RESEARCH REPORT ISJ 7: 89-106, 2010 ISSN 1824-307X RESEARCH REPORT The effect of oxidative stress on phagocytosis and apoptosis in the earthworm Eisenia hortensis SL Fuller-Espie, T Nacarelli, EL Blake, FM Bearoff Science Department, Cabrini College, 610 King of Prussia Road, Radnor, Pennsylvania 19087-3698, USA Accepted February 17, 2010 Abstract The effect of exogenous hydrogen peroxide (H202) on phagocytic function and apoptosis in coelomocytes from Eisenia hortensis was investigated. Treating coelomocytes with H202 (0.26 to 8.4 mM) evoked a significant increase in phagocytosis for one or more of the concentrations of H202 employed in 67 % of cases. Using annexin V-FITC we show that H202 induced apoptosis of coelomocytes in vitro. We found that 100 % of viable coelomocyte populations exhibited significant increases in phosphatidylserine translocation for one or more of the concentrations of H202 tested (8.4 to 67.6 mM). Using a fluorescent inhibitor of caspases, we revealed the presence of activated caspases observing increased caspase activity in 67 % of viable coelomocyte populations treated with 33.8mM H202, and in 100 % of cases treated with 67.6 mM H202. Agarose gel electrophoresis and the TUNEL assay showed DNA fragmentation in samples treated with 16.9 and 33.8 mM H202. In addition, endogenous H202 production during phagocytosis by hyaline amoebocytes was detected using a fluorogenic substrate. Thus, free radicals not only appear to facilitate phagocytosis and are produced during phagocytosis, but they also promote an oxidative-stress-induced apoptosis that may play an important function in regulating innate immune responses in E. hortensis. Key Words: phagocytosis; hydrogen peroxide; annexin V; DNA fragmentation; caspase, TUNEL assay Introduction When the production of reactive oxygen species (ROS) in microsomes, peroxisomes, mitochondria and the cytosol overwhelms a cell’s ability to either neutralize reactive intermediates or repair toxic effects from ROS, a state of oxidative stress is initiated. Toxic effects from ROS include damage to nucleic acids (mutagenesis) and carbohydrates, lipid peroxidation of cellular membranes, and enzyme inactivation (Imlay, 2003). Oxidative stress is also linked to the aging process (Larsen, 1993; Helfand and Rogina, 2003; Rattan, 2006; Csiszar et al., 2007). ROS encompass a wide array of oxidants including hydrogen peroxide (H202, the focus of this study), superoxide anions, hydroxyl radicals, peroxyl radicals and organic hydroperoxides (Dunford, 1987; Coffey et al., 1995; Panasenko et al., 2002). These oxidants are produced through various means including radiation, uncomplexed metals such as iron and copper, organic compounds such as quinones, uric acid and ___________________________________________________________________________ Corresponding author: SL Fuller-Espie Science Department Cabrini College 610 King of Prussia Road, Radnor, PA 19087-3698, USA Email: sfuller-espie@cabrini.edu homocysteines, certain classes of xenobiotics such as polycyclic aromatic hydrocarbons (PAH) going through redox cycling, and thermal stress (DiGuilioi et al., 1989; Livingstone et al., 1990; Sundaram et al., 1990; Livingstone et al., 1995; Abele et al., 2001; Tyagi et al., 2005; Valko et al., 2006; Strazzullo and Puig, 2007; Fato et al., 2008; Pichaud et al., 2008; Kell, 2009). They are also generated by intracellular enzymes including NADPH oxidase, xanthine oxidase and cytochrome P450 (Lewis, 2002; Bedard and Krause, 2007; Jankov et al., 2008). Paradoxically, ROS not only exert damaging effects in cells, but they also afford protecive effects, for example during immune defense for phagocytosis where ROS are toxic to phagocytized pathogens. Another benefit of ROS is their aility to participate in redox signaling (Thannickal and Fanburg, 2000; Forman and Torres, 2002; Wang, 2009). Because of the detrimental effects of ROS on cellular components, it is imperative that organisms possess cellular antioxidant defense mechanisms for the detoxification of ROS, often measured as the total oxyradical scavenging capacity (TOSC), an important biomarker of oxidative stress (Regoli, 2000; Gorbi and Regoli, 2003; Dovzhenko et al., 2005). ROS are neutralized by a variety of   89 mailto:sfuller-espie@cabrini.edu antioxidant processes aimed at stabilizing free radicals, terminating free radical reactions, and preventing the transfer of electrons from oxygen to organic molecules. One mechanism relies upon enzymatic detoxification of ROS by catalase, glutathione peroxidase, superoxide dismutase, thioredoxin reductase, peroxiredoxins and sulfiredoxin (Raes et al., 1994; Nordberg and Arnér, 2001; Flohé et al., 2003; Findlay et al., 2005). Nonenzymatic antioxidants also provide antioxidant defenses and include a diverse array of molecules including antioxidant quenchers comprising cellular proteins (e.g. transferrin, ferritin, metallothionein, ceruplasmin and others) that chelate pro-oxidant minerals (Cairo et al., 1995; Kang et al., 2001; Yamaji et al., 2004; Laukens et al., 2009). In addition, glutathione, selenium, phytochemicals, vitamin E, vitamin C and provitamin A compounds (e.g. beta carotene) also provide protective antioxidant defenses (Sies et al., 1992; Loo, 2003; Brenneisen et al., 2005; Ghezzi, 2005). The primary goal of this study was to investigate the in vitro effects of oxidative stress on cellular activities in the immune cells (coelomocytes) of the earthworm Eisenia hortensis (also known as the European nightcrawler) which reside in the coelomic cavity. Investigations of innate immunity in earthworms have identified three distinct subpopulations of coelomocytes (leukocyte equivalents): hyaline amoebocytes (large coelomocytes), granular amoebocytes (small coelomocytes) and chloragocytes (eleocytes), most likely diverging developmentally from a common progenitor cell (prohemocyte), as suggested by Hartenstein (2006). The immune functions of coelomocytes can be studied in vitro after harvesting the coelomic fluid, which is rich in coelomocytes, by extruding the coelomocytes through the dorsal pores of the body wall from experimentally-induced earthworms. The hyaline amoebocytes are the major phagocytic cells, the granular amoebocytes constitute the subpopulation exhibiting NK-like activity, and the eleocytes contain chloragosomes and do not participate in either phagocytic or NK-like activities, but they do secrete lytic substances (Cooper, 1996; Cossarizza et al., 1996; Adamowicz and Wojtaszek, 2001; Engelmann et al., 2002; Engelmann et al., 2005). Differences in granularity and size between coelomocytes permits amoebocytes (hyaline and granular) and eleocytes to be distinguished using flow cytometry methodology employing forward light scatter (FSC) and side light scatter (SSC) measurements (Cossarizza et al., 1996, 2005; Engelmann et al., 2004; Patel et al., 2007; Fuller-Espie et al., 2008). Selective analysis of subpopulations