complexi- both after one-day (1D) and two-week (2W) recove- ty) properties. Fluorescence FL1-H (emission 530 ry periods. After the 4-week recovery period (4W) nm; excitation 488 nm) was recorded. The resul- the total number of coelomocytes did not differ ting files were analysed for percentages of auto- significantly from the number of coelomocytes in fluorescent granulated eleocytes using WinMDI 2.8 worms after the shorter (1D and 2W) and longer software (Joe Trotter, http://facs.scripps.edu), (6W) periods after extrusion (Fig. 2a). Neverthe- 14 Klimek et al. (a) (d)

(b) (e)

Restoration duration time

Fig. 2. Kinetics of restoration of the coelomocyte system in Dendrobaena veneta. Comparisons of D. veneta coelomocytes re- Fig. 2 trieved from the intact control worms electrostimulated for the first time (black bars, 0D) with those retrieved at the selected time intervals after experimental depletion by previous electrostimulation (grey bars) performed 1 day (1D), 2 weeks (2W), 4 weeks (4W), or 6 weeks (6W) earlier. (a) number of colomocytes (CN); (b) number of amoebocytes (AN); (c) number of eleocytes (EN); (d) percentage of autofluorescent granular cells, the eleocytes (E, %), established by flow cytometry; (e) total riboflavin content (RF) in arbitrary units (AU) established by spectrofluorimetry; (f) riboflavin content per eleocyte (RF/EN). The data are presented as means ±SE; n = 6 worms per group. Means not sharing the same small letter at error bars are significantly different according to ANOVA.

less, after the 4-week recovery period the number number of eleocytes was still significantly reduced of amoebocytes (AN, Fig. 2b) and eleocytes (EN, (Fig. 2c). Fig. 2c) was still lower as compared with the re- In contrast, the percentages of autofluorescent spective intact worms. It was the 6-week recovery eleocytes established by flow cytometry were rela- period (6W) that was necessary for electrostimu- tively stable and similar for the worms recovering lated experimental worms to restore the number the eleocyte system after electrostimulation and of coelomocytes (CN, Fig. 2a), including amoebo- the intact control worms (Fig. 2d). The amount of cytes (AN, Fig. 2b), to the levels similar to those riboflavin in coelomocyte lysates was statistically characteristic of their intact counterparts (black significantly diminished only 1 day after expulsion bars in Figs 2a and 2b, respectively); however, the (Fig. 2e) and reached the level similar to that in Restoration of Earthworm Coelomocytes 15 the control worms already one week later, despite coelomocytes reached the level characteristic of of the long-term significant depletion of the num- undisturbed control worms as soon as 4 weeks af- ber of riboflavin-storing eleocytes (compare with ter expulsion, while in worms maintained at 10oC Fig. 2c). It can be explained by the fact that the only half the number of control cells, even 6 weeks amount of riboflavin per eleocyte (RF/EN, Fig. 2f) after expulsion, was observed. was significantly increased after electrostimula- In other studies on D. veneta maintained at tion-induced coelomocyte expulsion, and decre- 16oC the coelomocyte system was compared ased till the low level characteristic of the intact between undisturbed worms and their counter- worm eleocytes not sooner than 5 – 6 weeks of the parts subjected to coelomocyte expulsion 7 weeks 6-week recovery period (Fig. 2f). earlier (Po l a n e k et al. 2011). It turned out that the total number of coelomocytes, including amoebocytes, was similar for the control and experimen- Discussion tal worms, while the percentage and the number of eleocytes were significantly lowered in the expe- Pioneer experiments on the restoration of coelo- rimental worms. Such delayed restoration of the mocytes experimentally extruded by worm im- eleocyte system was observed both in worms fed mersion in 5% ethanol solution were performed ad libitum a mixed diet and in those deprived of on Lumbricus terrestris by Ey a mb e et al. (1991). external food supply. The 7-week food deprivation The results revealed that in adult worms kept at inhibited worm body weight gain without adverse 10oC the initial number and characteristics of co- effects on the number and composition of coelo- elomocytes, called in their paper the leukocytes, mocytes. The main novelty of the work of Po l a n e k was achieved not sooner than 6 weeks after extru- et al. (2011) consisted in the comparisons made sion, while at shorter time intervals after extru- between the level of riboflavin in undisturbed sion the total number of coelomocytes was signifi- worms and in those tested 7 weeks after coelomo- cantly lowered. The authors concluded that after cyte expulsion. In the experimental worms fed ad initial extrusion, sequential leukocyte collection libitum there was only a slight diminution of ribo- without affecting total and differential cell counts flavin in worms restoring the coelomocyte system, was possible at intervals of 6 weeks (Ey a mb e et al. both the total riboflavin content and that adju- 1991). sted per body weight (RF/BW), while the amount Lumbricus terrestris belongs to the earthworm of riboflavin adjusted per eleocyte number (RF/ species with a small contribution of chloragocyte- EN) was slightly increased. These tendencies were derived eleocytes among coelomocytes freely floa- much stronger in worms deprived of external food ting in the coelomic cavity which contains almost supply, which was statistically significant for total exclusively amoebocytes (Ch o l e w a et al. 2006; riboflavin content in the coelomocyte lysates. This Pl y t y c z et al. 2006). In contrast, Dendrobaena ve- suggests that the newly formed eleocytes freshly neta used for the present investigations belongs detached from the chloragogen tissue accumula- to lumbricid worms with coelomocytes consisting te more riboflavin per cell than the old eleocytes of amoebocytes plus an abundant population of which have inhabited the coelomic fluid for a long eleocytes (Ch o l e w a et al. 2006), the latter exhibi- time as freely floating cells. ting distinct autofluorescence (Pl y t y c z et al. 2006) The latter result has prompted us to repeat derived from moderate amounts of riboflavin ac- the studies on kinetics of restoration of D. vene- companied by another unidentified fluorophore ta coelomocytes, which has been described in (Cy g a l et al. 2007). In one of previous experiments the present paper. As illustrated in Fig. 2, during D. veneta coelomocytes were sequentially 8 times the experiments performed at 17oC, i.e. the tem- extruded day by day by electric shock without ad- perature intermediate between the temperatures verse effects on worm viability, which was followed used previously by Ol c h a w a et al. (2003), the re- by cell proliferation (Ho m a et al. 2008). storation of coelomocytes was faster than it was The kinetics of restoration of D. veneta coelo- at 10oC, but slower than at 22oC. The number of mocytes was preliminarily described by Ol c h a w a amoebocytes reached the control level within 6 et al. (2003). It turned out that this process was weeks, while the number of eleocytes was still be- temperature-dependent. At 22oC the number of low the control level, the result corresponding to 16 Klimek et al. that obtained during 7-week studies performed by of the earthworm Dendrobaena veneta (Oligochaeta). Soil. Po l a n e k et al. (2011). 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