Method Article

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

DOI:

10.3791/59367

⸱

July 29th, 2019

 ,  ,  ,  , 
1College of Environmental Sciences and Engineering, Tongji University, 2Shanghai Institute of Pollution Control and Ecological Security, 3Jiaxing Tongji Institute of Environment, 4College of Ecological Technique and Engineering, Shanghai Institute of Technology

In This Article

Summary

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Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

Abstract

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Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Introduction

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The application and waste management of chemicals is highly dependent upon the information of their effects at certain concentrations. Notably, time is another essential element between effects and concentrations. That is to say, chemicals, especially those at low concentrations in the actual environments, need time to provoke measurable effects1. Therefore, researchers arrange different lengths of the exposure duration in animal experiments, and even cover the whole life cycle. For example, mice were exposed to nicotine for 30, 90 or 180 days to study its toxic effects 2. Yet, such exposure durations are still not enough to elucidate the long-term effects of pollutants (e.g., persistent organic pollutants [POPs]) that can last over generations of organisms in the environment. Therefore, studies on effects over generations are gaining more and more attention.

There are two main aspects in generational effect studies. The first one is the trans-generational (TG) effect study which can robustly test whether chemical exposure to parents can result in any consequences on the offspring3. The second one is a multi-generational effect study which is more systematic with considerations in both exposure and residual effects. On the one hand, the multi-generational exposure (MGE) effects are used to illustrate adaptive responses in the animals to the long-term challenging environments. On the other hand, the multi-generational residual (MGR) effects are used to demonstrate the long-term residual consequences after exposure, since maternal exposure is accompanied with embryo exposure to the first offspring and germ-line exposure to the second offspring which makes the third offspring as the first generation completely out of exposure4.

Although mammals (e.g., mice) are model organisms in toxicity studies especially in relation to human beings, their application in studying generational effects is quite time-consuming, expensive and ethically concerning 5. Accordingly, organisms including crustacean Daphnia magna6, insect Drosophila melanogaster7 and zebrafish Danio rerio8, provide alternative choices. Yet, these organisms either lack similarities to human beings, or require specific equipment in studies.

Caenorhabditis elegans is a small free-living nematode (approximately 1 mm in length) with a short life-cycle (approximately 84 h at 20 °C)9. This nematode shares many biological pathways conservative to human beings, and therefore it has been widely employed to illustrate effects of various stresses or toxicants10. Notably, 99.5% of the nematodes are hermaphrodites making this organisms extremely suitable in studying generational effects, e.g., TG effects of heavy metals and sulfonamides3,11, MGE effects of gold nanoparticles and heavy metals12 and temperature13, MGR effects of sulfonamide14, and both MGE and MGR effects of gamma irradiation15 and lindane4. Furthermore, comparable results were found between the effects of chemicals (e.g., zearalenone) on the development and reproduction of mice and C. elegans16,17, which would provide an advantage to extrapolate effects from this small animal to human beings.

Both TG and MG effect studies are time consuming and need careful design and performance. Notably, differences existed in life-stage choices, exposure conditions and generation separation methods in the aforementioned studies. Such differences hindered the direct comparison among the results and hampered further interpretation of the results. Therefore, it is imperative to establish uniform protocols to guide TG and MG effects studies, and also to provide a bigger picture to reveal similar patterns of various toxicants or pollutants in long-term consequences. The over goal of the present protocols will demonstrate clear operation processes in studying trans- and multi-generational effects with C. elegans. The protocols will benefit researchers that are interested in studying the long-term effects of toxicants or pollutants.