is facilitated by specifying regions to identify particular subpopulations, and then gating on assigned regions, permitting the investigator to include only desired subpopulations and exclude irrelevant subpopulations from final analyses. Light and fluorescent microscopy have been used by researchers to study immune functions in earthworms (Adamowicz and Wojtaszek, 2001; Kalaç et al., 2002), however, these methods are more subjective than flow cytometry, and they impose restrictions on the number of cells included in analyses owing to time constraints. In contrast, flow cytometry is an objective, quantitative methodology that analyzes thousands of cells per second with the option of restricting analyses to predetermined subpopulations. This investigation focused specifically on phagocytic function and the induction of apoptosis in the amoebocytes of E. hortensis following exposure to exogenous H202. We evaluated the phagocytic uptake of Escherichia coli using flow cytometry and found that in vitro exposure to H202 (0.26 - 8.4 mM) enhanced phagocytosis. Using flow cytometric and agarose gel electrophoresis methodologies we also examined the effect of H202 on three events associated with apoptosis: 1) translocation of phosphatidylserine (PS) to the extracellular face of the plasma membrane using annexin-V binding; 2) caspase activation using a fluorescein-conjugated inhibitor of caspase activation (FLICA); and 3) DNA fragmentation. We present data supporting an apoptotic-like cell death in coelomocytes of E. hortensis resulting from in vitro exposure to exogenous H202 (8.4 - 67.6 mM). Finally, using the fluorogenic substrate DHR 123, a probe widely used to measure intracellular H202, we also show that H202 is generated in hyaline amoebocytes during phagocytosis of Bacillus megaterium and Pseudomonas stutzeri. Materials and Methods Cell culture supplies and chemical reagents Tissue culture plasticware was purchased from Fisher Scientific. Phosphate buffered saline (PBS) was purchased from Invitrogen. Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) was supplemented with either 10 % heat-inactivated fetal calf serum (Invitrogen) or Serum Supreme (Lonza BioWhittaker), plus 100 μg ml-1 ampicillin (Shelton Scientific), 10 μg ml-1 kanamycin (Shelton Scientific), 10 μg ml-1 tetracycline, 5 μg ml-1 chloramphenicol (Fluka Biochemika), 1× penicillin, streptomycin and amphotericin B, 1× nonessential amino acids (Invitrogen) and 1× L-glutamine (Invitrogen) to comprise Super DMEM (SDMEM). SDMEM supplemented with Serum Supreme was used for all exogenous H202 assays while SDMEM supplemented with fetal calf serum was used for endogenous H202 assays. Earthworm husbandry Eisenia hortensis (European nightcrawlers) was purchased from Vermitechnology Unlimited, Orange Lake, Florida, USA, who imports E. hortensis from Star Food, Holland, Scherpenzeelseweg 95, 3772ME Barneveld, The Netherlands. Species identity was determined by the United States Department of Agriculture, USDA Permit #52262 (Vermitechnology, personal communication). Short- term colonies were maintained at RT in the dark on moistened autoclaved pine woodchips sprinkled with Single Grain Rice Cereal or Rice with Bananas Cereal (Gerber) and covered with autoclaved, shredded and moistened paper towels. Habitats were changed twice weekly. Animals were euthanized by freezing at -20 oC.   90 Extrusion of coelomocytes Prior to experimentation, earthworms were first washed with distilled water on paper towels using a water bottle to remove wood chip fragments or food particles. They were then placed overnight on paper towels moistened with 2.5 μg ml-1 Fungizone (Fisher Scientific) in 100 mm Petri dishes to minimize fecal contamination during the extrusion process, and remove further any surface contaminants. To collect coelomocytes, earthworms were placed in either 100 mm Petri dishes or in multichannel pipette reservoirs containing 3 ml BD FACSFlow sheath fluid (BD Biosciences). The earthworms extruded their coelomocytes through their dorsal pores in response to this external stimulus without the need to use the alcohol extrusion method reported by others (Engelmann et al., 2005). The coelomocytes were then transferred to 0.5 ml Accumax (Innovative Cell Technology) in 15 ml conical test tubes for a 5 min incubation period at RT to reduce aggregation of cells. Finally, 5 ml PBS was added and the samples were centrifuged immediately at 150 x g, 5 min at 4 oC. After decanting the supernatant, the coelomocyte pellet was gently mixed by flicking the bottom of the centrifuge tube, and coelomocytes were resuspended in 0.5 ml SDMEM. Enumeration was carried out using a hemacytometer. Only hyaline amoebocytes (large coelomocytes) and hyaline granulocytes (small coelomocytes) were included in the cell count; eleocytes were not counted but did factor into a quality score. Samples with large numbers of eleocytes compared to large and small coelomocytes were not used in phagocytosis assays. Samples were adjusted to 3.8 x 105 or 5 x 105 (phagocytosis and annexin V assays, see below) or 1 x 106 (caspase assays) coelomocytes ml-1 in SDMEM. Bacteria for phagocytosis assays E. coli/GFP: Escherichia coli HB101 transformed with pGLO (BioRad) and expressing green fluorescent protein (GFP) were grown on tryptic soy agar containing 100 μg ml-1 ampicillin and 0.2 % (w/v) arabinose at 32 °C for 24 h. After washing the cells once in PBS, they were fixed chemically with 4 % (v/v) paraformaldehyde in PBS, 1 h at RT with periodic mixing, followed by three PBS washes. Centrifugation was carried out at 3273xg for 5 min at 4 oC. The final cell pellet was resuspended in PBS, bacteria were enumerated using a hemacytometer, and then stored in the dark at 4 °C. Bacillus megaterium and Pseudomonas stutzeri (Presque Isle Cultures) were grown overnight in tryptic soy broth at 37 oC in a shaking incubator. Absorbance was measured using a spectrophotometer (600 nm) and compared to a standard curve to determine concentration. Standard curves were generated by correlating absorbance with cell count using a hemacytometer. All bacteria were diluted in SDMEM to obtain the desired multiplicity of infection (m.o.i.). Phagocytosis: exogenous H202 pretreatment Phagocytosis assays were carried out in SDMEM. Coelomocytes (50,000 per well) were pretreated with or without H202 (0 - 8.4 mM final concentration) 5 % CO2, at 25 oC in 96-well, round- bottom plates in 200 μl SDMEM. Duplicate samples were used in every assay. Following H202 pretreatment, cells were centrifuged (150xg) and washed once with PBS. Finally, 200 μl E. coli/GFP was added to each well at a multiplicity of infection of 1000 bacteria:1 coelomocyte and incubated for 3 h at 30 °C. To control for non-specific binding of E. coli/GFP to the external surface of coelomocytes, 50 μM cytochalasin B (Sigma Aldrich) [an antibiotic that interferes with microfilament activity and thereby inhibits phagocytosis (Axline