Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

1. Culture E. coli OP50

1. Prepare 1 M sodium hydroxide solution by dissolving 4 g of sodium hydroxide in 100 mL water.
2. Prepare lysogeny broth (LB) medium by dissolving 10 g of tryptone, 5 g of yeast extract and 10 g of sodium chloride with 1 L of ultrapure water in a 1 L conical flask. Adjust the pH to 7.0 with 1 M sodium hydroxide solution.
3. Aliquot the LB liquid medium from step1.2 into 20 conical flasks (maximum allowable volume: 100 mL) with 50 mL medium in each. Cover the conical flasks with kraft paper.
4. Sterilize the LB liquid medium at 121 °C and 0.105 MPa for 20 min. Cool the LB medium down to room temperature.
5. Pipette 200 µL from the bacterial suspensions (see step 1.8) or pick a small colony from agars (in step 2.14) using an inoculating loop, place it in LB medium.
6. Incubate the LB medium with shaking at 150 rpm at 37 °C for 24-48 h. The LB medium will change from a brown transparent liquid to turbid khaki-colored suspension.
7. Use the bacterial suspensions from 80% of total flasks to provide E. coli OP50 as nematode food to be used in step 2.11.
8. Store the rest of the flasks containing the bacterial suspensions in a refrigerator at 4 °C. Pipette 200 µL of the LB suspensions on to the upper side of fresh LB medium (step 1.4) and repeat step1.6 for subsequent incubation.

2. Culture C. elegans

NOTE: Culture C. elegans using as per steps 2.1 to 2.11 based upon standard methods¹⁸.

1. Dissolve 22.8 g of K₂HPO₄•3H₂O in 100 mL of sterile distilled water.
2. Dissolve 6.8 g of KH₂PO₄ in 50 mL of sterile distilled water.
3. Mix solutions from steps 2.1 and 2.2, to prepare 1 M K₂HPO₄-KH₂PO₄ buffer (pH 6.0, 150 mL in total).
4. Prepare 1.0 M MgSO₄ by dissolving 1.232 g of MgSO₄•7H₂O in 5 mL of sterile distilled water, and sterilize it by filtering the solution through a 0.22 µm sterile disposable membrane filter into a sterile container.
5. Prepare 1 M CaCl₂ by dissolving 0.554 g of CaCl₂ in 5 mL of sterile distilled water, and sterilize it by filtering the solutions through a 0.22 µm sterile disposable membrane filter into a sterile container.
6. Prepare 1 M cholesterol solution by dissolving 0.025 g of cholesterol in 5 mL of absolute ethanol, and sterilize it by filtering the solution through a 0.22 µm sterile disposable membrane filter into a sterile container.
7. Prepare the nematode growth medium (NGM) agar by adding 17 g of agar powder, 2.5 g of peptone and 3 g of sodium chloride to a 1 L conical flask containing 1 L of ultrapure water. Add 25 mL of K₂HPO₄-KH₂PO₄ solution from step 2.3.
8. Sterilize the NGM agar from step 2.7 at 121 °C with 0.105 MPa for 20 min.
9. Cool the NGM agar to about 50 °C, add 1 mL of 1 M MgSO₄, 1 M CaCl₂, and 5 mg/mL cholesterol-ethanol solution from steps 2.4 to 2.6 into the medium and mix them thoroughly.
10. Pour ~10 mL NGM agar medium per dish into 100 sterile Petri dishes (6.0 cm diameter). Cool the medium in the Petri dishes to room temperature to form solid agar.
11. Pipette 170 µL of the bacterial suspensions from step 1.7 onto the aforementioned NGM agar using a sterile tip. Shake the NGM agar slightly to distribute the LB medium evenly over the NGM agar surface.
12. Inoculate the NGM agar with the top-side up at 37 °C for 8-12 h to form a bacterial lawn.
13. Use most of the agar with bacterial lawns from step 2.12 to culture nematodes in step 2.15 or 2.19.
14. Store 1 or 2 agars up-side down to avoid water evaporation and contamination in refrigerators at 4 °C so as to sustain the bacteria in of contamination.
15. When there are less than 2,000 nematodes on the stocking NGM agars (from earlier experiments or gifted from other labs), cut one sixth of the NGM agar that contains C. elegans with a sterile tip and transfer it onto newly prepared NGM agars with bacterial lawn (from step 2.13). Keep the NGM agars up-side down in a 22 °C incubator for the subsequent culture.
16. When there are more than 2,000 nematodes on the stocking NGM agar, flush the nematodes of the NGM agar with 2 mL of sterile water into centrifuge tubes. The input of 2 mL of water will result in an approximately 1.5 mL output.
17. Allow the nematodes to settle in the centrifuge tubes from for 30 min. Discard 1 mL of the supernatants by pipetting and add 1 mL of sterile water into each tube to wash the pellets (i.e., the nematodes).
18. Settle down the nematodes in the centrifuge tubes for 30 min. Discard 1 mL of the supernatants by pipetting. Add 1 mL of sterile water into each tube to resuspend the nematodes.
19. Distribute the nematode suspensions (approximately 1.5 mL in total) by pipetting 150 µL onto each new NGM agar with bacterial lawn (from step 2.13), making 8-10 new NGM agars in total.
20. Keep the NGM agars from step 2.19 up-side up in a 20 °C incubator overnight and then up-side down for the subsequent culture.
21. Repeat step 2.15 or step 2.16-2.20 every three days.

