Figure 1.
Morphological markers of life-stage and fertility in C. elegans.
Detailed descriptions of P. pacificus vulva and gonad development are available [38,39,42]. 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 in utero 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-2 female detail of uterus and vulva. fog-2 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.
Figure 2.
Collection sites for test sediment in the Midwest USA.
(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 C. elegans survivorship performance. The letter A designates the position of Chicago, IL, USA.
Figure 3.
Recovery of P0 adult nematodes and F1 progeny from eight un-spiked test sediments.
C. elegans – blue. P. pacificus – red. Box and whisker plots: box represents the range between the 25th and 75th 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, C. elegans groupings; capital red letters, P. pacificus 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.
Figure 4.
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).
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, C. elegans; red diamonds, P. pacificus. (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.
Figure 5.
Recovery of P0 adult nematodes and F1 progeny from nickel-spiked water.
C. elegans – blue. P. pacificus – red. (A) P0 recovery. (B) F1 recovery. (C) Fecundity ratio.
Figure 6.
Adult lifespan in nickel-spiked sediment – survivorship curves.
Recovery of Cel-fog-2 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.