and Reaven, 1974)] was added to control wells 45 min before the addition of E.coli/GFP. Following E. coli/GFP uptake, trypan blue (BioWhittaker) was used at a final concentration of 0.02 % (w/v) for 30 min at RT in the dark, for quenching purposes to reduce background fluorescence (Mosiman et al., 1997). The cells were transferred to flow cytometry tubes containing 100 μl FACS Flow buffer (BD Biosciences), placed on ice in the dark, and run immediately on the flow cytometer. Annexin V-FITC/PI assay Recombinant human annexin V-FITC (Invitrogen, ANNEXINV01)) and propidium iodide (PI) (Invitrogen, P3566) were used to detect PS translocation and to enable exclusion of dead cells from analyses. Using a 96-well, round-bottom plate, 5 x 104 (assay 1) or 3.8 x 104 (assay 2) coelomocytes in 50 μl SDMEM were added to appropriate experimental (H202-treated) and control (double negative autofluorescent background; single positive FITC; single positive PI; double positive annexin V/PI background) wells in triplicate. Experimental wells received 50 μl of H202 (final concentrations of 67.6, 33.8, 16.9, and 8.45 mM). Single positive FITC controls received 50 μl of H202 (270mM final). Single positive PI controls received 50 μl saponin (0.01 % final). The plate was incubated 6 h, 25 oC, 5 % CO2 before adding 100 μl PBS and centrifuging (5 min, 4 oC, 150xg). After removing the supernatant fraction, the wells were washed with 200 μl well-1 of PBS, and centrifuged again. The supernatant fraction was removed and the cells were resuspended in 200 μl SDMEM containing 1× binding buffer (0.01M HEPES, 0.14 mM NaCl, 2.5mM CaCl2) with or without annexin V- FITC (3.75 μl well-1) and/or PI (0.5 μl well-1). The autofluorescent background control did not receive annexin V-FITC or PI. The single positive PI control did not receive annexin V-FITC. The single positive FITC control did not receive PI. All other samples received both annexin V-FITC and PI. Samples were incubated for 5 min at RT and transferred to flow cytometry tubes containing 150 μl of FACS Flow sheath buffer containing 1× binding buffer. Samples were kept on ice protected from light and analyzed immediately by flow cytometry. Caspase assay Caspase activation was measured using a Vybrant® FAM Caspases Assay Kit (FITC) (Invitrogen/Molecular Probes) according to the manufacturer’s instructions. This assay utilized a fluorescent inhibitor of caspases known as FLICA™   91 which detects activation of caspase enzymes in cells undergoing apoptosis. Using a 96-well, V- bottom plate, 1 x 105 coelomocytes in 0.1 ml SDMEM were added to appropriate experimental (H202-treated) and control [untreated (0 mM) double negative autofluorescent background; single positive FITC; single positive PI; double positive caspase background] wells in duplicate. Experimental wells received 50 μl H202 (final concentrations of 33.8 mM for EW F1-F3; 16.9 mM, 33.8 mM and 67.9 mM for EW F4-F6); single positive PI control wells received 50 μl 0.03 % saponin in SDMEM; and single positive FITC control wells received 50 μl SDMEM. All control and experimental samples were incubated for 6 h, 25 °C, 5 % CO2. After centrifugation at 150xg (5 min, 4 oC), the supernatant fraction was removed and the wells were washed with 200 μl PBS. Again the plate was centrifuged and the supernatant fraction was removed. Untreated double-negative, autofluorescent control samples (FITC negative, PI negative) and single positive PI controls were resuspended in 100 μl of SDMEM. Untreated samples (caspase background) and H202- treated samples were resuspended in 90 μl SDMEM plus 10 μl of 10X FLICA reagent. Single positive FITC control wells were resuspended in 50 μl SDMEM, 10 μl of 10X FLICA reagent, and 40 μl of 10 % formaldehyde. The plate was incubated in the dark, 1 h, 25 °C, 5 % CO2, with gentle mixing every 20 min before adding 100 μl of 1× washing buffer and centrifuging as above. After removing the supernatant fraction, the cells were washed twice with 200 μl well-1 of 1× wash buffer. Following the last centrifugation and removal of the supernatant fraction, 200 μl well-1 of 1× wash buffer with or without PI was added to each well; double-negative and single positive FITC controls did not receive PI, all other samples received PI. Samples were placed on ice protected from light and analyzed immediately by flow cytometry. Cell volume measurements EW F4-F6 used in the caspase assay were also subjected to cell volume analysis using flow cytometry. Forward scatter measurements of PI- negative large coelomocytes treated in duplicate with 0, 16.9, 33.8 and 67.6 mM H202 were averaged and analyzed by Student’s t test to determine if differences observed in forward light scatter measurements were statistically significant compared to controls. Flow cytometry Fluorescence was measured using FL-1 (FITC, GFP and rhodamine 123) and FL-2 (PI) detectors of a FACSCalibur flow cytometer (BD Biosciences). Autofluorescent controls were used to set voltages for forward scatter (FSC), side scatter (SSC), FL-1 and FL-2 during instrument set-up. Single positive FITC and PI controls were used to adjust compensation settings (spectral overlap removal) for annexin V and caspase assays. LISTMODE data was acquired and analyzed using Cell Quest (BD Biosciences) and WinList5.0 (Verity Software House) software. Only coelomocytes corresponding to the large coelomocyte population (phagocytosis assays) or large and small coelomocyte populations combined (annexin V and caspase assays), as determined by appropriate granularity and size, were gated for further analyses. DNA fragmentation assay DNA purification was carried out according to Hermann et al. (1994) with some modifications. Briefly, coelomocytes from ten individual earthworms were extruded and plated at 1.5 x 105 coelomocytes well -1 in 200 μl. For each treatment, 10 wells were used (1.5 x 106 coelomocytes per treatment from 10 individual earthworms), one well for each earthworm extruded, and each earthworm exposed to all treatments of the assay. H202 was added at 0, 8.4, 16.9 or 33.8 mM and then incubated at 30 oC, 5 % CO2, 6 h. After the incubation period, the plate was centrifuged (150xg, 10 min, 4 oC), the supernatant was removed, and the cells were gently resuspended by vortexing. Then 40 μl of lysis buffer (1% NP-40, 20 mM EDTA, 50 mM Tris-HCl, pH 7.5) was added to each well before pooling the 10 wells (10 individual earthworms) for each treatment group into a single microcentrifuge tube and centrifuging the lysate at 1600xg, 5 min to pellet debris. The supernatant was transferred to a new tube and the pellet was re- extracted with 40 μl lysis buffer and respun. Supernatants were combined for each treatment group, and adjusted to 1 % SDS, 5 μg ml-1 RNAse (Fermentas Life Sciences) and incubated 2 h at 56oC before adding proteinase K (Fisher, BP1700- 50) (2.5 μg ml-1) and incubating 2 h at 37 oC. Then 0.5 volume 7.5 mM ammonium acetate and 2.5 volume