Temperature and copper effects on the nematode Caenorhabditis elegans

Experimental design

Culture conditions for test organism: C. elegans (N2 Bristol strain) from the C. elegans Genetics Centre, University of Minnesota) were cultivated in darkness at 20°C on nematode growth medium (NGM) agar plates and fed Escherichia coli of uracil deficient strain OP50. At least 2 weeks before the start of all experiments new cultures were initiated at the selected test temperatures to acclimatize the worms through several generations. Cultures were maintained by transferring a chunk of agar and associated eggs, juvenile and adult worms from an existing culture to freshly prepared NGM agar plates weekly.

Set-up of toxicity tests: Exposures were conducted at steady temperatures of 8, 12, 16, 20 and 24°C and variable temperature scenarios with daily fluctuations of +/- 4°C in the ranges 8-16°C (12°C average), 12-20°C (16°C average) and 16-24°C (20°C average) and daily fluctuations of +/- 8°C in the ranges 8-24°C (16°C average). Temperatures were changed with a rate of 4°C per hour. Separate toxicity tests with copper were conducted under all temperature regimes using concentration known to result in changes in measured traits. Copper concentrations used were 0, 1, 3, 8, 20 and 40 mg Cu/L agar. The highest concentrations equate to levels 2-3 orders of magnitude greater than those found in surface waters and soil pore waters from uncontaminated sites, but are comparable to soil pore water concentrations in heavily polluted soils.

A stock solution of CuCl2 in demineralized water (2 g Cu/L) was made and used throughout the experiment. This copper stock solution was added to the NGM agar while liquid and mixed. Batches of NGM were produced every five days during the course of the experiment to minimize immobilization of copper in the agar. All tests were conducted using synchronized cohorts of nematodes produced from adults selected from the main stock cultures acclimatized at each temperature scenario. To generate these cohorts, adult hermaphrodite worms were selected from the acclimatized cultures and placed on Petri dishes with copper spiked NGM and E. coli and left at the test temperatures for 4 hours (6 hours for 8°C to ensure sufficient eggs were laid). After this time, the adults were removed and the eggs laid in the 4 (6) hour period left to hatch. The offspring (the test organisms), once hatched and grown to the L4 larval stage on the copper spiked plates, were transferred to 12-well plates with copper spiked NGM and E. coli, with one individual per well. There were 12 replicate worms for each treatment, except the controls of 24°C constant, 8-16°C, 8-24°C and 16-24°C which had 36 replicates, and the 8°C test in which we could not always obtain 12 worms for each treatment, hence, between 6 and 12 worms were used.

During the test, the nematodes were moved to new wells in fresh plates every day. These transfers and the associated observations allowed calculation of the time taken until first egg was laid and subsequently daily egg production. The daily transfer and counting were repeated through both the reproductive and senescence stages until death (determined by the lack of response to being probed). Fertile eggs and hatched juveniles were counted as offspring, while visibly infertile eggs were excluded from brood size counts. At regular intervals, nematode length in micrometers was measured using a Nikon DS-Fi1 camera connected to a Nikon SMZ 800 stereomicroscope. Body length was measured using the program Nikon NIS Elements Imaging Software 3.2 to provide a description of growth pattern.

Copper analysis: The copper content in the water fraction of the agar was determined one and five days after production. This allowed us to distinguish dissolved copper and agar bound copper (which may not have been fully bioavailable). Samples of NGM from each produced batch were saved in centrifuge tubes and, after one or five days, centrifuged at 10000 G for five minutes to separate water and agar. A sample of the water fraction was removed and acidified. The analyses were performed on a graphite furnace AAS (Perkin Elmer Zeeman 5100, Waltham, MA) . Blanks, spiked reference samples and a standard reference material NIST 1577c (bovine liver) were included in the analysis to confirm validity.

Data structure: Data structure for columns.

Plate: Gives the TC-plate number. Treatments were randomised at different plates.

Replicate: Gives the replicate number

Cu: Gives the Cu-concentrations in mg/L agar

Day: Gives the day after the eggs are layed.

Length: Is given in µm (micrometers). Length measurements on individuals were started when the worms reached the L4 larval stage and were moved to individual wells.

Offspring: Is the number of fertile eggs and larvae counted. If there are no eggs a zero is written. When the nematode is dead, nothing is written in the cell henceforth. All times after the last zero, the worm is dead. The experiment is terminated when the last worm died, indicated by end of the orange area.

Statistics: Differences between temperature treatments for brood size, lifespan and maximal length of non-Cu exposed nematodes were tested by a one-way ANOVA with a Tukey post hoc analysis using the R statistical software version 2.12.0 (http://www.r-project.org/).

The average cumulative egg production as a function of time for each treatment was described by a three parameter log-logistic sigmoid model (equation 1) using the statistical software R version 2.12.0 within the DRC package..

y = d/(1+(x/e)^b) (Equation 1)

where d is the maximum number of eggs produced, e is the time when half the maximum number of eggs is produced, b is proportional to the slope of the sigmoid curve at e, y is the accumulated number of produced eggs and x is the time. From this model the estimated time to first egg (TFE) was calculated by solving x for y equal to 1/d. The DRC package returns a standard error with the estimate.