Reduction of total hemocyte count (THC) in Cr(VI) exposed <i>D. melanogaster</i> larvae.

<p>Graphical representation of total hemocyte number (%) in Cr(VI) exposed Oregon R⁺ larvae after immunostaining by Hemese (H2) antibody (A). Hemocyte population in <i>hml^Δ-Gal4 UAS-2xEGFP</i> larvae after Cr(VI) exposure (B). Graph representing total hemocyte count in Cr(VI) exposed <i>hml^Δ-Gal4 UAS-2xEGFP</i> larvae as determined by flow cytometry (C). Data represent mean ± SD (n = 3) (20 larvae in each replicate). Significant differences were ascribed as *<i>p</i><0.05; **<i>p</i><0.01 and ***<i>p</i><0.001 as compared to control. Representative confocal microscopic images of the hemocytes in control, 20.0 µg/ml Cr(VI) and 20.0 µg/ml Mo(VI) exposed Oregon R⁺ larvae for 48 h (D). Extreme right panel represents overlayed images of H2 (green) and DAPI (blue) stained cells. Scale bar: 20 µm.</p

Protective effect of <i>sod</i> over-expression on <i>Drosophila</i> cellular immune response after Cr(VI) exposure.

<p>Comparative hemocyte population in <i>He-Gal4</i>, <i>UAS-Sod</i>, <i>He-Gal4>UAS-Sod</i>, <i>UAS-Sod RNAi</i> and <i>He-Gal4>UAS-Sod RNAi</i> larvae after 48 h Cr(VI) exposure (A1). Representative microscopic images of hemocytes from control and in 20.0 µg/ml Cr(VI) exposed larvae for 48 h (A2). Phagocytic activity (%) of hemocytes of <i>He-Gal4</i>, <i>UAS-Sod</i>, <i>He-Gal4>UAS-Sod</i>, <i>UAS-Sod RNAi</i> and <i>He-Gal4>UAS-Sod RNAi</i> larvae after 48 h Cr(VI) exposure (B). Percent AV positive hemocytes in 48 h Cr(VI) exposed <i>He-Gal4</i>, <i>UAS-Sod</i>, <i>He-Gal4>UAS-Sod</i>, <i>UAS-Sod RNAi</i> and <i>He-Gal4>UAS-Sod RNAi</i> larvae (C1). Representative dot plots for Annexin V-FITC and PI staining in the hemocytes from <i>He-Gal4</i> control (a) and 20.0 µg/ml Cr(VI) exposed <i>He-Gal4</i> (b), <i>UAS-Sod</i> (c), <i>He-Gal4>UAS-Sod</i> (d), <i>UAS-Sod RNAi</i> (e) and <i>He-Gal4>UAS-Sod RNAi</i> (f) larvae for 48 h (C2). Comparative DEVDase activity in the hemocytes of <i>He-Gal4</i>, <i>UAS-Sod</i>, <i>He-Gal4>UAS-Sod</i>, <i>UAS-Sod RNAi</i> and <i>He-Gal4>UAS-Sod RNAi</i> larvae exposed to Cr(VI) for 48 h (D1). Representative confocal images of hemocytes from control and 48 h, 20.0 µg/ml Cr(VI) exposed larvae (D2). Comparative levels of O₂^.− (E1), H₂O₂ (E2) and ONOO⁻ (E3) generation in the hemocytes of 48 h Cr(VI) exposed <i>He-Gal4</i>, <i>UAS-Sod</i>, <i>He-Gal4>UAS-Sod</i>, <i>UAS-Sod RNAi</i> and <i>He-Gal4>UAS-Sod RNAi</i> larvae. Graphical representation of SOD activity (F1), CAT activity (F2), MDA content (F3), TrxR activitity (F4) and TAC (F5) in the hemocytes of <i>He-Gal4</i>, <i>UAS-Sod</i>, <i>He-Gal4>UAS-Sod</i>, <i>UAS-Sod RNAi</i> and <i>He-Gal4>UAS-Sod RNAi</i> larvae that were exposed to Cr(VI) for 48 h. Representation of the survival (%) of 20.0 µg/ml Cr(VI) exposed <i>Drosophila</i> larvae (<i>He-Gal4</i>, <i>UAS-Sod</i>, <i>He-Gal4>UAS-Sod</i>, <i>UAS-Sod RNAi</i> and <i>He-Gal4>UAS-Sod RNAi</i>) for 48 h following <i>Ecc</i>15 infection (G). Data represent mean ± SD (n = 3). Statistical significance was ascribed as *<i>p</i><0.05; **<i>p</i><0.01 and ***<i>p</i><0.001 as compared to control and <b>^$</b><i>p</i><0.05; <b>^$$</b><i>p</i><0.01 and <b>^$$$</b><i>p</i><0.001 in comparison to respective <i>He-Gal4</i>. Scale bar: 20 µm.</p

DEVDase (caspase 3-like) activity in the hemocytes of <i>Drosophila</i> larvae exposed to Cr(VI).