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Protocol

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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 (Tegg).
  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 Tegg, 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.
Examples for MGE (F0 to F3) effects on reproduction and lifespan with 3 groups (one control and two exposure treatments).
DayNGM agar number for MGE studyExplanation
LifespanReproduction
030 (F0 exposure)10 replicates for each group, marked as F0-1-1-0 to F0-3-10-0, with the last digit to show the survival days.
130 (F0 survive 1 d)F0-1-1-0 to F0-3-10-0 should be changed to F0-1-1-1 to F0-3-10-1.
230 (F0 survive 2 d)F0-1-1-1 to F0-3-10-1 should be changed to F0-1-1-2 to F0-3-10-2.
No need to transfer F0 nematodes until 3 d.
330 (F0 survive 3 d, cleared after nematodes transfer and collection)After 3 d, F0 nematodes are mature and 36 new NGM agars (with 2 nematodes on each agar) are used to observe their survival and reproduction.
36 (F0-1-1-3 to F0-3-12-3)Preliminary experiments should be performed to arrange the number of F0 nematodes, assuring at least 200 offspring for succeeding multi-generational operations.
Notably, if MGR effects are studied, the F0 nematodes should be transferred onto clear NGM agars without chemical exposure, and it should be noted as T1 start.
Most of the F0 nematodes are collected to measure chemical and genetic indices and the 30 agars in F0 are cleared.
436 (F0-1-1-4 to F0-3-12-4)36 (F1-1-1-1 to F1-3-12-1)Measurement on lifespan and reproduction requires transfer every day.
Parent nematodes on F0-1-1-3 to F0-3-12-3 are picked onto new NGM agars marked as F0-1-1-4 to F0-3-12-4.
The remaining offspring nematodes (i.e., F1 in MGE, or T1 in MGR) in F0-1-1-3 to F0-3-12-3 agars have grown for 1 d, and the markers are changed to F1-1-1-1 to F1-3-12-1. These agars are also used to monitor the lifespan of F1 with daily transfer.
536 (F0-1-1-5 to F0-3-12-5)36 (F1-1-1-2 to F1-3-12-2)Nematodes on F1-1-1-1 to F1-3-12-1 agars have grown for 2 d and become easily observable and the nematodes are counted, and the markers are changed to F1-1-1-2 to F1-3-12-2.
36 (F0-1-1-4 to F0-3-12-4)The offspring nematodes in F0-1-1-4 to F0-3-12-4 agars have grown for 1 d.
636 (F0-1-1-6 to F0-3-12-6)36 (F0-1-1-4 to F0-3-12-4, cleared after counted)Nematodes on F1-1-1-2 to F1-3-12-2 agars have grown for 3 d and the markers are changed to F1-1-1-3 to F1-3-12-3. Notably, the F1 nematodes start to reproduce F2 on this day, F1 nematodes should be transferred onto new NGM agars making F2-1-1-0 to F1-3-12-0. For MGR studies, T2 start today.
36 (F1-1-1-3 to F1-3-12-3)36 (F0-1-1-5 to F0-3-12-5)This can be delayed by chemical exposure, and therefore flexible changes should be performed in each experiment to ensure enough nematodes for subsequent generations.
36 (F2-1-1-0 to F1-3-12-0)The offspring nematodes on F0-1-1-4 to F0-3-12-4 agars have grown for 2 d, and the agars are cleared after the nematodes are counted.
The offspring nematodes on F0-1-1-5 to F0-3-12-5 agars have grown for 1 d.
736 (F0-1-1-7 to F0-3-12-7)36 (F0-1-1-5 to F0-3-12-5, cleared after counted)The offspring nematodes on F0-1-1-5 to F0-3-12-5 agars have grown for 2 d, and the agars are cleared after the nematodes are counted.
36 (F1-1-1-4 to F1-3-12-4)36 (F0-1-1-6 to F0-3-12-6)The overall nematode number in F1-1-1-1 to F1-3-12-1 agars, F0-1-1-4 to F0-3-12-4 agars and F0-1-1-5 to F0-3-12-5 are used to calculate the initial reproduction of F0.