3. Prepare synchronized eggs and L3 larvae of C. elegans

1. Flush off the gravid nematodes and newly produced eggs from the NGM agars into sterile centrifuge tubes, with 2 mL of sterile water on each NGM agar resulting in approximately 1.5 mL of output.
2. Settle down the nematodes in the centrifuge tubes for 30 min, and then discard 85% of the supernatants by pipetting.
3. Prepare sodium hypochlorite solutions by dissolving 0.6 g of NaOH and 5 mL of NaOCl (4-6% active gradients, see the Table of Materials for details) with 25 mL of water to bring the NaOH to 0.5 M and NaOCl to 1%.
4. Mix the pellets from step 3.2 (mark the volume as V₀) with 7-fold V₀ of sodium hypochlorite solutions from (step 3.3, i.e., volume ratio of 1:7)¹⁹.
5. Shake the centrifuge tubes every 2 min for 10-15 min to lyse the larvae and adult nematodes; the color of nematode suspensions will turn from turbid to clear.
6. Centrifuge the tubes at 700 x g for 3 min at 20 °C, and then discard the supernatants by pipetting.
7. Resuspend the pellets in 5-fold V₀ of sterile water to wash the age-synchronized eggs. Centrifuge at 700 x g for 3 min at 20 °C, and then discard the supernatants.
8. Repeat step 3.7 twice.
9. Add 1-fold V₀ of sterile water into the tubes to resuspend the age-synchronized eggs.
10. Distribute the eggs suspensions by pipetting 50 µL onto each new NGM agars with bacterial lawn from step 2.13. Keep the NGM agars top-side up in a 20 °C incubator for 30 min, allowing the water to evaporate or be adsorbed by the bacterial lawn. Then, make the NGM agars up-side down for the subsequent culture. Mark the time as the egg time (T_egg).
11. Prepare K-medium by dissolving 3 g of NaCl and 2.36 g of KCl in 1 L of water. Sterilize the medium at 121 °C and 0.105 MPa for 20 min and cool it down to room temperature.
12. When the time reaches 36 h after T_egg, the nematodes will reach the L3 larvae stage (L3 nematodes)²⁰. Flush the nematodes off the NGM agars into centrifuge tubes, with 2 mL of sterile water on each NGM agar resulting in approximately 1.5 mL of output.
13. After a settling for 30 min, replace 85% of the supernatants (by pipetting) with K-medium from 2 h to digest the food in the guts³.
14. Discard the supernatants. Use K-medium (from step 3.11) to adjust the nematode suspensions to about 200 nematodes per 100 µL for subsequent experiments.

