Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

This protocol describes how to raise worms and compost microcosms, enabling the in-lab exploration of host-microbiome interactions in a natural-like context. This method offers an alternative to isolating wild worms from nature or using synthetic microbial communities of limited microbial diversity. This protocol is straightforward and can be performed in any lab and is even suitable for beginners.

To begin, obtain compost or garden soil from any convenient source and store it inside the laboratory in a standard kitchen plastic container with holes cut in the lid for aeration. Plug the holes with cotton wool to keep fruit flies and other invertebrates out. Enrich the compost or soil with chopped produce or a mixture of different produce in the soil to produce a ratio of two to one by mass.

Mix the compost once a day and add M9 medium as required to maintain moisture without making it muddy. For each microcosm, add 10 grams of enriched compost to a 30 milliliter glass beaker covered with tinfoil. Add 30 grams of enriched compost to each 50 milliliter tube.

Fill the tube with M9 and vortex. Centrifuge the tubes at 560 G for five minutes at room temperature. Using a serological pipette, remove the supernatants without disturbing the pellet and combine them in a new 50 milliliter tube.

Concentrate the bacterial extract by centrifuging the tube at maximum speed for 15 minutes at room temperature. Resuspend the pellets in enough M9 to have 200 microliters for each microcosm and 200 microliters for a plate that will serve as a visible proxy for microcosms. Add 200 microliters of the concentrated microbial extract to the autoclaved compost beaker and the visible proxy NGM plate.

Add 500 to 1000 L1 larvae to each microcosm and the proxy plate. Line a cylindrical PVC pipe with tissue paper. This cylinder should have a one millimeter nylon mesh glued at its bottom in the funnel.

Place the cylinder in the Baermann funnel sitting in a flask. Add 20 milliliters of M9 to the worm microcosm. Agitate the mixture and then pour the mixture from the beaker into the tissue-paper-lined cylinder in the Baermann funnel setup.

Submerge the compost in the funnel entirely by adding more M9.Unfasten the clamp to release the filtrate containing the harvested worms into a 50 milliliter tube. Then concentrate the worms by centrifuging the tube at 560 G for two minutes at room temperature. Repeat these steps and pull the filtrates from different rounds if more worms are desired.

Remove the supernatant with a serological pipette while leaving 15 milliliters in the tube. Transfer the remaining liquid to a 15 milliliter tube and centrifuge again for one minute to further concentrate the worms. Remove 14 milliliters of the supernatant with a serological pipette.

Simultaneously, collect one gram of the remaining microcosm soil into a 1.5 milliliter tube while processing the soil samples containing the environmental bacterial community as described in the manuscript. Transfer one milliliter of the concentrated harvested worms to a 1.5 milliliter tube using a glass pipette. Incubate for two minutes to allow the worms to settle at the bottom of the tube.

Remove the supernatant while leaving 100 microliters of the pellet undisturbed at the bottom of the tube. Wash the pellet with 1.5 milliliters of M9 plus Triton X, allowing the worms to settle at the bottom each time. Transfer the washed worms in a volume of 100 microliters to a new 1.5 milliliter tube using a glass pipette.

To paralyze the worms, add 100 microliters of 25 millimolar levamisole hydrochloride and incubate for five minutes at room temperature. Add 200 microliters of 4%bleach solution and incubate for two minutes. Remove the supernatant leaving the bottom-most 150 microliters undisturbed, and wash the worm pellet thrice with M9 plus Triton X.These surface-sterilized worms can be stored at minus 20 degrees Celsius until use and can be used later for downstream applications.

Comparing the worm gut microbiomes and environmental communities using principle coordinate analysis based on unweighted or weighted UniFrac distance showed distinct clustering of worm gut microbiomes away from those in their respective environments. While principle coordinate analysis based on unweighted UniFrac distances did not distinguish between soil and worm microbiomes, clustering based on weighted distances revealed a clear separation of worm gut and compost microbiomes. In this study, worms raised in compost microcosms were used for 16S sequencing, but also can be used to test the effects of different environmental microbiomes on host resistance to adverse conditions.

Alternatively, novel bacterial taxa can be isolated from worms harvested from compost microcosms in order to expand the known taxonomic and functional diversity of worm gut commensals. After its development, the microcosms pipeline facilitated the assessment of the contributions of environmental versus host genetic factors in shaping the gut microbiome.