36 (F2-1-1-1 to F2-3-12-1)The offspring nematodes on F0-1-1-6 to F0-3-12-6 agars have grown for 1 d.
The F2 nematodes on F2-1-1-0 to F1-3-12-0 have grown for 1 d and their markers are changed into F2-1-1-1 to F2-3-12-1.
836 (F0-1-1-8 to F0-3-12-8)36 (F0-1-1-6 to F0-3-12-6, cleared after counted)The offspring nematodes on F0-1-1-6 to F0-3-12-6 agars have grown for 2 d, and the agars are cleared after the nematodes are counted.
36 (F1-1-1-5 to F1-3-12-5)36 (F0-1-1-7 to F0-3-12-7)The offspring nematodes of F0 on F0-1-1-7 to F0-3-12-7 agars have grown for 1 d.
36 (F2-1-1-2 to F2-3-12-2)36 (F1-1-1-4 to F1-3-12-4)The offspring nematodes of F1 on F1-1-1-4 to F1-3-12-4 agars have grown for 1 d.
36 (F2-1-1-1 to F2-3-12-1, changed to F2-1-1-2 to F2-3-12-2 after counted)The F2 nematodes on F2-1-1-1 to F2-3-12-1 have grown for 2 d, the nematodes are counted and their markers are changed into F2-1-1-2 to F2-3-12-2.
936 (F0-1-1-9 to F0-3-12-9)36 (F0-1-1-7 to F0-3-12-7, cleared after counted)The offspring nematodes of F0 on F0-1-1-7 to F0-3-12-7 agars have grown for 2 d, and the agars are cleared after the nematodes are counted.
36 (F1-1-1-6 to F1-3-12-6)36 (F1-1-1-4 to F1-3-12-4, cleared after counted)The offspring nematodes of F1 on F1-1-1-4 to F1-3-12-4 agars have grown for 2 d, and the agars are cleared after the nematodes are counted.
36 (F2-1-1-3 to F2-3-12-3)36 (F0-1-1-8 to F0-3-12-8)The offspring nematodes of F0 on F0-1-1-8 to F0-3-12-8 agars have grown for 1 d.
36 (F3-1-1-0 to F3-3-12-0)36 (F1-1-1-5 to F1-3-12-5)The offspring nematodes of F1 on F1-1-1-5 to F1-3-12-5 agars have grown for 1 d.
The F2 nematodes on F2-1-1-2 to F2-3-12-2 have grown for 3 days and their markers are changed to F2-1-1-3 to F2-3-12-3. The F2 nematodes start to reproduce today and they are transferred to 36 new NGM agars are needed and marked as F3-1-1-0 to F3-3-12-0. For MGR studies, T3 start today.
1036 (F0-1-1-10 to F0-3-12-10)36 (F0-1-1-8 to F0-3-12-8, cleared after counted)The offspring nematodes of F0 on F0-1-1-8 to F0-3-12-8 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F1-1-1-7 to F1-3-12-7)36 (F1-1-1-5 to F1-3-12-5, cleared after counted)The offspring nematodes of F1 on F1-1-1-5 to F1-3-12-5 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F2-1-1-4 to F2-3-12-4)36 (F0-1-1-9 to F0-3-12-9)The overall nematode number in F2-1-1-1 to F2-3-12-1, F1-1-1-4 to F1-3-12-4 agars and F1-1-1-5 to F1-3-12-5 are used to calculate the initial reproduction of F1.
36 (F3-1-1-1 to F3-3-12-1)36 (F1-1-1-6 to F1-3-12-6)The offspring nematodes of F0 on F0-1-1-9 to F0-3-12-9 agars have grown for 1 d.
The offspring nematodes of F1 on F1-1-1-6 to F1-3-12-6 agars have grown for 1 d.
The offspring nematode on F3-1-1-0 to F3-3-12-0 agars have grown for 1 d and the markers are changed into F3-1-1-1 to F3-3-12-1.
Notably, the reproduction of F0 nematodes will significantly decrease after the first several days. Therefore, the nematode transfer is not strictly required to be daily after D10 and can be performed every 2 days. Yet, the survival still requires daily observation.
The same rule also applies in F1 (T1, T1’), F2 (T2, T2’) and F3 (T3, T3’).