4. Use C. elegans for trans-generational effect study

1. Prepare chemical solutions with 5 concentration levels and one solvent or absolute control, i.e., 6 groups in total.
2. Add 100 µL of control or chemical solutions with 10 wells as replicates in each group (i.e., 60 wells in total) into the middle area of a 96-well sterile microplate to avoid edge effects.
3. Dilute the nematode suspensions from step 3.14 with K-medium from step 3.11 by 10-fold. Add 100 µL nematode suspensions to each of the 60 wells from step 4.2. Mark the time as t₀.
4. When the time reaches 24 h after t₀, count the living and dead nematodes in the wells. Calculate the median lethal concentration (LC₅₀).
5. Prepare a series of 5 concentrations below 10% of the LC₅₀ values.
6. Add 100 µL of control or chemical solutions (from step 4.5) with 10 wells as replicates in each group (i.e., 60 wells in total) into the middle area of a 96-well sterile microplate to avoid edge effects.
7. Add 100 µL of K-medium containing approximately 200 L3 nematodes (from step 3.14) to each of the 60 wells from step 4.6. Mark the time as T₀.
8. Perform the exposure for 24 to 96 h since T₀.
9. After the exposure, collect nematodes from five wells in each group into 1.5 mL centrifuge tubes by pipetting (i.e., 6 tubes in total).
10. Settle the nematodes for 30 min, discard the supernatants by pipetting, and resuspend and wash nematodes at the bottom with 1 mL of sterilized water.
11. Settle the nematodes for 30 min, discard the supernatants by pipetting and use the nematodes at the bottom for the indicator measurements (see section 7) of the exposed parent generation marked as F0.
12. Collect, settle and wash (by resuspending) the nematodes from the remaining five wells in each group in step 4.9 according to step 4.10.
13. Settle the nematodes from step 4.12 for 30 min. Discard the supernatants by pipetting and add 100 µL of sterile water to resuspend the nematodes. Transfer the nematode suspensions evenly onto three newly prepared NGM agars with bacterial lawn from step 2.13.
14. Incubate the nematodes for 36 h to become gravid and perform age-synchronization according to steps 3.1-3.9. Incubate the synchronized eggs on respective NGM agars with bacterial lawn from step 2.13 for 36 h.
15. Flush the nematodes on the NGM agars from step 4.14 into six centrifuge tubes.
16. Settle the nematodes for 30 min, and discard the supernatants by pipetting. Resuspend and wash the pellets with 1 mL of sterilized water.
17. Settle the nematodes for 30 min, and discard the supernatants by pipetting. Use the nematodes at the bottom for the indicator measurements (see section 7) of the offspring generation marked as T1.

5. Use C. elegans for multi-generational exposure (MGE) effect study

1. Mix 99.0 mL NGM agars (from step 2.9) with 1 mL of control or chemical solutions (low and high concentrations in the present protocol as examples, i.e., three groups in total).
2. Pour the approximately 10 mL of NGM agar medium per dish from step 5.1 into 100 sterile Petri dishes (6.0 cm diameter). Cool the medium in the Petri dishes to room temperature to form solid agar.
3. Pipette bacterial suspensions on to the agar as per step 2.11.
4. Set aside the upper lids of the Petri dishes and expose the bacterial lawn to UV light (145 µW/cm²) in the biosafety cabinet for 15 min.
5. Pick a small colony using an inoculating loop, place it in LB medium from agars in step 1.4. Incubate the LB medium with shaking at a speed of 150 rpm at 37 °C for 24 h to confirm the negligle bacterial growth, validating the killing step 5.4.
6. Pipette the age-synchronized eggs from step 3.9 onto the agars (from step 5.4). Mark the start of the exposure to the parent generation F0 and mark the as Day 0 (D0).
7. Incubate the agars for 3 d at 20 °C. Then (i.e., on D3), use the mature nematodes to measure effects in F0 (see section 7).
8. Also on D3, pick F0 mature nematodes onto new NGM agars (from step 5.4) using a glass rod, whose end is fitted with a man-made fiber wire bent into a ring.
9. On D4, pick out and discard the mature F0 nematodes from NGM agars. Mark the newly hatched offspring nematodes within these 24 h (from D3 to D4) as F1 to experience second-generation exposure.
10. On D6, measure indices (see section 7) of the F1 mature nematodes that have experienced exposure for 3 days.
11. On D9, repeat steps 5.8-5.10, use F1 nematodes to reproduce F2 worms and measure effects on F2 nematodes.
12. On D12, repeat step 5.11, use F2 nematodes to reproduce F3 worms and measure effects on F3 nematodes. Through the same way, reproduce offspring and measure MGE effects on the nth offspring generation (Fn).

