Using <em>Caenorhabditis elegans </em>for Studying Trans- and Multi-Generational Effects of Toxicants

Zhuo Li; Fangting Ai; Jing Zhang; Zhenyang Yu; Daqiang Yin

doi:10.3791/59367

JoVE Journal > Environment

Environment

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

Published: July 29, 2019

doi:

10.3791/59367

Zhuo Li^1,2, Fangting Ai³, Jing Zhang⁴, Zhenyang Yu^1,2, Daqiang Yin^1,2

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

Summary

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

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

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.

Protocol

1. Culture E. coli OP50

Prepare 1 M sodium hydroxide solution by dissolving 4 g of sodium hydroxide in 100 mL water.
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.
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.
Sterilize the LB liquid medium at 121 °C and 0.105 MPa for 20 min. Cool the LB medium down to room temperature.
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.
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.
Use the bacterial suspensions from 80% of total flasks to provide E. coli OP50 as nematode food to be used in step 2.11.
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.

Dissolve 22.8 g of K₂HPO₄•3H₂O in 100 mL of sterile distilled water.
Dissolve 6.8 g of KH₂PO₄ in 50 mL of sterile distilled water.
Mix solutions from steps 2.1 and 2.2, to prepare 1 M K₂HPO₄-KH₂PO₄ buffer (pH 6.0, 150 mL in total).
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.
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.
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.
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.
Sterilize the NGM agar from step 2.7 at 121 °C with 0.105 MPa for 20 min.
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.
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.
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.
Inoculate the NGM agar with the top-side up at 37 °C for 8-12 h to form a bacterial lawn.
Use most of the agar with bacterial lawns from step 2.12 to culture nematodes in step 2.15 or 2.19.
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.
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.
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.
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).
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.
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.
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.
Repeat step 2.15 or step 2.16-2.20 every three days.

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

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.
Settle down the nematodes in the centrifuge tubes for 30 min, and then discard 85% of the supernatants by pipetting.
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%.
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.
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.
Centrifuge the tubes at 700 x g for 3 min at 20 °C, and then discard the supernatants by pipetting.
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.
Repeat step 3.7 twice.
Add 1-fold V₀ of sterile water into the tubes to resuspend the age-synchronized eggs.
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).
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.
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.
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.
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

Prepare chemical solutions with 5 concentration levels and one solvent or absolute control, i.e., 6 groups in total.
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.
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.
When the time reaches 24 h after t₀, count the living and dead nematodes in the wells. Calculate the median lethal concentration (LC₅₀).
Prepare a series of 5 concentrations below 10% of the LC₅₀ values.
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.
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.
Perform the exposure for 24 to 96 h since T₀.
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).
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.
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.
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.
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.
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.
Flush the nematodes on the NGM agars from step 4.14 into six centrifuge tubes.
Settle the nematodes for 30 min, and discard the supernatants by pipetting. Resuspend and wash the pellets with 1 mL of sterilized water.
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

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).
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.
Pipette bacterial suspensions on to the agar as per step 2.11.
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.
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.
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).
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).
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.
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.
On D6, measure indices (see section 7) of the F1 mature nematodes that have experienced exposure for 3 days.
On D9, repeat steps 5.8-5.10, use F1 nematodes to reproduce F2 worms and measure effects on F2 nematodes.
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

Repeat steps 5.1-5.7. On D3, pick F0 mature nematodes onto new NGM agars without added chemicals (from step 2.13).
On D4, pick out and discard the mature F0 nematodes. Mark the newly hatched offspring nematodes within these 24 h as T1 nematodes.
On D6, measure indices (see section 7) of the T1 mature nematodes that have grown for 3 days.
On D9, repeat steps 6.2-6.3, use T1 nematodes to reproduce T2 nematodes and measure effects in T1 nematodes.
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

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).
Day	NGM agar number for MGE study		Explanation
Day	Lifespan	Reproduction	Explanation
0	30 (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.
1	30 (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.
2	30 (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.
2	30 (F0 survive 2 d)		No need to transfer F0 nematodes until 3 d.
3	30 (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.
4	36 (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.
5	36 (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.
5	36 (F0-1-1-5 to F0-3-12-5)	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.
6	36 (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.
7	36 (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.
8	36 (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.
9	36 (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.
10	36 (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’).
11	36 (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.
12	36 (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.
13	36 (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.
14	36 (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.
15	36 (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.
16	36 (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.
17	36 (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.
18	36 (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.
19	36 (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.
20	36 (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.
21	36 (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.
22	36 (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.
23	36 (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.
24	36 (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.
24	36 (F3-1-1-15 to F3-3-12-15)	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.
25	36 (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.
25	36 (F3-1-1-15 to F3-3-12-15, over)	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.
26		36 (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.