1136 (F0-1-1-11 to F0-3-12-11)36 (F0-1-1-9 to F0-3-12-9, cleared after counted)The offspring nematodes of F0 on F0-1-1-9 to F0-3-12-9 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F1-1-1-8 to F1-3-12-8)36 (F1-1-1-6 to F1-3-12-6, cleared after counted) The offspring nematodes of F1 on F1-1-1-6 to F1-3-12-6 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F2-1-1-5 to F2-3-12-5)36 (F0-1-1-10 to F0-3-12-10)The offspring nematodes of F0 on F0-1-1-10 to F0-3-12-10 agars have grown for 1 d.
36 (F3-1-1-2 to F3-3-12-2)36 (F1-1-1-7 to F1-3-12-7)The offspring nematodes of F1 on F1-1-1-7 to F1-3-12-7 agars have grown for 1 d.
36 (F2-1-1-4 to F2-3-12-4)The offspring nematodes of F2 on F2-1-1-4 to F2-3-12-4 agars have grown for 1 d.
36 (F3-1-1-1 to F3-3-12-1, changed to F3-1-1-2 to F3-3-12-2 after counting)The nematodes on F3-1-1-1 to F3-3-12-1 agars have grown for 2 d, the nematodes are counted and the markers are changed into F3-1-1-2 to F3-3-12-2.
1236 (F0-1-1-12 to F0-3-12-12)36 (F0-1-1-10 to F0-3-12-10, cleared after counted)The offspring nematodes of F0 on F0-1-1-10 to F0-3-12-10 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F1-1-1-9 to F1-3-12-9)36 (F1-1-1-7 to F1-3-12-7, cleared after counted)The offspring nematodes of F1 on F1-1-1-7 to F1-3-12-7 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F2-1-1-6 to F2-3-12-6)36 (F2-1-1-4 to F2-3-12-4, cleared after counted)The offspring nematodes of F2 on F2-1-1-4 to F2-3-12-4 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F3-1-1-3 to F3-3-12-3)36 (F0-1-1-11 to F0-3-12-11)The offspring nematodes of F0 on F0-1-1-11 to F0-3-12-11 agars have grown for 1 d.
36 (F4-1-1-0 to F4-3-12-0)36 (F1-1-1-8 to F1-3-12-8)The offspring nematodes of F1 on F1-1-1-8 to F1-3-12-8 agars have grown for 1 d.
36 (F2-1-1-5 to F2-3-12-5)The offspring nematodes of F2 on F2-1-1-5 to F2-3-12-5 agars have grown for 1 d.
The nematodes on F3-1-1-2 to F3-3-12-2 agars have grown for 3 d and the markers are changed into F3-1-1-3 to F3-3-12-3. The F3 nematodes start to reproduce today and they are transferred to 36 new NGM agars are needed and marked as F4-1-1-0 to F4-3-12-0. For MGR studies, the offspring of F3 (i.e., T1’) start today.
1336 (F0-1-1-13 to F0-3-12-13)36 (F0-1-1-11 to F0-3-12-11, cleared after counted)The offspring nematodes of F0 on F0-1-1-11 to F0-3-12-11 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F1-1-1-10 to F1-3-12-10)36 (F1-1-1-8 to F1-3-12-8, cleared after counted)The offspring nematodes of F1 on F1-1-1-8 to F1-3-12-8 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F2-1-1-7 to F2-3-12-9)36 (F2-1-1-5 to F2-3-12-5, cleared after counted)The offspring nematodes of F2 on F2-1-1-5 to F2-3-12-5 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F3-1-1-4 to F3-3-12-4)36 (F0-1-1-12 to F0-3-12-12)The overall nematode number in F3-1-1-1 to F3-3-12-1, F2-1-1-4 to F2-3-12-4 agars and F2-1-1-5 to F2-3-12-5 are used to calculate the initial reproduction of F2.
36 (F4-1-1-1 to F4-3-12-1)36 (F1-1-1-9 to F1-3-12-9)The offspring nematodes of F0 on F0-1-1-12 to F0-3-12-12 agars have grown for 1 d.
36 (F2-1-1-6 to F2-3-12-6)The offspring nematodes of F1 on F1-1-1-9 to F1-3-12-9 agars have grown for 1 d.
The offspring nematodes of F2 on F2-1-1-6 to F2-3-12-6 agars have grown for 1 d.
The offspring nematodes of F3 on F4-1-1-0 to F4-3-12-0 have grown for 1 d, and markers are changed into F4-1-1-1 to F4-3-12-1.