<p>Graphical representation of DEVDase activity (%) in the hemocytes of Oregon R⁺ larvae after their exposure to Cr(VI) (A). Data represent mean ± SD (n = 3) (50 larvae in each replicate). Significance in comparison to control was ascribed as *<i>p</i><0.05; **<i>p</i><0.01 and ***<i>p</i><0.001. Representative confocal images of hemocytes from control and 20.0 µg/ml Cr(VI) exposed Oregon R⁺ larvae for 48 h (B). Extreme right panel represents overlayed images of H2 (green), DAPI (blue) and cleaved caspase-3 (red) antibody stained cells. Scale bar: 20 µm.</p

Effect of NAC, L-NAME, SNP on THC and apoptosis in <i>Drosophila</i> hemocytes after Cr(VI) exposure.

<p>Graphical representation of total hemocyte number (%) in 20.0 µg/ml of Cr(VI) exposed Oregon R⁺ larvae along with 10 mg/ml N-acetylcysteine (NAC) or 100 mM N-nitro-L-arginine methyl ester (L-NAME) or 50 µM sodium nitroprusside (SNP) after 24 and 48 h (A1). Representative confocal images of hemocytes from control, 20.0 µg/ml Cr(VI), 20.0 µg/ml Cr(VI) with 10 mg/ml NAC, 20.0 µg/ml Cr(VI) with 100 mM L-NAME and 20.0 µg/ml Cr(VI) with 50 µM SNP exposed Oregon R⁺ larvae after 48 h (A2). Scale bar: 20 µm. Twenty larvae were taken for each replicate. Graphical representation of percent AV positive hemocytes in <i>Drosophila</i> larvae exposed to 20.0 µg/ml Cr(VI) along with 10 mg/ml NAC or 100 mM L-NAME or 50 µM SNP for 24 and 48 h respectively (B1). Dot plots showing Annexin V-FITC and PI staining in the hemocytes of control (a), 20.0 µg/ml Cr(VI) (b), 20.0 µg/ml Cr(VI) with 10 mg/ml NAC (c), 20.0 µg/ml Cr(VI) with 100 mM L-NAME (d) and 20.0 µg/ml Cr(VI) with 50 µM SNP (e) exposed Oregon R⁺ larvae after 48 h (B2). Fifty larvae were taken for each replicate. Data represent mean ± SD (n = 3). Significant differences were ascribed as **<i>p</i><0.01; ***<i>p</i><0.001 in comparison to control and <b>^#</b><i>p</i><0.05; <b>^##</b><i>p</i><0.01 and <b>^###</b><i>p</i><0.001 as compared to 20.0 µg/ml Cr(VI) exposure.</p

Increased apoptosis in the hemocyte population of Cr(VI) exposed Oregon R⁺ larvae.

<p>Quantitative graph of percent AV positive hemocytes in Cr(VI) exposed <i>Drosophila</i> larvae (A). Bar graphs represent mean ± SD (n = 3) (50 larvae in each replicate). Significance was ***<i>p</i><0.001 in comparison to control. Representative dot plots for Annexin V-FITC and PI staining in the hemocytes of control (a) and 20.0 µg/ml Cr(VI) exposed (b) larvae for 48 h (B).</p

Poor resistance of Cr(VI) exposed Oregon R⁺ larvae against <i>Ecc</i>15 infection.

<p>Survival (%) of <i>Drosophila</i> larvae that were exposed to Cr(VI) for 24 (A) and 48 (B) h followed by <i>Ecc</i>15 infection indicating resistance of an organism. Each survival curve in the graph represents mean survival of larvae from three independent experiments having 100 larvae in each and statistical significance was ascribed as **<i>p</i><0.01 and ***<i>p</i><0.001 as compared to control.</p

Schematic representation of Cr(VI)-induced alterations on cellular immunity in <i>D. melanogaster</i>.

<p>Cr(VI) altered cellular innate immune response through O₂^.−/ONOO<b>⁻</b> mediated oxidative stress in the hemocytes of exposed organism. The induction of oxidative stress leads to caspase-3 activation vis a vis apoptosis in the hemocytes which results into reduction in total hemocyte population in exposed organism. The altered immunity was manifested by decreased resistance of these organisms against pathogenic <i>Ecc</i>15 infection. Cr(VI) induced suppression of cellular immunity was subsequently modulated/rescued by over-expressing <i>sod</i> in <i>Drosophila</i> hemocytes.</p

Generation of free radicals in the hemocytes of <i>Drosophila</i> larvae exposed to Cr(VI).

<p>Graphs showing O₂⁻ generation (A), H₂O₂ generation (B) and ONOO⁻ generation (C) in the hemocytes from control and Cr(VI) exposed Oregon R⁺ larvae. Values are mean ± SD (n = 3) (50 larvae in each replicate). Statistically significant differences were ascribed as *<i>p</i><0.05; **<i>p</i><0.01 and ***<i>p</i><0.001 in comparison to control.</p

Inhibited phagocytic activity of the hemocytes of Cr(VI) exposed <i>Drosophila</i> larvae.

<p>Graph showing inhibition of phagocytic activity (%) in the hemocytes of Cr(VI) exposed Oregon R⁺ larvae. Bar graphs represent mean ± SD (n = 3) (10 larvae in each replicate). Statistical significance was *<i>p</i><0.05; **<i>p</i><0.01 and ***<i>p</i><0.001 as compared to control.</p