Recovery of P0 adult nematodes and F1 progeny from eight un-spiked test sediments.

<p><i>C. elegans</i> – blue. <i>P. pacificus</i> – red. Box and whisker plots: box represents the range between the 25^th and 75^th percentile (interquartile range). The line within the box represents the median. The whiskers indicate minimum and maximum values, except where circles and stars represent outliers (>1.5 times interquartile range from median) or extreme outliers (>3 times interquartile range from median), respectively. Letters above the box and whisker plots represent significant groupings based upon Tukey post-hoc comparison tests (p< 0.05). Lower case blue letters, <i>C. elegans</i> groupings; capital red letters, <i>P. pacificus</i> groupings. (A) Recovery of added P0 animals. (B) Recovery of L1/J2 progeny. (C) Fecundity Index, total progeny recovered / total live P0 adult nematodes recovered.</p

Adult lifespan in nickel-spiked sediment – survivorship curves.

<p>Recovery of <i>Cel-fog-2</i> females from WB spiked nickel sediments, WB-0 (blue circles), WB-2 (green triangle), WB-3 (black asterisks), and WB-5 (red squares). Survivability decreases as nickel increases. Yellow-orange dotted line represents a 50% recovery. WB-0 and WB-2 show a 50% reduction around day 16. WB-3 shows a 50% reduction around day 7. WB-5 showed a 50% reduction around day 3.</p

Collection sites for test sediment in the Midwest USA.

<p>(1) SR – Spring River, Jasper County, Missouri(2). STJ – St. Joseph River, Michigan(3). P30 – USGS Pond 30 Missouri(4). DOW – Dow Creek Michigan(5). RR2 – Raisin River Site 2, Michigan(6). STM – South Tributary of Mill Creek, Michigan(7). RR3-0 Raisin River Site 3, Michigan(8). WB – West Bearskin Lake Minnesota. Sites are given in order presented in Tables 1-3 and based upon <i>C. elegans</i> survivorship performance. The letter A designates the position of Chicago, IL, USA.</p

Recovery of P0 adult nematodes and F1 progeny from nickel-spiked sediments (i.e. WB-0 through WB-5 and SR-0 through SR-5).

<p>Box-and-whisker plots are formatted and labeled as in Figure 3. Scatterplots: P0 recovery for individual wells plotted against sediment nickel concentration. Blue circles, <i>C. elegans</i>; red diamonds, <i>P. pacificus</i>. (A-D, I) WB Ni(II)-spiked sediment series. (A) P0 recovery and sediment treatment. (B) P0 recovery and sediment nickel concentration. (C) F1 recovery and sediment treatment. (D) F1 recovery and sediment nickel concentration. (E-H, J) SR Ni(II)-spiked sediment series. (E) P0 recovery and sediment treatment. (F) P0 recovery and sediment nickel concentration. (G) F1 recovery and sediment treatment. (H) F1 recovery and sediment nickel concentration. (I and J) Fecundity ratio and sediment treatment.</p

Recovery of P0 adult nematodes and F1 progeny from nickel-spiked water.

<p><i>C. elegans</i> – blue. <i>P. pacificus</i> – red. (A) P0 recovery. (B) F1 recovery. (C) Fecundity ratio.</p

Morphological markers of life-stage and fertility in <i>C. elegans</i>.

<p>Detailed descriptions of <i>P. pacificus</i> vulva and gonad development are available [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077079#B38" target="_blank">38</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077079#B39" target="_blank">39</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077079#B42" target="_blank">42</a>]. Panels include a DIC micrograph on top and a cartoon of the micrograph below. For body orientation, anterior is to the left and dorsal to the top. Black arrows mark the position of the vulva, the egg- laying organ. Asterisks in the cartoons denote developing embryos <i>in </i><i>utero</i> and are not shown in the micrograph for clarity. White triangles denote the position of oocytes. Tissues are denoted by color: gut, blue; uterus and spermatheca, purple; germ line (including the germ cells, oocytes, and developing embryos), red. Ovals and circles depict easily seen nuclei within tissues. (A) Young adult hermaphrodite. The entire posterior gonad arm is shown. The red arrow outlines the path of gonad arm extension starting proximal to the vulva and terminating with the arrowhead at the distal tip. As an adult, this animal has made sperm, oocytes, and contains embryos, has two fully reflexed and inflated gonadal arms, and a fully everted vulva with a slit like morphology. The black asterisk denotes an egg in the spermatheca that has just been fertilized but has not developed an eggshell yet. In contrast, L4 larvae never contain embryos as they only begin to produce gametes late in the L4-stage. The L4 gonad arms are smaller and not as inflated, and early in L4 have only reached the dorsal side of the animal, but do not reach the center above the vulva until late in L4. (B) Wild type early L4-larva hermaphrodite detail of uterus and vulva. The vulva has a characteristic “Christmas tree” like shape, it is not everted. The uterus is empty and un-inflated. Gametes have not been produced, the gonad arms are skinny and contain relatively few germ cells making the gonad arms difficult to capture in the same focal plane as the vulva. (C) Wild type adult hermaphrodite detail of uterus and vulva. The uterus is full of multicellular embryos. The gold asterisk denotes an embryo with a clearly visible eggshell. The eggshell is present as an oval around the ball of cells. The embryo and shell are separated by a slim cleared liquid-filled space. (D) Adult fog-<i>2</i> female detail of uterus and vulva. <i>fog-2</i> females do not produce sperm and contain no embryos. Hence the uterus and spermatheca remain unexpanded. Unfertilized oocytes stack up in the gonad arms and become compressed, giving a “piano key” phenotype. Eventually pressure may push an oocyte into the uterus and the oocyte will be laid, but without an eggshell. Laid oocytes have a “mushy” appearance and remain single celled until decomposition. The edge of a laid embryo has a refractory appearance due to the eggshell.</p

Dual Electrochemical and Physiological Apoptosis Assay Detection of in Vivo Generated Nickel Chloride Induced DNA Damage in Caenorhabditis elegans

Environmental nickel exposure is known to cause allergic reactions, respiratory illness, and may be responsible for some forms of cancer in humans. Nematodes are an excellent model organism to test for environmental toxins, as they are prevalent in many different environments. Nickel exposure has previously been shown to impact nematode life processes. In this study, Caenorhabditis elegans nematodes exposed to NiCl₂ featured high levels of programmed cell death (PCD) in a concentration-dependent manner as measured by counting apoptotic corpses in the nematode germ line. A green fluorescent protein (GFP) reporter transgene was used that highlights cell corpse engulfment by fluorescence microscopy. Analysis of the reporter in a <i>p53</i> mutant strain putatively indicates that the PCDs are a result of genomic DNA damage. In order to assay the potential genotoxic actions of NiCl₂, DNA was extracted from nematodes exposed to increasing concentrations of NiCl₂ and electrochemically assayed. In vivo damaged DNA was immobilized on pyrolytic graphite electrodes using the layer-by-layer (LbL) technique. Square-wave voltammograms were obtained in the presence of redox mediator, ruthenium trisbipyridine (Ru­(bpy)₃²⁺), that catalytically oxidizes guanines in DNA. Oxidative peak currents were shown to increase as a function of NiCl₂ exposure, which further suggests that the extracted DNA from nematodes exposed to the nickel was damaged. This report demonstrates that our electrochemical biosensor can detect damage at lower Ni concentrations than our physiological PCD assay and that the results are predictive of physiological responses at higher concentrations. Thus, a biological model for toxicity and animal disease can be assayed using an electrochemical approach