1436 (F0-1-1-14 to F0-3-12-14)36 (F0-1-1-12 to F0-3-12-12, cleared after counted)The offspring nematodes of F0 on F0-1-1-12 to F0-3-12-12 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F1-1-1-11 to F1-3-12-11)36 (F1-1-1-9 to F1-3-12-9, cleared after counted)The offspring nematodes of F1 on F1-1-1-9 to F1-3-12-9 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F2-1-1-8 to F2-3-12-8)36 (F2-1-1-6 to F2-3-12-6, cleared after counted)The offspring nematodes of F2 on F2-1-1-6 to F2-3-12-6 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F3-1-1-5 to F3-3-12-5)36 (F4-1-1-1 to F4-3-12-1, cleared after counted)The offspring nematodes of F3 on F4-1-1-1 to F4-3-12-1 have grown for 2 d, and agars are cleared after the nematodes are counted. For MGR studies, T1’ nematodes have grown for 2 d, and will start to reproduce T2’ on the next day (D15), and T2’ will start to reproduce T3’ on D18. The lifespan of wild type C. elegans is exampled as 15 days. Then, the ending of T3’ lifespan will be on D33.
36 (F0-1-1-13 to F0-3-12-13)The offspring nematodes of F0 on F0-1-1-13 to F0-3-12-13 agars have grown for 1 d.
36 (F1-1-1-10 to F1-3-12-10)The offspring nematodes of F1 on F1-1-1-10 to F1-3-12-10 agars have grown for 1 d.
36 (F2-1-1-7 to F2-3-12-7)The offspring nematodes of F2 on F2-1-1-7 to F2-3-12-7 agars have grown for 1 d.
36 (F3-1-1-4 to F3-3-12-4)The offspring nematodes of F3 on F3-1-1-4 to F2-3-12-4 agars have grown for 1 d.
1536 (F0-1-1-15 to F0-3-12-15)36 (F0-1-1-13 to F0-3-12-13, cleared after counted)The offspring nematodes of F0 on F0-1-1-13 to F0-3-12-13 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F1-1-1-12 to F1-3-12-12)36 (F1-1-1-10 to F1-3-12-10, cleared after counted)The offspring nematodes of F1 on F1-1-1-10 to F1-3-12-10 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F2-1-1-9 to F2-3-12-9)36 (F2-1-1-7 to F2-3-12-7, cleared after counted)The offspring nematodes of F2 on F2-1-1-7 to F2-3-12-7 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F3-1-1-6 to F3-3-12-6)36 (F3-1-1-4 to F3-3-12-4, cleared after counted)The offspring nematodes of F3 on F3-1-1-4 to F3-3-12-4 agars have grown for 2d, and agars are cleared after the nematodes are counted.
36 (F0-1-1-14 to F0-3-12-14)The offspring nematodes of F0 on F0-1-1-14 to F0-3-12-14 agars have grown for 1 d.
36 (F1-1-1-11 to F1-3-12-11)The offspring nematodes of F1 on F1-1-1-11 to F1-3-12-11 agars have grown for 1 d.
36 (F2-1-1-8 to F2-3-12-8)The offspring nematodes of F2 on F2-1-1-8 to F2-3-12-8 agars have grown for 1 d.
36 (F3-1-1-5 to F3-3-12-5)The offspring nematodes of F3 on F3-1-1-5 to F2-3-12-5 agars have grown for 1 d.
1636 (F0-1-1-15 to F0-3-12-15, over)36 (F0-1-1-14 to F0-3-12-14, cleared after counted)The lifespan of wild type C. elegans is exampled as 15 days. Therefore, F0 should have all died before Day 16 since the exposure.
36 (F1-1-1-13 to F1-3-12-13)36 (F1-1-1-11 to F1-3-12-11, cleared after counted)The offspring nematodes of F0 on F0-1-1-14 to F0-3-12-14 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F2-1-1-10 to F2-3-12-10)36 (F2-1-1-8 to F2-3-12-8, cleared after counted)The offspring nematodes of F1 on F1-1-1-11 to F1-3-12-11 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F3-1-1-7 to F3-3-12-7)36 (F3-1-1-5 to F3-3-12-5, cleared after counted)The offspring nematodes of F2 on F2-1-1-8 to F2-3-12-8 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F0-1-1-15 to F0-3-12-15)The offspring nematodes of F3 on F3-1-1-5 to F3-3-12-5 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F1-1-1-12 to F1-3-12-12)The overall nematode number in F4-1-1-1 to F4-3-12-1, F3-1-1-4 to F3-3-12-4 agars and F3-1-1-5 to F3-3-12-5 are used to calculate the initial reproduction of F3.
