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.
