TITLE: DIY Intervention Testing in Daphnia: A simplified way to test lifespan interventions at home
AUTHORS: Cora Anderson, Benyamin Matei-Dediu, Edouard Debonneuil, Rachael A Jonas-Closs, Leonid Peshkin

[DESCRIPTION]
This guide is for amateur science enthusiasts who are looking to conduct safe and easy animal experiments at home. This can be done with the aim of educating yourself and others about ecology, aquaculture, and pharmaco-biology. Additionally, by using a safe and accessible organism, you could contribute effort and data to the community science movement. We provide accessible ways for obtaining Daphnia, keeping daphnids at home, conducting experiments, troubleshooting, recording parameters of health and lifespan, and reporting the results. You will be able to begin immediately and build up gradually. A typical experiment might take 4-8 hours a week and last 1-2 months. The basic setup cost is around $50, depending on how food and equipment are sourced. We strongly encourage the reader to contact us with any suggestions and feedback to help further improve and develop this guide.

[STEPS]
SECTION: Grandmother culture generation, your Daphnia reserve
1. First steps

 

SECTION: Grandmother culture generation, your Daphnia reserve
2. Once the water begins to turn, green Daphnia may be added. 
 
 
SECTION: Grandmother culture generation, your Daphnia reserve
3. Once the grandmother culture is established and stabilized the only regular water quality maintenance required is to compensate for evaporation by adding rain or distilled water to the original level.
9. Feeding

 

SECTION: Mother generation, the experimental set up
4. Once your grandmother culture is stabilizied, you can start the experimental set up. 

 

SECTION: Data collection workflow
14. 
 

SECTION: Mother generation, the experimental set up
6. Identify and remove three large females, preferably ones with neonates visible in the brood chamber.
SECTION: Mother generation, the experimental set up
7. Isolate each of these females in their own container.

SECTION: Mother generation, the experimental set up
8. Feed these females spirulina every other day and complete 100% water changes on the other days.
 

SECTION: Start testing drugs !
19. 
 
 



9.1. Create a mixture of 1 tsp (3g) freeze-dried spirulina in 1/3c (75mL) water (scale up proportionally). 

 

9.2. Mix again and immediately pour into a silicone tray for making small ice cubes and then freeze.
9.3. A few hours before feeding the required amount of cubes can be thawed and remixed to ensure a constant concentration of food is used for the duration of your experiments. 

 
 
9.4. Check if there's uneaten food collects at the bottom, if so, try feeding less.
 


10. Water Changes and Removal of offspring 

10.1.  

SECTION: Daughter cultures, the execution of the experiment
11. Before starting the experiment, these conditions have to be checked:

The “mother generation” containers contain 1-2 mothers that can have clutches of 5-30 neonates each. (3.d)
10 mL per individual Daphnia so the volume of experimental containers will be the number of individuals multiplied by 10mL to make sure you have the correct number for your container or the correct container for the number. (3.b) We recommend only filling the volume of the container ⅔ of the way so that the media is not easily spoiled when the containers are being moved.
In longevity experiments, adequate statistical power for conditions that may extend the life span by 10% or more typically require at least 50 individuals per group when followed throughout their lifespan (more is better, statistically). Consider this when setting up the mothers for your experiment.

 

SECTION: Daughter cultures, the execution of the experiment
12. Synchronizing age
 
 

SECTION: Daughter cultures, the execution of the experiment
13. Removal of offspring without water changes

 

SECTION: Data collection workflow
15. Life span
SECTION: Data collection workflow
16. Fecundity

 

SECTION: Data collection workflow
17. Physical changes
SECTION: Data collection workflow
18. 
Behavioral changes
 

SECTION: Start testing drugs !
21. Find your drug's toxic dose in literature

SECTION: Start testing drugs !
22.  Short-term toxicity test: Find your EC048
SECTION: Start testing drugs !
23.  Prolonged toxicity test: Finding your EC07 Days 

Do the same steps as for your EC048h but you leave your experimetal group exposed to the drug for 7 days.

SECTION: Start testing drugs !
24.  Full scale longevity testing

 

SECTION: Grandmother culture generation, your Daphnia reserve
1.1. Before introducing Daphnia, take a large spring water jug and cut off the top in order to have a light transparent bucket with a side handle. 

 



SECTION: Grandmother culture generation, your Daphnia reserve
1.2. Add a drop/granule of any plant mineral fertilizer to generate “green water” which will serve both as food and provide autogenous biological filtration. 
10.2. Check the container to ensure that no neonates have been transferred accidentally.

