Preparing clean DNA from Daphnia

Daphnia with bacteria

Daphnia are usually associated with large numbers of microorganisms. These EM pictures show bacteria on our main study organism, Daphnia magna. Picture by Frida Ben-Ami.

The problem

Using shotgun sequencing approaches requires DNA samples with a high degree of purity, as otherwise many reads will be of other species, mainly microbes from the microbiota and gut content. If Daphnia are not cleaned before DNA extraction, the shotgun sequences may contain up to 80% of reads from bacteria. This means you pay 5 times more for the same amount of Daphnia reads, not mentioning the bioinformatic nightmare which comes along when you have to exclude the reads from a highly diverse (up to 500 species!) microbiota. Here is a protocol used in our lab for several applications which in the best case has reduced contaminants to less than 1% of the sequenced reads. Since the protocol is labour intensive and requires some experience with handling Daphnia, you may decide to take short cuts, but this comes at a cost of having more contamination.

The principle

The basic idea of the protocol is to reduce all sources of non-Daphnia DNA associated with your Daphnia sample. These are microbes growing on the body surface (see picture) and microbes, food and detritus in the gut. Furthermore, you want to reduce microbes in the culture medium. The protocol combines different methods to achieve this.

The method

In order to eliminate surface bacteria adult and well-conditioned females are maintained in antibiotic medium throughout one molting cycle (about 3.5 days for D. magna at 20C). The antibiotic medium contains a mix of equal amounts of Streptomycin, Tetracycline, and Ampicillin, disolved at 50mg/L in the Daphnia medium (we use ADaM, but other medium will do as well). The antibiotic solution has to be vigorously mixed for 20-30 min due to low solubility of tetracycline. Antibiotic solution, protected from light, can be stored in the fridge for 15 days. All of the antibiotics are light sensitive thus their effects significantly decline within 24 hours. For that reason, animals should be transferred every day into the fresh antibiotic medium. And to keep the medium clean during those 24 hours it is recommended to keep not more than 3 animals in the small jars (80 - 100 mL) jars. A large source of contamination comes from the gut content. For that reason the animal's gut should be emptied prior to DNA extraction. This is achieved by feeding Daphnia with sterile beads (Sephadex G-25, cross-linked dextran gel) beads during the antibiotic treatment. A stock mixture of the beads is prepared by adding about 0.5g of beads (it looks like a powder) per 100 mL of sterile Daphnia medium. If kept sterile and stored in the fridge, this mixture can be used during the whole treatment. Starving without feeding with beads will not reduce the gut content efficiently, as gut content will remain in the lower part of the gut. Therefore, it is important that animals have constant access to the beads in their medium. Since Sephadex beads sink to the bottom of the jar, it is recommended to feed the animals several times a day (3 times works well) with small amounts (200-400 uL) of the stock. After 3 days there should be no visible food leftovers in the intestine. Animals that have empty guts and that have molted at least once are ready to be sampled for DNA extraction.

Materials used

What Comment concentration
Teracycline >98%, use hydrochloride form 50 mg/L
Streptomycin use as Strep. sulphate 50 mg/L
Ampicillin use as sodium salt 50 mg/L
Sephadex beads G-25, dry bead, cross-linked dextran gel, diameter 20 to 50 um about 5g/L

Some notes on how improve your treatment:

  • Keep animals in the dark. The antibiotics (in particular Tetracyclin) are light sensitive.
  • Produce sterile ADaM to begin with. Either autoclave or sterile filter the medium (0.45um or 0.2um filter).
  • Keeping several small jars with few Daphnia worked better for us then mass cultures.
  • We used this protocol with Daphnia magna. Other species may be more sensitive and may not be able to handle triple antibiotic treatment.

This protocol was developed by Isabele Colson, Jarkko Routtu and Marinela Dukic

PS: The better you work, the less DNA you will have in the end. Don't be surprised!