of absolute ethanol was added to precipitate the DNA. DNA pellets were collected by centrifugation (14,000xg), rinsed with ice cold 70 % ethanol and air dried. Pellets were resuspended in 21 μl TE (10 mM Tris-Cl, 1 mM EDTA, pH 8,0) at 37 oC, 5 min before adding 4 μl loading dye (6x Blue/Orange Loading Dye, Promega, G1881) containing 1:100 SYBR® Safe gel stain (10,000X concentration in DMSO, Invitrogen). Molecular weight markers (10 μl well-1) (exACTGene, Fisher BioReagents, BP257110) containing SYBR® Safe gel stain (2 μl well-1) and DNA samples from each treatment group were electrophoresed in a 1.5 % agarose gel containing 1:10,000 SYBR® Safe gel stain in 1 x TBE (89 mM Tris base, 89 mM boric acid, 2 mM EDTA, pH 8.3), 120V until first dye front was ~ 2 cm from bottom of gel. Gels were photographed using a Gel-Documentation System (Bio-Rad). TUNEL assay A Flow TACS Apoptosis Detection Kit (Trevigen, Inc.) was used according to the manufacturer’s instruction except 150,000 cell ml-1 were used in 0.5 volume recommended, and optional PI was not included. Coelomocytes were incubated in 0.2 ml SDMEM for 12 h at 25oC, 5% CO2 with or without H2O2 (33.8 mM) before washing and fixing the cells. Flow cytometry measured FL-1 signals from gated amoebocytes. Samples were run in duplicate and subjected to statistical analysis by Student’s t test.   92 Phagocytosis: endogenous H202 production values were statistically significant as exhibited as a p-value less than or equal to 0.05. All data is based on averages of either triplicate (exogenous H202/phagocytosis and annexin V assays) or duplicate (endogenous H202/phagocytosis, caspase and TUNEL assays) samples. Bacteria were introduced to coelomocytes (50,000 per well in 96 V-bottom plate in duplicate) at a m.o.i. ranging from 10:1 to 1000:1. Following 90 min incubation at 30 oC, DHR 123 (Invitrogen, D- 632, 1 mM stock in DMSO) was added (1 μM final, 1:1000). After 10 min incubation at RT, samples were placed on ice, protected from light, and run on the flow cytometer immediately. Negative controls (no DHR 123) were incubated with 1:1000 DMSO to control for carrier effect of DMSO. Fluorescence was measured using the FL-1 detector. Results Flow cytometry: exogenous phagocytosis assay Figure 1 illustrates how our data was collected and analyzed to determine specific phagocytosis by hyaline amoebocytes from E. hortensis when using exogenous H202. The left panel of the top row is a dot plot representing a typical coelomocyte profile obtained on the flow cytometer when analyzing forward scatter (FSC) (abscissa) versus side scatter (SSC) (ordinate) properties of earthworm Statistical Analysis Data analysis and graphs were generated using Microsoft Excel 2007. The Student’s t-test assuming unequal variance was utilized with a 95 % confidence interval to determine if the experimental Fig. 1 Representative flow cytometry profile from phagocytosis assays. Top row: left panel shows a typical coelomocyte profile of FSC (size) (abscissa) versus SSC (granularity) (ordinate) of extruded earthworm coelomocytes where R1 = hyaline amoebocytes, R2 = granular amoebocytes, and R3 = eleocytes; right panel shows FSC (abscissa) versus FL-1 (relative fluorescence intensity) (ordinate) of R1-gated, untreated coelomocytes cultured without E. coli/GFP. Bottom row: FSC (abscissa) versus FL-1 (ordinate) of R1-gated coelomocytes cultured with E. coli/GFP without pretreatment (left panel) and with pretreatment (right panel) of H202. FSC = forward scatter; SSC = side scatter; FL-1 = relative fluorescence intensity of GFP; UR = upper right; LR = lower right.   93 Fig. 2 Phagocytosis is enhanced by pretreating earthworm coelomocytes with H202. Asterisks indicate p ≤ 0.05. Top row: Earthworms EW-P1 – EW-P4 were pretreated with 0, 1.1, 2.1, 4.2 and 8.4 mM H202. Middle and bottom rows: Earthworms EW-P5 – EW-P12 were pretreated with 0, 0.26, 0.53, 1.1, 2,1, 4.2 and 8.4 mM H202. coelomocytes. R1 depicts large coelomocytes (hyaline amoebocytes), R2 depicts small coelomocytes (granular amoebocytes), and R3 depicts chloragocytes (eleocytes). For analysis purposes and the determination of percent specific phagocytosis, FSC (abscissa) versus FL-1 (ordinate) dot plots were gated on hyaline amoebocytes (R1), the phagocytic cell population. The FSC versus FL-1 dot plots were partitioned into quadrants moving the horizontal bar (left to right) such that all of the events fell within the two right quadrants. The vertical bar (up and down) for FL-1 was established based on the negative control population (i.e., autofluorescence). Relative fluorescence intensity values for FL-1 delineate positive events (upper right quadrant - UR) and negative events (lower right quadrant - LR). The right panel of the top row shows FSC versus FL-1 for an untreated sample in the absence of E. coli/GFP, the negative control population. Note that the vertical bar was placed above the majority of events. The left panel of the bottom row shows an untreated sample in the presence of E. coli/GFP, while the right panel of the bottom row shows an H202-treated sample in the presence of E. coli/GFP. Note the shift of the coelomocyte population from the LR (negative events) to the UR (positive events) quadrants between these two dot plots as fluorescence intensity increases due to phagocytic uptake of E. coli/GFP when pretreated with H202. In this example percent positive events in UR increases from 7.54 to 13.23 % when coelomocytes were pretreated with H202. Effects of exogenous H202 on phagocytosis Having established the data analysis protocol, we studied the effect of H202 at concentrations ranging from 0.26 - 8.4 mM on the phagocytosis of E. coli/GFP by earthworm coelomocytes. Percent specific phagocytosis of E. coli/GFP was determined by subtracting the percent positive events (UR) of negative controls (absence of E. coli/GFP) in FL-1 from each of the experimental samples (presence of E. coli/GFP with or without H202). The average of duplicates of controls (0 mM H202) versus H202- treated samples were plotted and statistical significance was determined using the Student’s t test. Figure 2 displays the results obtained for   94 A B C D E Fig. 3 Representative flow cytometry profile of annexin V-FITC/PI assay using EW-A7 as an example for data collection. Coelomocytes were pretreated with 0 (spontaneous apoptosis) (A), 8.4 (B), 16.9 (C), 33.8 (D) and 67.6 (E) mM H202. Left hand column: FSC (abscissa) versus SSC (ordinate) of total, ungated coelomocytes population. Region 1 (R1) depicts the amoebocytes population (hyaline and granular amoebocytes). Middle column: FSC (abscissa) versus FL-2 (PI) (ordinate) of R1 gated amoebocytes (excluding eleocytes). Region 6 (R6) depicts PI- negative (FL-2 negative), viable amoebocyte population. Right hand column: FL-1 (abscissa) versus cell number (ordinate) of amoebocytes gated on R1 and R6 (i.e. only viable amoebocytes that have not taken up PI). Region 7 (R7) corresponds to annexin V negative amoebocytes while region 8 (R8) corresponds to annexin V positive (early apoptotic) amoebocytes. FSC = forward scatter; SSC = side scatter; FL-1 = relative fluorescence intensity of FITC, FL-2 = relative fluorescence intensity of PI.   95 Table 1 Percent early apoptotic cells binding annexin V-FITC in untreated and H202-treated samples. Only cells residing in R1 and R6 were gated for annexin V-FITC analysis. R1 included hyaline and granular amoebocytes (not eleocytes) and R6 included PI-negative cells. Background autofluorescence of untreated samples not receiving annexin V-FITC or PI was subtracted from all values for each indicated earthworm sample before averaging triplicate data and performing statistical analyses. Results indicate data obtained from two assays performed by two independent researchers. Percent positive values for annexin V-FITC (± SD) are shown for spontaneous apoptosis (0 mM H202) and two-fold serial dilutions from 67.6 to 8.4 mM H202. Statistically significant values above spontaneous apoptosis levels are indicated as: * = p ≤ 0.05; ** = p ≤ 0.005; *** = p ≤ 0.0005 as determined by Student’s t test. Earthworm Sample Spontaneous Apoptosis 0 mM 8.4 mM 16.9 mM 33.8 mM 67.6 mM EW-A1 46.23 (±2.42) 31.75 (±1.33) 42.27 (±0.95) 62.81 (±2.39)*** 82.86 (±1.48)*** EW-A2 54.68 (±2.07) 52.82 (±0.74) 64.91 (±2.17)** 66.70 (±1.10)** 81.72 (±1.40)*** EW-A3 31.76 (±1.67) 31.88 (±0.49) 39.51 (±1.13)** 49.99 (±1.26)*** 62.03 (±1.45)*** A ss ay 1 EW-A4 47.01 (±0.57) 36.99 (±1.22) 44.93 (±1.00) 49.78 (±5.04) 63.24 (±1.76)** EW-A5 63.11 (±4.80) 50.40 (±0.96) 58.90 (±1.22) 79.79 (±2.35)* 85.99 (±0.67)* EW-A6 72.68 (±1.68) 69.51 (±0.63) 70.75 (±1.30) 71.56 (±2.65) 80.43 (±2.40)** EW-A7 31.95 (±0.51) 59.35 (±1.48)*** 62.06 (±1.45)*** 67.56 (±2.45)** 75.95 (±2.04)*** A ss ay 2 EW-A8 38.75 (±2.25) 27.83 (±0.08) 27.25 (±1.23) 37.60 (±0.59) 46.88 (±1.12)* coelomocytes from 12 earthworms pretreated in vitro with H202 prior to phagocytosis. Earthworms 1, 2, 3 and 4 (EW-P1-P4) were pretreated in the range of 1.1 - 8.4 mM H202, while EW-P5-P12 were pretreated in the range of 0.26 - 8.4 mM H202. Eight of the 12 earthworms tested (67 %) exhibited statistically significant enhancement of phagocytosis to at least one of the concentrations employed. At doses above 8.4 mM, inhibitory effects on phagocytosis were observed (data not shown). Flow cytometry detection of early apoptosis For the next two experiments, which were aimed at investigating the effects of H202 on PS translocation and caspase activation in amoebocytes of E. hortensis, it was important to be able to discriminate between necrotic/late apoptotic amoebocytes and amoebocytes undergoing early apoptosis to ensure that analyses were restricted to amoebocytes with intact plasma membranes. To do this, we utilized PI in addition to the fluorescein- tagged reporters of PS translocation and caspase activation. PI exhibits a sufficiently large Stokes shift compared to the fluorescein permitting simultaneous detection of fluorescein-labelled moieties and nuclear DNA providing the appropriate optical filters and compensation adjustments for spectral overlap are utilized. In our case, we used a FACSCalibur flow cytometer which employes FL-1 for fluorescein detection and FL-2 for PI detection. PI is membrane impermeant and is thus excluded from viable cells, making this fluorescent counterstain an ideal marker for identifying necrotic/late apoptotic cells in a population when used together with a second fluorescent dye which has minimal spectral emission overlap. Figure 3A illustrates the gating strategy used for the analysis of both the PS translocation and caspase activation experiments. First the coelomocytes were analyzed for FSC versus SSC (Fig. 3A - left panel) and region (R1) was set around the amoebocytes. Next a dot plot of FSC versus FL- 2 gated on the R1 population was created and quadrants were established according to procedure described in Fig. 1 (Fig. 3A - middle panel) positioning the vertical bar at a location that clearly delineated live from dead cells based on the saponin-treated, PI-stained single-positive control employed for compensation purposes. The quadrants corresponded to regions 2-5 (R2-R5). Next another region (R6) was drawn around the PI- negative cell population, serving to identify viable cells (this would include non-apoptotic, non-necrotic and early-apoptotic cells whose membranes were still intact). Finally, a single parameter histogram measuring fluorescein (FL-1) was generated and gated on R1 (amoebocytes) and R6 (PI-negative cells). Therefore, only events that satisfied both prerequisites of belonging to the amoebocytes pool (hyaline and granular) as well as being viable were quantified in the R1/R6 gated FL-1 histogram (Fig. 3A - right panel). This strategy permitted the exclusion of necrotic/late apoptotic cells from the final analysis.   96 Table 2 Relative fluorescence intensity (RFI) of early apoptotic, annexin V-FITC-positive cells in untreated and H202-treated samples. Only cells residing in R1 and R6 were gated for annexin V analysis. R1 included hyaline and granular amoebocytes (not eleocytes) and R6 included PI-negative cells. Triplicate data was averaged and subjected to statistical analyses. Results indicate data obtained from two assays performed by two independent researchers. RFI values (geometric mean) above background detected by FL-1 (± SD) are shown for spontaneous apoptosis (0 mM H202) and two-fold serial dilutions from 67.6 to 8.4 mM H202. Statistically significant values exceeding spontaneous apoptosis levels are indicated as: * = p ≤ 0.05; ** = p ≤ 0.005; *** = p ≤ 0.0005 as determined by Student’s t test. Earthworm Sample Spontaneous Apoptosis 0 mM 8.4 mM 16.9 mM 33.8 mM 67.6 mM EW-A1 14.75 (±0.59) 10.65 (±0.42) 11.22 (±0.08) 14.03 (±0.66) 19.69 (±0.94)** EW-A2 11.99 (±0.16) 12.56 (±0.44) 12.58 (±0.02)* 13.91 (±0.21)*** 21.66 (±1.50)** EW-A3 15.12 (±0.57) 10.72 (±0.48) 11.70 (±0.47) 13.53 (±0.57) 14.90 (±0.41) A ss ay 1 EW-A4 12.79 (±0.12) 10.19 (±0.06) 11.20 (±0.12) 13.33 (±0.53) 18.02 (±0.39)** EW-A5 29.88 (±1.92) 17.81 (±1.46) 18.40 (±0.19) 30.45 (±3.50) 81.52 (±5.72)** EW-A6 25.55 (±2.74) 19.13 (±0.40) 22.40 (±0.70) 28.61 (±0.28) 45.16 (±1.65)** EW-A7 12.23 (±1.61) 14.46 (±0.24) 23.22 (±0.40)** 26.48 (±0.82)*** 31.84 (±0.65)*** A ss ay 2 EW-A8 10.31 (±0.18) 10.35 (±0.35) 11.72 (±0.75)* 13.58 (±0.56)** 19.37 (±2.04)* PS translocation: annexin V-FITC binding Cells undergoing early apoptosis can be easily identified using annexin V, an anticoagulant protein that exhibits a high degree of specificity for PS, and when conjugated to a reporter molecule, such as fluorescein isothiocyanate (FITC), can be used an indicator of early apoptosis. PS is a phospholipid that is normally retained on the inner leaflet of the plasma membrane, however, in cells undergoing apoptosis, it is translocated to the outer leaflet and is exposed