 

10.3. Once individuals in this group begin to show signs of sexual maturity you may split them and house them individually or in pairs. 

 

SECTION: Daughter cultures, the execution of the experiment
12.1. Try to pull females at the same egg stage to have a better chance of synchronizing neonate emergence.
SECTION: Daughter cultures, the execution of the experiment
12.2. Overfeed these mothers and perform water changes at least every 3 days (so waste wont build up from the increased feeding), this will lead to larger, more frequently produced and healthier clutches.

 

SECTION: Daughter cultures, the execution of the experiment
12.3. Let the clutches reach sexual maturity to confirm that they are all female. 

 

SECTION: Daughter cultures, the execution of the experiment
12.4. 
 

SECTION: Daughter cultures, the execution of the experiment
12.5. Reach the  ideal density in the experimental groups of 10mL of water per daphnid. 
 

SECTION: Daughter cultures, the execution of the experiment
13.1.  
SECTION: Data collection workflow
18.1. Check if the Daphnia are swimming differently, less or more.
SECTION: Data collection workflow
18.2. Check if an experimental group increased or decreased feeding rate compared to the control.
SECTION: Data collection workflow
18.3. Check if the Daphnia are producing ephippia/resting eggs.
SECTION: Data collection workflow
18.4. Check if the Daphnia started staying on the bottom of the jar or if they are occupying any specific vertical area in the jar. 
SECTION: Data collection workflow
18.5. Check if the Daphnia are still responding to light.
SECTION: Start testing drugs !
20. Selecting and storing drugs

 


SECTION: Start testing drugs !
20.1. Search online for a drug's water solubility. 
SECTION: Start testing drugs !
20.2. If your drug is in tablet form then thoroughly crush it before trying to get it into solution.

 

SECTION: Mother generation, the experimental set up
5. How to choose containers for the experiment

 

SECTION: Daughter cultures, the execution of the experiment
13.2.  
SECTION: Daughter cultures, the execution of the experiment
13.3.  
 

SECTION: Daughter cultures, the execution of the experiment
13.4.  
SECTION: Data collection workflow
17.1. 

 

SECTION: Data collection workflow
17.2. Check the color of the gut tract.

 

SECTION: Data collection workflow
17.3. Check the general shape of the Daphnia.

 

SECTION: Data collection workflow
17.4. Check if there's an abnormal proportion of males.

 

SECTION: Data collection workflow
15.1. Start with Daphnia born on the same day and ensure that the neonates from each mother are split evenly between groups, refer to step    to see how to achieve this best. 

SECTION: Data collection workflow
15.2.  
 

SECTION: Start testing drugs !
21.1. Check this paper if your drugs has a known EC50 and LC50 (or LD50) in Daphnia :
https://doi.org/10.1016/j.scitotenv.2020.143038
SECTION: Start testing drugs !
21.2. If your drug isn't listed, check the litterature for an available LC/EC 50 in Daphnia.
SECTION: Start testing drugs !
21.3. If you can't find it in the literature for Daphnia, check for another invertebrate or aquatic model (i.e. C. elegans and zebrafish).
SECTION: Start testing drugs !
21.4. If there's still no information, it is recommended that you perform at-home toxicology bioassays to help find your starting dose.
SECTION: Start testing drugs !
22.1. Collect a large group of neonates and split them evenly between seven containers.
 

SECTION: Start testing drugs !
22.2. Make 2 of these containers the controls and have the other five contain the LC50:
50% the concentration of the LC50
25% the concentration of the LC50
10% the concentration of the LC50
5% the concentration of the LC50
1% of the concentration of the LC50

SECTION: Start testing drugs !
22.3. Lightly feed your neonates and leave them in their solutions for 48 hours.
SECTION: Start testing drugs !
22.4. Check the containers after 48h and count the number of immobilized neonates. Your EC048 is the highest concentration at which there is no difference from the experimental to control group.
SECTION: Start testing drugs !
23.1. Chekc for animal reproductive maturation (when do they start producing ephippia or neonates) and note the day that each group becomes reproductive.
 

SECTION: Start testing drugs !
23.2. If the dose has animals reaching sexual maturity within 4 days of the control then it is an appropriate dose for a longevity experiment. Continue to maintain experimental conditions for the lifespan of the animal recording whichever findings are relevant to your investigation see section.
 

SECTION: Discussion and Future work
25. If you want to go a step further, you can do a smurf assay. 
For more information check: https://bio-protocol.org/pdf/bio-protocol2722.pdf
SECTION: Discussion and Future work
26. Sharing data is an important part of this open science project, guidelines will be provided soon so stay informed !