6. Use C. elegans for multi-generational residual (MGR) effect study

1. Repeat steps 5.1-5.7. On D3, pick F0 mature nematodes onto new NGM agars without added chemicals (from step 2.13).
2. On D4, pick out and discard the mature F0 nematodes. Mark the newly hatched offspring nematodes within these 24 h as T1 nematodes.
3. On D6, measure indices (see section 7) of the T1 mature nematodes that have grown for 3 days.
4. On D9, repeat steps 6.2-6.3, use T1 nematodes to reproduce T2 nematodes and measure effects in T1 nematodes.
5. On D12, repeat steps 6.4, use T2 nematodes to reproduce T3 nematodes and measure effects in T2 nematodes. Through the same way, reproduce offspring and measure MGR effects on the nth offspring generation (Tn) nematodes of F0, or the nth offspring (Tn') nematodes of Fn from step 5.12.

7. Measure indicators

1. Measure locomotion behavior.
   1. Flush the nematodes off the NGM agars using sterile water and collect them into centrifuge tubes. Settle the nematodes for 30 min, discard the supernatants and use the nematodes in the pellets for effect measurement.
   2. Resuspend the nematodes in the pellets with 1 mL of sterile water and pipette them onto NGM agars without bacterial lawn from step 2.10.
   3. Use a dissecting microscope to score the nematodes for the (number of) body bending frequency (BBF) which refers to the times the posterior bulb of the pharynx changes direction along the vertical direction of the traveling path within a 60 s interval.
   4. Use the dissecting microscope to score reversal movement (RM) which refers to the times when the traveling direction changes over 90° including backward turns and Omega turn (OT) in an interval of 60 s. The OT refers to the movement when the head of the nematode touches or almost touches its tail making the nematode shape like the Greek letter Omega (Ω).
      NOTE: At least 6 nematodes were examined for each treatment in each experimental replicate.

Table 1: List of markers and their definitions.

