Name: using caenorhabditis elegans for studying trans- and multi-generational effects of toxicants
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1. Culture E. coli OP50
<ol>
	<li>Prepare 1 M sodium hydroxide solution by dissolving 4 g of sodium hydroxide in 100 mL water.</li>
	<li>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.</li>
	<li>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 conica...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenerational+comparison+of+developmental+and+reproductive+toxicities+in+zearalenone+exposed+Caenorhabditis+elegans.">Transgenerational comparison of developmental and reproductive toxicities in zearalenone exposed Caenorhabditis elegans.</a> Asian Journal of Ecotoxicology. 11 (4), 61-68 (2016).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+dlegans.">The genetics of Caenorhabditis dlegans.</a> Genetics. 77, 71-94 (1974).</li><li>Emmons, S., Klass, M., Hirsch, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+the+constancy+of+DNA+sequences+during+development+and+evolution+of+the+nematode+Caenorhabditis+elegans.">An analysis of the constancy of DNA sequences during development and evolution of the nematode Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences of the United States of America. 76, 1333-1337 (1979).</li><li>Van Gilst, M. 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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...

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 &#176;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.

<table border="0" cellpadding="0" cellspacing="0" style="width:684px;" width="684">
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		<tr height="37">
			<td height="37" style="height:37px;width:191px;">&#160;agar powder</td>
			<td style="width:200px;">OXOID, Thermo Fisher Scientific, UK</td>
			<td style="width:123px;">9002-18-0</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:191px;">79nnHT Fast Real-Time PCR System</td>
			<td>&#160;Applied Biosystems&#160;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">96-well sterile microplate</td>
			<td style="width:200px;">Costar&#65292;Corning&#65292;America</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Autoclave sterilizer</td>
			<td style="width:200px;">Tomy, Tomy Digital Biology, Japan</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">Biosafety cabinet</td>
			<td style="width:200px;">LongYue, Shanghai longyue instrument equipment co. Ltd, China</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">calcium chloride</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>10043-52-4</td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">centrifuge&#160; 5417R</td>
			<td style="width:200px;">Eppendorf, Ai Bende (Shanghai) International Trade Co., Ltd, Germany</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Centrifuge tubes</td>
			<td style="width:200px;">Axygen, Aixjin biotechnology (Hangzhou) co. Ltd, America</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">cholesterol</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td style="width:123px;">57-88-5</td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">Dimethyl sulfoxide</td>
			<td style="width:200px;">VETEC, Sigmar aldrich (Shanghai) trading co. Ltd, America</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">disodium hydrogen phosphate</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td style="width:123px;">7558-79-4</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">ethanol</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td style="width:123px;">64-17-5</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Filter</td>
			<td style="width:200px;">Thermo, Thermo Fisher Scientific, America</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">incubator</td>
			<td style="width:200px;">YiHeng17, Shanghai yiheng scientific instrument co. Ltd, China</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">inoculating loop</td>
			<td style="width:200px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">K2HPO4&#8226;3H2O</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>16788-57-1</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">kraft paper</td>
			<td style="width:200px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Mcroplate Reader</td>
			<td style="width:200px;">Boitek, Boten apparatus co. Ltd, America</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MgSO4&#8226;7H2O</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>10034-99-8</td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">Microscopes XTL-BM-9TD</td>
			<td style="width:200px;">BM, Shanghai BM optical instruments manufacturing co. Ltd, China&#160;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Petri dishes</td>
			<td style="width:200px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">Pipette</td>
			<td style="width:200px;">Eppendorf, Ai Bende (Shanghai) International Trade Co., Ltd, Germany</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Potassium chloride</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">potassium dihydrogen phosphate</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>7778-77-0</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Qiagen RNeasy kits</td>
			<td style="width:200px;">Qiagen Inc., Valencia, CA, United States</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:191px;">QuantiTect SYBR Green RT-PCR kits</td>
			<td style="width:200px;">Qiagen Inc., Valencia, CA, United States</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:191px;">RevertAid First Strand cDNA Synthesis Kit</td>
			<td style="width:200px;">Thermo Scientific, Wilmington, DE, United States</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">sodium chloride</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">sodium hydroxide</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">sodium hypochlorite solution</td>
			<td style="width:200px;">Aladdin, Shanghai Aladdin biochemical technology co. Ltd, China</td>
			<td style="width:123px;">7681-52-9</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">tryptone</td>
			<td style="width:200px;">OXOID, Thermo Fisher Scientific, UK</td>
			<td>73049-73-7</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">yeast extract</td>
			<td style="width:200px;">OXOID, Thermo Fisher Scientific, UK</td>
			<td>119-44-8</td>
			<td style="width:171px;"></td>
		</tr>
	</tbody>
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using caenorhabditis elegans for studying trans- and multi-generational effects of toxicants

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.

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

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.