on the surface of the cell. Once exposed on the surface, it is accessible to annexin V binding. Early apoptotic cells maintain the integrity of their plasma membrane and are thus impermeable to PI. We conducted experiments to determine the effect of H202 on PS translocation. We treated coelomocytes with H202 in the range of 0 to 67.6 mM and after washing incubated them annexin V- FITC and PI. Figure 3 illustrates a representative set of flow cytometry obtained for earthworm 7 (EW- A7). As the concentration of H202 increased, the degree of PS translocation also increased (right panels). The left hand panels illustrate that H202 affected the morphology of the coelomocytes; as H202 concentration increased, granularity (SSC) decreased (note tightening of SSC signal on ordinate). The middle panels shows that H202 induced cell necrosis/late apoptosis at 33.8 mM and above. In two separate assays performed by two independent researchers on separate days, eight earthworms were analyzed. Tables 1-3 report the overall findings of these experiments. Table 1 shows % annexin V-FITC binding of viable amoebocytes; 100 % (8/8), 63 % (5/8), 38 % (3/8) and 12.5 % (1/8) exhibited statistically significant PS translocation when exposed to 67.6, 33.8, 16.9 and 8.4 mM H202, respectively. Note that untreated amoebocytes exhibit signs of spontaneous apoptosis, perhaps attributed to a stimulus delivered during isolation and/or in vitro culturing. Table 2 illustrates the increase in relative fluorescence intensity (RFI) of annexin V-FITC compared to baseline controls; 88 % (7/8), 38 % (3/8), and 38 % (3/8) revealed statistically significant increases in annexin V-FITC levels on the cell surface of viable amoebocytes following exposure to 67.6, 33.8 and 16.9 mM H202, respectively. There was no significant increase in RFI in any of the earthworms when exposed to 8.4 mM H202. Table 3 reveals that necrosis/late apoptosis correlates with increasing concentration of H202 but only at the highest concentrations used; 100 % (8/8) and 63 % (5/8) of samples had statistically significant increases of necrosis/cell death compared to controls when exposed to 67.6 and 33.8 mM H202, respectively. No significant difference in necrosis/cell death was observed at concentrations of 16.9 and 8.4 mM H202. Caspase activation in coelomocytes To detect the events associated with signal transduction in cells undergoing apoptosis, we measured the activation of caspases in coelomocytes exposed to H202 by using a reporter reagent called FAM-VAD-FMK FLICA where 1) FAM is the carboxyfluorescein group which fluoresces and is detectable in the FL-1 detector of the flow cyometer, 2) VAD is the three amino acid (valine, alanine, aspartic acid) generic probe that binds to most caspases (including caspases -1, -3, -4, -5, -6, -7, -8, and -9) and 3) FMK is the fluoromethyl ketone moiety which anchors the FAM-VAD-FMK reagent   97 Table 3 Percent late apoptotic/necrotic cells in untreated and H202-treated samples. Only cells residing in R1 were gated for PI analysis. R1 included hyaline and granular amoebocytes (not eleocytes). Triplicate data was averaged and subjected to statistical analyses. Results indicate data obtained from two assays performed by two independent researchers. Percent PI-positive cells detected by FL-2 (± SD) are shown for spontaneous apoptosis (0 mM H202) and two-fold serial dilutions from 67.6 to 8.4 mM H202. Statistically significant PI values exceeding spontaneous apoptosis levels are indicated as: * = p ≤ 0.05; ** = p ≤ 0.005; *** = p ≤ 0.0005 as determined by Student’s t test. Earthworm Sample Spontaneous Apoptosis 0 mM 8.4 mM 16.9 mM 33.8 mM 67.6 mM EW-A1 24.55 (±1.19) 18.47 (±2.07) 25.93 (±1.00) 39.12 (±1.57)*** 58.95 (±1.07)*** EW-A2 14.03 (±2.71) 10.79 (±0.39) 11.43 (±0.40) 12.48 (±1.13) 49.93 (±1.80)*** EW-A3 25.03 (±2.09) 18.79 (±0.63) 19.70 (±0.72) 22.85 (±0.20) 44.87 (±0.44)** A ss ay 1 EW-A4 24.85 (±0.14) 19.43 (±1.80) 20.90 (±1.01) 29.75 (±10.7) 45.75 (±2.89)** EW-A5 50.61 (±1.86) 34.53 (±1.39) 39.47 (±2.29) 54.49 (±2.26)* 72.34 (±1.07)*** EW-A6 35.93 (±2.79) 34.84 (±1.62) 35.96 (±2.17) 43.69 (±2.22)* 52.78 (±3.94)** EW-A7 15.48 (±2.05) 11.34 (±1.15) 16.11 (±1.15) 21.15 (±1.47)* 45.75 (±1.71)*** A ss ay 2 EW-A8 20.17 (±3.34) 20.76 (±0.31) 23.69 (±4.15) 29.33 (±1.34)* 57.27 (±1.23)*** to activated caspases in the cell via a covalent cysteine linkage. Unbound FAM-VAD-FMK reagent is washed from the cell while bound reagent is anchored in the cell and fluoresces during flow cytometry, hence enabling the detection of activated caspases in cells undergoing the initial stages of apoptosis. We tested six earthworms in two separate assays. Figure 4 shows the results of these assays where coelomocytes were pretreated with 0 (A), 16.9 (B), 33.8 (C) and 67.6 (D) mM H202. The same strategy for excluding necrotic/late apoptotic cells described in Fig. 3 was used in these experiments, i.e., relevant cells used for analysis were obtained by gating R1 (amoebocytes) and R6 (PI-negative) positive coelomocytes (Fig. 4). A marked increase in necrosis/cell death was observed between 33.8 and 67.6 mM H202 (Fig. 4C- D). Again we observed changes in cellular morphology (as detected by FSC versus SSC profiles) as the concentration of H202 increased, particularly at 67.6 mM (Fig. 4, middle panels). Statistically significant increases in caspase activation were detected in 100 % (3/3) and 67 % (4/6) of samples exposed to 67.6 and 33.8 mM H202, respectively (Fig. 5). Morphological changes in coelomocytes EW F4-F6 were subjected to further analysis to determine if morphological changes associated with a decrease in cell volume, another hallmark of cells undergoing apoptosis, was occurring in response to H202 treatment. Treated and untreated coelomocytes incubated with FAM-VAD-FMK FLICA and PI (as described in caspase assay above) were gated to select for PI-negative (viable and early apoptotic), large coelomocytes. Gated cells were analyzed in a single-parameter histogram measuring forward light scatter (FSC). Duplicate samples were averaged and p values were determined by Student’s t test. Figure 6 shows a decrease in cell volume; in all cases except one (16.9 mM H202 for EWF5), decreases in cell volume of H202-treated samples were statistically significant (p < 0.05) compared to untreated samples. The difference in cell volume was much more pronounced in the large coelomocytes (Fig. 6) than in the small coelomocytes (data not shown). These results show that in earthworms undergoing biochemical changes (caspase activation), morphological alterations (cell volume) are also occurring. DNA fragmentation Another hallmark of apoptosis is the fragmentation of DNA which occurs when an endonuclease (caspase-activated deoxyribonuclease, CAD) is activated as a consequence of programmed cell death induction. Genomic DNA is cleaved at sites between nucleosomes at ~ 200 base pairs (bp) intervals (Nagata, 2000). In this study, total