36 (F2-1-1-9 to F2-3-12-9)The offspring nematodes of F0 on F0-1-1-15 to F0-3-12-15 agars have grown for 1 d.
36 (F3-1-1-6 to F3-3-12-6)The offspring nematodes of F1 on F1-1-1-12 to F1-3-12-12 agars have grown for 1 d.
The offspring nematodes of F2 on F2-1-1-9 to F2-3-12-9 agars have grown for 1 d.
The offspring nematodes of F3 on F3-1-1-6 to F2-3-12-6 agars have grown for 1 d.
1736 (F1-1-1-14 to F1-3-12-14)36 (F0-1-1-15 to F0-3-12-15, cleared after counted, over)The offspring nematodes of F0 on F0-1-1-15 to F0-3-12-15 agars have grown for 2 d, and agars are cleared after the nematodes are counted. There will be no more F0 offspring.
36 (F2-1-1-11 to F2-3-12-11)36 (F1-1-1-12 to F1-3-12-12, cleared after counted)The offspring nematodes of F1 on F1-1-1-12 to F1-3-12-12 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F3-1-1-8 to F3-3-12-8)36 (F2-1-1-9 to F2-3-12-9, cleared after counted)The offspring nematodes of F2 on F2-1-1-9 to F2-3-12-9 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F3-1-1-6 to F3-3-12-6, cleared after counted)The offspring nematodes of F3 on F3-1-1-6 to F3-3-12-6 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F1-1-1-13 to F1-3-12-13)The offspring nematodes of F1 on F1-1-1-13 to F1-3-12-13 agars have grown for 1 d.
36 (F2-1-1-10 to F2-3-12-10)The offspring nematodes of F2 on F2-1-1-10 to F2-3-12-10 agars have grown for 1 d.
36 (F3-1-1-7 to F3-3-12-7)The offspring nematodes of F3 on F3-1-1-7 to F2-3-12-7 agars have grown for 1 d.
1836 (F1-1-1-15 to F1-3-12-15)36 (F1-1-1-13 to F1-3-12-13, cleared after counted)The offspring nematodes of F1 on F1-1-1-13 to F1-3-12-13 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F2-1-1-12 to F2-3-12-12)36 (F2-1-1-10 to F2-3-12-10, cleared after counted)The offspring nematodes of F2 on F2-1-1-10 to F2-3-12-10 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F3-1-1-9 to F3-3-12-9)36 (F3-1-1-7 to F3-3-12-7, cleared after counted)The offspring nematodes of F3 on F3-1-1-7 to F3-3-12-7 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F1-1-1-14 to F1-3-12-14)The offspring nematodes of F1 on F1-1-1-14 to F1-3-12-14 agars have grown for 1 d.
36 (F2-1-1-11 to F2-3-12-11)The offspring nematodes of F2 on F2-1-1-11 to F2-3-12-11 agars have grown for 1 d.
36 (F3-1-1-8 to F3-3-12-8)The offspring nematodes of F3 on F3-1-1-8 to F2-3-12-8 agars have grown for 1 d.
In MGR studies, T2’ will start to reproduce T3’ today. The lifespan of wild type C. elegans is exampled as 15 days. Then, the ending of T3’ lifespan will be on D33.
1936 (F1-1-1-15 to F1-3-12-15, over)36 (F1-1-1-14 to F1-3-12-14, cleared after counted)The offspring nematodes of F1 on F1-1-1-14 to F1-3-12-14 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F2-1-1-13 to F2-3-12-13)36 (F2-1-1-11 to F2-3-12-11, cleared after counted)The offspring nematodes of F2 on F2-1-1-11 to F2-3-12-11 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F3-1-1-10 to F3-3-12-10)36 (F3-1-1-8 to F3-3-12-8, cleared after counted)The offspring nematodes of F3 on F3-1-1-8 to F3-3-12-8 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F1-1-1-15 to F1-3-12-15)The offspring nematodes of F1 on F1-1-1-15 to F1-3-12-15 agars have grown for 1 d.
36 (F2-1-1-12 to F2-3-12-12)The offspring nematodes of F2 on F2-1-1-12 to F2-3-12-12 agars have grown for 1 d.
36 (F3-1-1-9 to F3-3-12-9)The offspring nematodes of F3 on F3-1-1-9 to F2-3-12-9 agars have grown for 1 d.