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

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

The presented protocol will facilitate studies on the trans and the multi-generational effects of proteins that can exist for a long time in the environment. We employed a free-living nematode with 99.5 as hermaphrodites to save time and effort in studying trans and multi-generational effects. This technique can be used to demonstrate the long-term impacts of end drugs because the nematode in the present protocol shares many conservative mechanisms or pathways with humans.This method can provide insight to the fields of pharmaceutical synthesis and environmental management because they need feedback of potential hazards of chemicals. This method can be applied to Daphnia magna with more focus on aquatic toxicity. Also Drosphila melanogaster to further explore influence on gender.He or she would be stuck with idea that studying effects over generations is time and energy-consuming. Don't get frightened at first impression. Following the protocol will earn you fruitful outcome.To culture E.coli, pipette 200 microliters from the bacterial suspensions into 50 milliliters of LB medium in a flask. Or use an inoculating loop to pick a small colony from NGM agars and place it in the LB medium. To culture C.elegans, under a microscope, count the nematodes on the stacking NGM agars prepared earlier.When the number reaches close to 2, 000, use a sterile tip to cut 1/6 of the NGM agar and transfer it onto newly prepared NGM agars with bacterial lawn. Keep the NGM agars upside down in a 22 degree Celsius incubator for the subsequent culture. After about 36 hours, the nematodes become gravid.Use two milliliters of sterile water to flush off the gravid nematodes and newly-produced eggs on each NGM agar into sterile centrifuge tubes. Place the tubes on a rack for 30 minutes to settle down the nematodes, and then discard 85%of the supernatants by pipetting. It's very important to judge whether the nematodes are gravid enough for synchronization.Otherwise, the age-synchronization operation would be in vain. Mix the pellets with seven volumes of sodium hypochlorite solution and shake the centrifuge tubes every two minutes for five to eight times to lyse the larvae and adult nematodes. Centrifuge the tubes at 700 times G for three minutes at 20 degrees Celsius and then discard the supernatants by pipetting.Re-suspend the pellets in five volumes of sterile water to wash the age-synchronized eggs. Centrifuge at 700 times G for three minutes at 20 degrees Celsius. Repeat the wash by sterile water, twice.Next, add one volume of sterile water into the tubes to re suspend the age-synchronized eggs. Distribute the egg suspensions by pipetting fifty micro liters onto each new NGM agars with bacterial lawn. Keep the NGM agars top side up in a 20 degree Celsius incubator for 30 minutes, allowing the water to evaporate or be absorbed by the bacterial lawn.Then, turn over the NGM agars, upside down, for the subsequent culture, and mark the time as the egg time. 36 hours after the egg time, the nematodes reach the L3 larvae stage. Use two milliliters of sterile water to flush the nematodes off each NGM agar into centrifuge tubes.After settling for 30 minutes, replace 85%of the supernatants with K Medium to digest the food in the guts for two hours. Then, discard the supernatants. Use K Medium to adjust the nematode suspensions to about 200 nematodes per 100 microliters for subsequent experiments.To avoid edge effects, in the middle area of a 96-well sterile microplate, add 100 microliters of prepared control or chemical solutions into 10 wells, as replicates in six groups. Add 100 microliters of the prepared nematode K Medium to each of the 60 wells. Mark the time as T0.Perform the exposure for 24 to 96 hours.After the exposure, use a pipette to collect nematodes from five wells in each group, into six 1.5 milliliter centrifuge tubes. Settle the nematodes for 30 minutes and then pipette to discard the supernatants. Wash the nematodes at the bottom with one milliliter of sterilized water.Settle the nematodes for 30 minutes. Discard the supernatants, and use the nematodes at the bottom for indicator measurements of the exposed parent generation, marked as F0.Collect the nematodes from the remaining five wells in each group. Settle and wash the nematodes as previously.Next, add 100 microliters of sterile water to re-suspend the nematodes in each tube, and transfer the nematode suspensions evenly onto three newly prepared NGM agars with bacterial lawn. Incubate the nematodes for 36 hours to become gravid, and perform age-synchronization, as previously. After incubating the synchronized eggs on NGM agars with bacterial lawn for 36 hours, flush the nematodes into six centrifuge tubes.After settling and washing with one milliliter of sterilized water, use the nematodes at the bottom for indicator measurements of the offspring generation marked as T1.Mix 99 milliliters of the warm NGM agars with one milliliter of control or chemical solutions. After preparing the agars with bacteria suspensions according to the manuscript, in the bio-safety cabinet, set aside the upper lids of the petri dishes and expose the bacterial lawn to UV light for 15 minutes. Pipette the age-synchronized eggs onto the UV-treated agars.Mark the start of the exposure to the parent generation, F0, as day zero. Incubate the agars for three days at 20 degrees Celsius. Then, use the mature nematodes to measure effects in F0.Also, on day three, use a glass rod fitted with a man-made fiber wire bent into a ring to pick F0 mature nematodes, and slide onto the surface of new UV-treated NGM agars.On day four, pick out and discard the mature F0 nematodes from NGM agars. On day six, use the F1 mature nematodes that have experienced exposure for three days for indices measurement. In this protocol, the trans-generational effects of cadmium, copper, lead, and zinc on the body-bending frequency showed that the inhibitions in the nematode parent, F0, after prenatal exposure were less than in their progeny, T1.The multi-generational residual effects of sulfamethoxazole on the nematode lifespan and reproduction showed that the reproduction was significantly affected in germ-line exposure, and the toxicities persisted in non-exposed generations from T3 to T6 generations.Multi-generational exposure and multi-generational residual effects of lindane on the nematode biochemical and genetic indices showed obesogenic effects with disturbances in the insulin signal regulation. Moreover, the changes between sgk-1 and akt-1 signaling indicated that nematodes from different exposure generations showed different response strategies for tolerance and avoidance. Check the living status of the nematodes before each operation, and modify following steps accordingly.Other effects, for example, obesogenic effects of chemicals, can be included to further answer the increasing prevalence of obesity over generations. Based on the method, further mechanisms can be explored. For example, the heritable epigenetic signals between generations.The chlorine solutions have strong oxidatative potentials, and avoid direct contact of skin to it.

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