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.
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.
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.
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.

Representative Results

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 genetic indices measurements⁴^,¹⁴.

TG effects of heavy metals on locomotion behavior of C. elegans

The TG effects of cadmium (Cd), copper (Cu), lead (Pb) and zinc (Zn) on the body bending frequency (BBF) were studied in the nematode parent (F0) after maternal exposure and their progeny (T1)³. The effects of metals on BBF showed that the inhibitions in T1 were greater than in F0, demonstrating more severe toxicities of heavy metals on the locomotion behavior in the embryo-exposed offspring than in the directly exposed parent. The TG effects of heavy metals at environmentally realistic concentrations demonstrated that maternal exposure can multiply the hazards of heavy metal pollution in succeeding generations. See Figure 1.

Figure 1: The effects of cadmium (Cd), copper (Cu), lead (Pb) and zinc (Zn) on the body bending frequency of the nematode parent (F0, blank) after prenatal exposure and their progeny (T1, shaded). Error bar = standard error; * = significantly different from the control, p < 0.05; # = significantly different from the lower concentration, p < 0.05; + implies significantly different effects in F1 than in F0, p < 0.05. This figure has been modified from Yu et al.³ with permission. Please click here to view a larger version of this figure.

MGR effects of sulfamethoxazole (SMX) on the nematode lifespan and reproduction

The MGR effects of sulfamethoxazole (SMX) on the nematode lifespan and reproduction¹⁴ were studied on the gestating parent (F0), embryo-exposed offspring (T1), germline-exposed offspring (T2), the first non-exposed offspring (T3) and the three following generations (T4-T6). Results showed that the reproduction (a total brood size as 49% of the control) were significantly affected in germline exposure (T2), and the toxicities persisted in non-exposed generations from T3 to T6 generations (Figure 2). Our findings raised new concerns regarding the long-term influences of antibiotics themselves besides their effects on antibiotic resistance.

Figure 2: Brood size (in (A), expressed in percentage of control) and initial reproduction (in (B)) of C. elegans in the exposed parent and its offspring (F0, T1 to T6, from left to right at each concentration). Error bar = standard error; a = significantly different from the control by ANOVA (p < 0.05); b = significantly different from the control and from the earlier generation at the same concentration (p < 0.05); c = significantly different from the control and from the lower concentration in the same generation (p < 0.05); d = significantly different from the control and from the earlier generation at the same concentration and the lower concentration in the same generation (p < 0.05); e = significantly different from the earlier generation at the same concentration and the lower concentration in the same generation (p < 0.05); f = significantly different from the earlier generation at the same concentration (p < 0.05). This figure has been modified from Yu et al.¹⁴ with permission.

MGE and MGR effects of lindane on the nematode biochemical and genetic indices

The MGE and MGR effects of lindane (a persistent organic pollutant [POP]) were studied on key biochemicals in the lipid metabolisms and genetic expression changes in the related insulin-like pathway⁴. Results showed that lindane showed obesogenic effects with disturbances in the insulin signal regulation (Figure 3). Moreover, the changes between sgk-1 (F0, F3, T1' and T3') and akt-1 (T1 and T3) signaling indicated that nematodes from different exposure generations showed different response strategies for tolerance and avoidance.

Figure 3: Changes of expression levels of key genes in insulin-like signal pathway in nematodes with different exposure experiences. →: positive regulation; : negative regulation; ↑: expression up-regulation; ↓: expression down-regulation. This figure has been modified from Chen at al.⁴ with permission. Please click here to view a larger version of this figure.

Discussion

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 eyes or microscopes. When the scale of C. elegans on the agar exceeds 75% in area, or the culture time exceeds one week, perform a new round of inoculation to avoid over-growth or population decline of C. elegans. Before the process of the synchronization, use a microscope to observe the growth of C. elegans, and continue the process when nematode eggs are widely distributed on the agar. In addition, if solvents (e.g., dimethyl sulfoxide [DMSO]) are used, their concentrations in the stock solutions should be lower than 1% to ensure that their final concentrations do not exceed 0.5% (v/v) to avoid the adverse effects of the solvents themselves. In TG effect studies, the duration of exposure over 24 h is necessary to ensure that the exposure time covers the embryo formation of the next generation, and the duration should be within 96 h to facilitate the subsequent generation separation. Use small amounts of nematodes (usually within 20) for measuring the lifespan and reproduction. On the other hand, use large amounts of nematodes (usually more than 500) for measuring biochemical and genetic regulation indices. Hence, in order to ensure sufficient number of samples, perform preliminary experiments to roughly estimate how many offspring the mature F0 nematodes can reproduce within the first 24 h since they start reproduction. Then determine the number of F0 nematodes required to ensure there are at least 200 offspring for the proceeding multi-generational studies.