2036 (F2-1-1-14 to F2-3-12-14)36 (F1-1-1-15 to F1-3-12-15, cleared after counted)The offspring nematodes of F1 on F1-1-1-14 to F1-3-12-14 agars have grown for 2 d, and agars are cleared after the nematodes are counted. There will no more F1 offspring.
36 (F3-1-1-11 to F3-3-12-11)36 (F2-1-1-12 to F2-3-12-12, cleared after counted)The offspring nematodes of F2 on F2-1-1-12 to F2-3-12-12 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F3-1-1-9 to F3-3-12-9, cleared after counted)The offspring nematodes of F3 on F3-1-1-9 to F3-3-12-9 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F2-1-1-13 to F2-3-12-13)The offspring nematodes of F2 on F2-1-1-13 to F2-3-12-13 agars have grown for 1 d.
36 (F3-1-1-10 to F3-3-12-10)The offspring nematodes of F3 on F3-1-1-10 to F2-3-12-10 agars have grown for 1 d.
2136 (F2-1-1-15 to F2-3-12-15)36 (F2-1-1-13 to F2-3-12-13, cleared after counted)The offspring nematodes of F2 on F2-1-1-13 to F2-3-12-13 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F3-1-1-12 to F3-3-12-12)36 (F3-1-1-10 to F3-3-12-10, cleared after counted)The offspring nematodes of F3 on F3-1-1-10 to F3-3-12-10 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F2-1-1-14 to F2-3-12-14)The offspring nematodes of F2 on F2-1-1-14 to F2-3-12-14 agars have grown for 1 d.
36 (F3-1-1-11 to F3-3-12-11)The offspring nematodes of F3 on F3-1-1-11 to F2-3-12-11 agars have grown for 1 d.
2236 (F2-1-1-15 to F2-3-12-15, over)36 (F2-1-1-14 to F2-3-12-14, cleared after counted)The offspring nematodes of F2 on F2-1-1-14 to F2-3-12-14 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F3-1-1-13 to F3-3-12-13)36 (F3-1-1-11 to F3-3-12-11, cleared after counted)The offspring nematodes of F3 on F3-1-1-11 to F3-3-12-11 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F3-1-1-12 to F3-3-12-12)The offspring nematodes of F3 on F3-1-1-12 to F2-3-12-12 agars have grown for 1 d.
2336 (F3-1-1-14 to F3-3-12-14)36 (F2-1-1-15 to F2-3-12-15, cleared after counted)The offspring nematodes of F2 on F2-1-1-15 to F2-3-12-15 agars have grown for 2 d, and agars are cleared after the nematodes are counted. There will be no more F2 offspring.
36 (F3-1-1-12 to F3-3-12-12, cleared after counted)The offspring nematodes of F3 on F3-1-1-12 to F3-3-12-12 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F3-1-1-13 to F3-3-12-13)The offspring nematodes of F3 on F3-1-1-13 to F2-3-12-13 agars have grown for 1 d.
2436 (F3-1-1-15 to F3-3-12-15)36 (F3-1-1-13 to F3-3-12-13, cleared after counted)The offspring nematodes of F3 on F3-1-1-13 to F3-3-12-13 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F3-1-1-14 to F3-3-12-14)The offspring nematodes of F3 on F3-1-1-14 to F2-3-12-14 agars have grown for 1 d.
2536 (F3-1-1-15 to F3-3-12-15, over)36 (F3-1-1-14 to F3-3-12-14, cleared after counted)The offspring nematodes of F3 on F3-1-1-14 to F3-3-12-14 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
36 (F3-1-1-15 to F3-3-12-15)The offspring nematodes of F3 on F3-1-1-15 to F2-3-12-15 agars have grown for 1 d.
2636 (F3-1-1-15 to F3-3-12-15, cleared after counted)The offspring nematodes of F3 on F3-1-1-15 to F3-3-12-15 agars have grown for 2 d, and agars are cleared after the nematodes are counted.
Notably, in MGR studies, the first non-exposed offspring of F3 (i.e., T3’) would be born on D18. The lifespan of wild type C. elegans is exampled as 15 days. Then, the ending of T3’ lifespan will be on D33.
Both MGE and MGR studies will cover more days when the nematode lifespan is longer.