coelomocytes (including amoebocytes and eleocytes) were pretreated with H202 prior to the purification and electrophoresis of DNA. In the first assay (Fig. 7, right hand gel), H202 was used at 0 and 33.8 mM. DNA fragmentation consistent with apoptosis was   98 Fig. 4 Representative flow cytometry profile of caspase (FLICA/PI) assay using EW-F6 as an example for data collection. Coelomocytes were pretreated with 0 (spontaneous apoptosis) (A), 16.9 (B), 33.8 (C), and 67.6 (D) mM H202. Left hand column: FSC (abscissa) versus SSC (ordinate) of total, ungated coelomocyte population. Region 1 (R1) depicts the amoebocyte population (hyaline and granular amoebocytes). Middle column: FSC (abscissa) versus FL-2 (PI) (ordinate) of R1 gated amoebocytes (excluding eleocytes). Region 6 (R6) depicts PI-negative (FL-2 negative), viable amoebocyte population. Right hand column: FL-1 (abscissa) versus cell number (ordinate) of amoebocytes gated on R1 and R6 (i.e. only viable amoebocytes that have not taken up PI). Region 7 (R7) corresponds to fluorescein negative amoebocytes while region 8 (R8) corresponds to fluorescein positive (early apoptotic) amoebocytes. FSC = forward scatter; SSC = side scatter; FL-1 = relative fluorescence intensity of fluorescein, FL-2 = relative fluorescence intensity of PI.   99 Fig. 5 Caspase activation in untreated and H202-treated coelomocytes. Top row: Percent caspase positive amoebocytes of EW-F1-F3 treated with 0 mM (spontaneous apoptosis) or 33.8 mM H202. Bottom row: Percent caspase positive amoebocytes of EW-F4-F6 treated with 0, 16.9, 33.8 and 67.6 mM H202. Asterisks denote p ≤ 0.05 compared to control. observed compared to the untreated control. In a second assay (Fig. 7, two left hand gels) H202 was used at 0, 33.8, 16.9 and 8.4 mM. Laddering was observed only when coelomocytes were exposed to 33.8 and 16.9 mM H202. These results indicate that initiation of the stereotypical internucleosomal degradation of genomic DNA characteristic of apoptosis is occurring in earthworm coelomocytes in response to oxidative stress. TUNEL assay A terminal dUTP nick-end labeling (TUNEL) method was used to enzymatically verify DNA fragmentation in H2O2-treated coelomocytes. This method involved labeling the 3’-hydroxyl DNA ends generated during DNA fragmentation using a terminal deoxynucleotidyl transferase (TdT) and biotin-labeled UTP. Labeled DNA was detected by incubating with FITC-conjugated streptavidin and subjecting the samples to flow cytometric analysis. Amoebocytes identified on forward versus side scatter dot plots were gated for FL-1 (FITC) analysis. Four earthworms were treated with 33.8 mM H2O2, however, only three exhibited any demonstrable difference in labeling compared to untreated controls(EW T1, EW T2 & EW T4); two of these (EW T1 & EW T2) were statistically significant (p < 0.006). Figure 8 shows the results from the three responding earthworms. Note the increase in relative fluorescence intensity compared to untreated controls. A nuclease control was included for verification purposes (data not shown). These results complement the agarose gel electrophoresis results and illustrate that flow cytometry combined with the TUNEL assay is a useful and rapid method to detect DNA fragmentation in earthworm coelomocytes. H202 production during phagocytosis Our final investigation was aimed at determining whether hyaline amoebocytes of E. hortensis undergo a respiratory burst and produce endogenous H202 upon phagocytosis of the soil bacteria P. stutzeri (gram negative) and B. megaterium (gram positive). We used DHR 123, a fluorogenic molecular probe (excitation 505 nm; emission 534 nm) to detect the generation of H202. DHR 123 is oxidized by H202 and is converted consequently to it fluorescent derivative rhodamine 123, which can be measured intracellularly by the flow cytometer. Flow cytometry analysis involved: 1) setting a region around the hyaline amoebocytes (as described in Fig. 1, top row, left hand dot plot); 2) generating a FL-1, single-parameter histogram gated on this region; and 3) establishing a boundary (markers) to separate negative events from positive events (as described in Fig. 4, right hand histograms). The boundary was established based   100 Fig. 6 Cell volume changes in untreated and H202-treated coelomocytes. EW F4-F6 samples from the caspase assay were gated on PI-negative events falling in the region corresponding to the large coelomocyte population. H202 concentration (abscissa) versus geometric mean of forward side scatter (ordinate) are plotted for EW F4 (diamonds), EW F5 (squares) and EW F6 (triangles). With the exception of EW F5 at 16.9 mM, p values < 0.05 compared to controls (untreated) were obtained. on the negative control sample which was incubated in the absence of DHR 123 (but with an equivalent amount of DMSO compared to experimental samples) to control for autofluorescence. Any events exceeding the FL-1 boundary marker were considered positive (percent positive hyaline amoebocytes). Eight earthworms were extruded and three (EW 2, 7 and 8) were selected for the phagocytosis assay based on the quality of the coelomocytes (i.e., higher proportion of amoebocytes compared to eleocytes and adequate cell numbers). Figure 9 shows the results of these three earthworms and illustrates that DHR 123 is oxidized to rhodamine 123 during phagocytosis of P. stutzeri and B. megaterium. Statistical significance was determined based on comparison between control (DHR 123 only) and experimental (bacteria plus DHR 123) samples. P. stutzeri generated statistically significant results in 100 % and 67 % of cases at a m.o.i. of 100:1 and 10:1, respectively. B. megaterium generated statistically significant results in 67 % of cases at a m.o.i. of 100:1 and 10:1. A second assay employing P. stutzeri at a m.o.i. of 1000:1 revealed enhanced H2O2 production in 67% of cases (4 out of 6 earthworms) compared to controls (data not shown). Discussion This study demonstrates that exogenous H202 (0.26 - 8.4 mM) caused an increase in phagocytosis by E. hortensis; 67 % of earthworms tested exhibited statistically significant enhancement of percent specific phagocytosis for one or more of the concentrations of H202 used. Bejarano et al. (2007), Gamaley et al. (1994) and Takeda et al. (1998) examined the effects of oxygen free radicals in cultured human neutrophils, murine macrophages and rat ameboid microglia, respectively, and found that exposure to exogenous H202 also caused an increase in phagocytic function. It is interesting to propose that in our model H202 may be acting as a second messenger involved in evoking a significant elevation of phagocytic function. We are interested in determining if calcium mobilization from agonist- sensitive intracellular stores or influx across the plasma membrane accompanies H202-enhanced phagocytosis, an effect reported by others (Redondo et al., 2004; Bejarano et al., 2007). The use of a calcium