As compared with earlier reports of TG studies with C. elegans, the present experimental protocol was more considerate of the choice of life stage. In C. elegans, sperms are formed at L4 stage to fertilize the later-formed oocytes²³. Accordingly, the exposure covering the spermiogenesis and oocytogenesis period will provide a particular window to study the TG effects on the offspring. The age-synchronized eggs are used for multi-generational effect studies to ensure that the exposure covers the overall period from the beginning of each life cycle. Compared with earlier multi-generational studies, the present experimental protocol facilitated the measurement of effects over multiple generations instead of only 1-2 generations. Moreover, the present protocol considered both MGE and MGR effects, which is more systematic than earlier studies that only measured MGE or MGR effects.

Notably, there are still some issues to be considered in the present experimental protocol. The present protocol employs wild-type C. elegans whose generation time is around 60 h and lifespan is 20 days. This makes the overall duration of the experiment fairly long (e.g., MGE effects study on lifespan over 3 generations requires at least 30 days). In order to shorten the time, researchers can choose mutant C. elegans, such as the short-lived mutant nematodes. Another issue is the killing treatment on the bacteria, the live status of which is necessary to keep nematodes healthy ²⁴. Also, the UV killing process might introduce changes in the chemicals²⁵. Therefore, other treatments on the bacteria should be considered, and careful monitoring on the chemical changes during the preparation or exposure process may be necessary, especially for unstable compounds. At the same time, there are limitations in studying the sex differences in toxic effects because most of the fact that C. elegans is hermaphrodite. Further improvements to investigate the sex contribution in the TG, MGE or MGR effects are needed. In summary, we anticipate that the proposed protocol will be great significance for using C. elegans to study TG, MGE and MGR effects of toxicants.

Disclosures

The authors have nothing to disclose.

Materials

agar powder	OXOID, Thermo Fisher Scientific, UK	9002-18-0
79nnHT Fast Real-Time PCR System	Applied Biosystems
96-well sterile microplate	Costar，Corning，America
Autoclave sterilizer	Tomy, Tomy Digital Biology, Japan
Biosafety cabinet	LongYue, Shanghai longyue instrument equipment co. Ltd, China
calcium chloride	Sinopharm chemical reagent company Ltd, China	10043-52-4
centrifuge 5417R	Eppendorf, Ai Bende (Shanghai) International Trade Co., Ltd, Germany
Centrifuge tubes	Axygen, Aixjin biotechnology (Hangzhou) co. Ltd, America
cholesterol	Sinopharm chemical reagent company Ltd, China	57-88-5
Dimethyl sulfoxide	VETEC, Sigmar aldrich (Shanghai) trading co. Ltd, America	67-68-5
disodium hydrogen phosphate	Sinopharm chemical reagent company Ltd, China	7558-79-4
ethanol	Sinopharm chemical reagent company Ltd, China	64-17-5
Filter	Thermo, Thermo Fisher Scientific, America
incubator	YiHeng17, Shanghai yiheng scientific instrument co. Ltd, China
inoculating loop
K₂HPO₄•3H₂O	Sinopharm chemical reagent company Ltd, China	16788-57-1
kraft paper
Mcroplate Reader	Boitek, Boten apparatus co. Ltd, America
MgSO₄•7H₂O	Sinopharm chemical reagent company Ltd, China	10034-99-8
Microscopes XTL-BM-9TD	BM, Shanghai BM optical instruments manufacturing co. Ltd, China
Petri dishes
Pipette	Eppendorf, Ai Bende (Shanghai) International Trade Co., Ltd, Germany
Potassium chloride	Sinopharm chemical reagent company Ltd, China	7447-40-7
potassium dihydrogen phosphate	Sinopharm chemical reagent company Ltd, China	7778-77-0
Qiagen RNeasy kits	Qiagen Inc., Valencia, CA, United States
QuantiTect SYBR Green RT-PCR kits	Qiagen Inc., Valencia, CA, United States
RevertAid First Strand cDNA Synthesis Kit	Thermo Scientific, Wilmington, DE, United States
sodium chloride	Sinopharm chemical reagent company Ltd, China	7647-14-5
sodium hydroxide	Sinopharm chemical reagent company Ltd, China	1310-73-2
sodium hypochlorite solution	Aladdin, Shanghai Aladdin biochemical technology co. Ltd, China	7681-52-9
tryptone	OXOID, Thermo Fisher Scientific, UK	73049-73-7
yeast extract	OXOID, Thermo Fisher Scientific, UK	119-44-8

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Li, Z., Ai, F., Zhang, J., Yu, Z., Yin, D. Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants. J. Vis. Exp. (149), e59367, doi:10.3791/59367 (2019).

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