Table 1: List of markers and their definitions.

  1. 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.
  2. 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.
  3. 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.
  4. 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.

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Results

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Here, we describe protocols for studying effects of chemicals over generations using C. elegans in trans-generational (TG), multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. Our own research results are presented as examples. One study presents the TG effects of heavy metals on locomotion behavior3. The other two studies present MGE and MGR effects of sulfomethoxazole and lindane on the reproduction and biochemical and...

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Discussion

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In order to successfully conduct the described protocol, the following suggestions should be taken into consideration. Perform the overall experimental operations in a sterile environment. Improper operation may result in contamination of the E. coli strains, e.g., fungi and mites may hinder the normal growth of C. elegans and therefore affect the experimental results. In the section describing cultivating C. elegans, observe the growth scale of C. elegans on the NGM agars by naked eye...

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Disclosures

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The authors are grateful for the financial supports by National Science and Technology Major Project for Water Pollution Control and Treatment (2017ZX07201004), and International Science & Technology Cooperation Program of China (No. 2016YFE0123700).

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
 agar powderOXOID, Thermo Fisher Scientific, UK9002-18-0
79nnHT Fast Real-Time PCR System Applied Biosystems 
96-well sterile microplateCostar?Corning?America
Autoclave sterilizerTomy, Tomy Digital Biology, Japan
Biosafety cabinetLongYue, Shanghai longyue instrument equipment co. Ltd, China
calcium chlorideSinopharm chemical reagent company Ltd, China10043-52-4
centrifuge  5417REppendorf, Ai Bende (Shanghai) International Trade Co., Ltd, Germany
Centrifuge tubesAxygen, Aixjin biotechnology (Hangzhou) co. Ltd, America
cholesterolSinopharm chemical reagent company Ltd, China57-88-5
Dimethyl sulfoxideVETEC, Sigmar aldrich (Shanghai) trading co. Ltd, America67-68-5
disodium hydrogen phosphateSinopharm chemical reagent company Ltd, China7558-79-4
ethanolSinopharm chemical reagent company Ltd, China64-17-5
FilterThermo, Thermo Fisher Scientific, America
incubatorYiHeng17, Shanghai yiheng scientific instrument co. Ltd, China
inoculating loop
K2HPO4•3H2OSinopharm chemical reagent company Ltd, China16788-57-1
kraft paper
Mcroplate ReaderBoitek, Boten apparatus co. Ltd, America
MgSO4•7H2OSinopharm chemical reagent company Ltd, China10034-99-8
Microscopes XTL-BM-9TDBM, Shanghai BM optical instruments manufacturing co. Ltd, China 
Petri dishes
PipetteEppendorf, Ai Bende (Shanghai) International Trade Co., Ltd, Germany
Potassium chlorideSinopharm chemical reagent company Ltd, China7447-40-7
potassium dihydrogen phosphateSinopharm chemical reagent company Ltd, China7778-77-0
Qiagen RNeasy kitsQiagen Inc., Valencia, CA, United States
QuantiTect SYBR Green RT-PCR kitsQiagen Inc., Valencia, CA, United States
RevertAid First Strand cDNA Synthesis KitThermo Scientific, Wilmington, DE, United States
sodium chlorideSinopharm chemical reagent company Ltd, China7647-14-5
sodium hydroxideSinopharm chemical reagent company Ltd, China1310-73-2
sodium hypochlorite solutionAladdin, Shanghai Aladdin biochemical technology co. Ltd, China7681-52-9
tryptoneOXOID, Thermo Fisher Scientific, UK73049-73-7
yeast extractOXOID, Thermo Fisher Scientific, UK119-44-8

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Caenorhabditis elegansTrans Generational EffectsMulti Generational EffectsAge SynchronizationSodium Hypochlorite SolutionNGM Agar CultureLocomotion Behavior AnalysisReproduction Lifespan MeasurementBiochemical Gene ExpressionEpigenetic Signals

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