Use of transgenic GFP reporter strains of the nematode Caenorhabditis elegans to investigate the patterns of stress responses induced by pesticides and by organic extracts from agricultural soils

As a free-living nematode, C. elegans is exposed to various pesticides used in agriculture, as well as to persistent organic residues which may contaminate the soil for long periods. Following on from our previous study of metal effects on 24 GFP-reporter strains representing four different stress-response pathways in C. elegans (Anbalagan et al. 2012), we now present parallel data on the responses of these same strains to several commonly used pesticides. Some of these, like dichlorvos, induced multiple stress genes in a concentration-dependent manner. Unusually, endosulfan induced only one gene (cyp-34A9) to very high levels (8-10-fold) even at the lowest test concentration, with a clear plateau at higher doses. Other pesticides, like diuron, did not alter reporter gene expression detectably even at the highest test concentration attainable, while others (such as glyphosate) did so only at very high concentrations. We have also used five responsive GFP reporters to investigate the toxicity of soil pore water from two agricultural sites in south-east Spain, designated P74 (used for cauliflower production, but significantly metal contaminated) and P73 (used for growing lettuce, but with only background levels of metals). Both soil pore water samples induced all five test genes to varying extents, yet artificial mixtures containing all major metals present had essentially no effect on these same transgenes. Soluble organic contaminants present in the pore water were extracted with acetone and dichloromethane, then after evaporation of the solvents, the organic residues were redissolved in ultrapure water to reconstitute the soluble organic components of the original soil pore water. These organic extracts induced transgene expression at similar or higher levels than the original pore water. Addition of the corresponding metal mixtures had either no effect, or reduced transgene expression towards the levels seen with soil pore water only. We conclude that the main toxicants present in these soil pore water samples are organic rather than metallic in nature. Organic extracts from a control standard soil (Lufa 2.2) had negligible effects on expression of these genes, and similarly several pesticides had little effect on the expression of a constitutive myo-3::GFP transgene. Both the P73 and P74 sites have been treated regularly with (undisclosed) pesticides, as permitted under EU regulations, though other (e.g. industrial) organic residues may also be present

Over-expression of superoxide dismutase ameliorates Cr(VI) induced adverse effects via modulating cellular immune system of Drosophila melanogaster.

The evolutionarily conserved innate immune system plays critical role for maintaining the health of an organism. However, a number of environmental chemicals including metals are known to exert adverse effects on immune system. The present study assessed the in vivo effect of a major environmental chemical, Cr(VI), on cellular immune response using Drosophila melanogaster and subsequently the protective role of superoxide dismutase (SOD) based on the comparable performance of the tested anti-oxidant enzymes. The immuno-modulatory potential of Cr(VI) was demonstrated by observing a significant reduction in the total hemocyte count along with impaired phagocytic activity in exposed organism. Concurrently, a significant increase in the percentage of Annexin V-FITC positive cells, activation of DEVDase activity, generation of free radical species along with inhibition of anti-oxidant enzyme activities was observed in the hemocytes of exposed organism. In addition, we have shown that ONOO(-) is primarily responsible for Cr(VI) induced adverse effects on Drosophila hemocytes along with O2(-). While generation of O2(-)/ONOO(-) in Cr(VI) exposed Drosophila hemocytes was found to be responsible for the suppression of Drosophila cellular immune response, Cr(VI) induced alteration was significantly reduced by the over-expression of sod in Drosophila hemocytes. Overall, our results suggest that manipulation of one of the anti-oxidant genes, sod, benefits the organism from Cr(VI) induced alteration in cellular immunity. Further, this study demonstrates the applicability of D. melanogaster to examine the possible effects of environmental chemicals on innate immunity which can be extrapolated to higher organisms due to evolutionary conservation of innate immune system between Drosophila and mammals

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

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

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

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