quelator (e.g., dimethyl BAPTA) would help to reveal the role, if any, that calcium mobilization plays in phagocytosis in earthworms, and to facilitate a better understanding of the physiological role of oxygen free radicals in calcium homeostasis. We also plan to examine the effect of pre-treating earthworm amoebocytes with catalase, an enzyme facilitating H202 decomposition into water and oxygen, prior to the addition of exogenous H202 in phagocytosis assays. In parallel with the invertebrate studies of Blanco et al. (2005) which examined the effects of H202 on coelomocytes of Themiste petricola (the sipunculan marine worm), we also revealed that H202 induced apoptosis-like cell death in coelomocytes of E. hortensis. Using PI, we were able to discriminate between viable coelomocytes (nonapoptotic and early apoptotic) and nonviable coelomocytes (necrotic/late apoptotic); PI-positive cells were excluded from flow cytometric analyses due to DNA labeling. Note that eleocytes were not included in   101 Fig. 7 DNA fragmentation of H202-treated coelomocytes. These three gels represent two independent DNA fragmentation assays where earthworm coelomocytes were treated with H202 for 6 h and DNA was isolated and electrophoresed in 1.5 % agarose gels in TBE buffer. Lanes M (molecular weight markers in bp are indicated for the nine distinct bands of the 100 bp ladder), Lanes C (untreated cells, 0 mM H202), Lanes 1 (33.8 mM H202), Lane 2 (16.9 mM H202), and Lane 3 (8.4 mM H202). The two left-hand gels are from the same assay and same population of coelomocytes. The right-hand gel is from a separate assay with a different population of coelomocytes. these analyses owing to relatively high level of autofluorescence exhibited by this subpopulation. We observed PS translocation and caspase activation when amoebocytes were treated with H202 in the range of 8.4 - 67.6 mM and 16.9 - 67.6 mM, respectively, under the conditions specified. It is interesting to note that relatively high concentrations of H202 were required to induce apoptosis, suggestive of high TOSC in earthworm coelomocytes. We also observed dose-dependent changes in FSC when amoebocytes were pre- treated with H202, a phenomenon consistent with apoptotic volume decrease (AVD) (Maeno et al., 2000; Okada and Maeno, 2001) when cells undergoing apoptosis experience cell shrinkage and subsequent cell fragmentation (apoptotic bodies). Exposure of PS on the surface of infected coelomocytes may be one mechanism by which phagocytes recognize and remove cellular reservoirs of pathogens during innate immune responses. Fadok et al. (1992), for example, showed that murine macrophages rapidly recognize and remove apoptotic lymphocytes following exposure of PS on the outer leaflet of the plasma membrane of apoptotic cells, thereby prevent potential tissue damage from lysis of these cells in vivo. Caspases are highly conserved and have been identified in invertebrates; for example the CED-3 protein of the nematode Caehorhabditis elegans was the first to be characterized. The gene encoding CED-3 is known to exhibit homology to murine and human caspase-1 (formerly known as interleukin-1 beta converting enzyme) (Yuan et al., 1993). Several caspases known to participate in apoptosis have also been identified in Drosophila melanogaster Fig. 8 TUNEL analysis of H202-treated coelomocytes. Coelomocytes treated with 0 mM H2O2 (filled graph line) (control) or 33.8 mM H2O2 (unfilled graph line) were chemically fixed, and the fragmented DNA ends were labeled with biotin- conjugated UTP and stained with streptavidin FITC. The gated amoebocyte population was analyzed for FL-1 (FITC) fluorescence intensity (abscissa) versus cell number (ordinate). EW T1 and EW T2 exhibited statistical significance (p < 0.006) between untreated and treated amoebocytes.   102 Fig. 9 Endogenous H202 is produced during phagocytosis. The percent hyaline amoebocytes oxidizing DHR 123 to rhodamine 123 for each treatment is shown (percent positive hyaline amebocyes). Phagocytosis assays using P. stutzeri and B. megaterium at m.o.i. of 100:1 and 10: 1 compared to control (DHR 123) shows statistically significant (p ≤ 0.05) increases in rhodamine 123 production (*) for EW 2, 7 and 8. (reviewed in Boyce et al., 2004). In addition, the crystal structure of Sf-caspase-1 from Spodoptera frugiperda has been resolved and its overall fold found to be exceedingly similar to active caspases from humans (Forsyth et al., 2004). Using a fluorescent inhibitor of poly-caspases, our results show that H202 induces caspase activation in earthworm amoebocytes. It would be worthwhile to try to dissect the caspase signaling pathway in E. hortensis using inhibitors known to target caspases with specificity for particular caspase subfamilies (Matsura et al., 1999). We also evaluated oligonucleosomal DNA fragmentation using agarose gel electrophoresis to resolve DNA fragments. A dose-dependent laddering pattern characteristic of apoptotic cells was observed when coelomocytes were treated with 16.9 and 33.8 mM, but not 8.4 mM H202. Perhaps a longer incubation period would have resulted in DNA laddering at the lower concentration. The presence of discrete ~200 bp DNA fragments is consistent with apoptosis and not necrosis as the latter would result in random DNA fragmentation causing smears on an agarose gel rather than discrete repetitive oligonucleosomal fragments generated by endonuclease cleavage between nucleosomes (Walker et al., 1988). In addition, we conducted TUNEL assays in conjunction with flow cytometry which provided enzymatic verification of DNA fragmentation in H2O2-treated coelomocytes, and a more rapid procedure for detecting DNA fragmentation in apoptotic cells. We are also interested in examining mitochondrial transmembrane potential using a mitochondria- specific probe such as JC-1 which incorporates into mitochondria and undergoes a shift in fluorescence emission spectra during membrane depolarization (Cossarizza et al., 1995). Finally, our results show that endogenous H202 is produced by hyaline amoebocytes during phagocytosis of gram positive (B. megaterium) and gram negative (P. stutzeri) bacteria at m.o.i. ranging from 10:1 to 1000:1. Although further investigation is required in vivo, it is tempting to speculate that during infection in the earthworm, the production of H202 facilitates physiological changes in coelomocytes that induce appropriate innate immune responses needed not only to eradicate pathogen load through ingestion (phagocytosis), but also to eliminate infected and damaged cells, and control cell numbers through a clearance mechanism (apoptosis). Engulfment of apoptotic cells would prevent the lytic release of toxic molecules (e.g. enzymes, proteases and oxidizing molecules) and spare or minimize surrounding tissue from inflammatory-type damage, thus enabling immune mechanisms to function effectively. 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