2. Measure reproduction and lifespan in MGE effect studies.
   1. On D0, repeat step 5.6. Mark the agars (3 groups with 10 replicates in each) as Fx–a–b–c, where x refers to generation number, a refers to the group number (1 for control, 2 for the low concentration and 3 for the high concentration); b refers to the replicate from 1 to 10; and c refers to the exposure duration (0 indicates the start). For D0, marks the agars as F0-1-1-0 to F0-3-10-0. Readers can refer to Table 1 for detailed information.
   2. On D1, check the nematode growth on the agars, and change the markers from F0-1-1-0 to F0-3-10-0 to F0-1-1-1 to F0-3-10-1.
   3. On D2, check the nematode growth on the agars, and change the markers F0-1-1-2 to F0-3-10-2.
   4. On D3, pick 24 F0 nematodes from each group onto 12 new NGM agars with two NGM agar from step 5.4. Mark the agars as F0-1-1-3 to F0-3-12-3.
   5. On D4, transfer the two parent nematodes from F0-1-1-3 to F0-3-12-3 by picking onto new NGM agars from step 5.4. Mark the new NGM agars as F0-1-1-4 to F0-3-12-4. Change the markers of F0-1-1-3 to F0-3-12-3 to F1-1-1-1 to F1-3-12-1 to represent the offspring of F0 (i.e., F1) within the first day since F0 start to reproduce.
   6. On D5, use a dissecting microscope to count the nematodes on F1-1-1-1 to F1-3-12-1 agars where the nematodes have grown 2 days. Allow F1-1-1-1 to F1-3-12-1 agars to reproduce F2 for successive generations.
   7. Transfer the two parent nematodes from F0-1-1-4 to F0-3-12-4 agars by picking onto new NGM agars from step 5.4. Mark NGM agars as F0-1-1-5 to F0-3-12-5. Change the marks of F0-1-1-4 to F0-3-12-4 to F1-1-1-2 to F1-3-12-2 to represent the offspring of F0 (i.e., F1) within the second day since F0 start to reproduce.
   8. On D6, count the nematodes on F1-1-1-2 to F1-3-12-2 agars. Transfer the parent nematodes from F0-1-1-5 to F0-3-12-5 agars to F0-1-1-6 to F0-3-12-6. Change the markers of F0-1-1-5 to F0-3-12-5 to F1-1-1-3 to F1-3-12-3 to represent the offspring of F0 (i.e., F1) within the third day since F0 start to reproduce.
   9. The nematodes on F1-1-1-1 to F1-3-12-1 agars have grown for 3 days. Use them to reproduce F2 in succeeding MGE effect studies. Through the same way, use F2 nematodes to reproduce F3 to continue the MGE effect studies.
   10. Through the same way, transfer the two parent nematodes daily and count the offspring nematodes the next day, until the parent nematodes in 6 NGM agars (i.e., half of the total agars in each group) stop reproduction.
   11. Calculate the total offspring number over the whole reproduction duration as the total brood size. Use the offspring number within the first 3 days to represent the initial reproduction of the parents.
   12. Use the day when the parent nematode reproduction stops in 6 NGM agars to estimate the reproduction duration. Use the days that each individual parent has survived as its lifespan.
   13. Obtain the initial reproduction, reproduction duration, total brood size and lifespan of F1 in the same way as done for F0. Similarly, by repeating the aforementioned procedure, reproduction and lifespan information of F2 (to Fn) can be obtained.
3. Measure reproduction and lifespan in MGR effect studies.
   1. Perform step 7.2.4 with the NGM agars from step 5.4 changed to those from step 2.13.
4. Measure biochemical indices.
   1. Flush the nematodes off the NGM agars with sterile water and collect them into centrifuge tubes. Settle the nematodes for 30 min and discard the supernatants. Use the nematodes in the pellets for effect measurement.
   2. Add 1 mL of ice-cold phosphate buffered saline (PBS, pH 7.0) into the nematodes (pellets) in the 1.5 mL centrifuge tubes to wash the nematodes.
   3. Centrifuge at 10,000 x g for 5 min at 4 °C, and carefully discard the supernatants with a pipette.
   4. Snap freeze the pellets by liquid nitrogen or in a -80 °C freezer.
   5. Homogenize the pellets using pestles in an ice bath. Use 200 µL ice-cold PBS to wash the residual liquids on the pestle into the centrifuge tube before taking out the pestle.
   6. Centrifuge at 10,000 x g for 5 min at 4 °C again, and use the supernatants to determine the activities or amounts of biochemicals with the commercial kits (see the Table of Materials for detail).
   7. Measure the amounts of the total protein (TP) in samples, and use the results as the denominator in representing other biochemical indicators, so that the difference among nematode numbers among samples can be eliminated.
5. Measure gene expression.
   1. Repeat steps 7.4.1 to 7.4.4. Isolate the total RNA from the nematode samples using a commercial RNA extraction kit (see the Table of Materials for details) according to the manufacturer's instructions²¹.
   2. Use the RNA to synthesize cDNA according to the manufacturer's instructions²¹.
   3. Analyze the cDNA sample in the real-time polymerase chain reaction (RT-PCR) using SYBR Green RT-PCR kits according to the manufacturer's instructions (see Table of Materials)²¹.
   4. Quantify the relative expression levels of the chosen genes by the 2^-ΔΔCT method²², and treat the expression levels of gdp-2 (or other reference gene) as negative reference.