Figure A1: The experimental procedure of the evolution experiment in
detail.
The procedure of the evolution experiment can be seen in Figure S1 and
will be the following:
- On the first day of each generation, worms were bleached(Stiernagle,
2006). During this step worms were removed from the plates by washing
with M9 buffer. Afterwards a 50:50 mixture of 10% NaClO and 5M NaOH
was used to bleach all the remaining bacteria in the solution and to
release eggs from bleached adult worms. Only eggs survive this step
and were then left in the M9 buffer over-night. All bleached eggs
hatched overnight, but do not develop any further and thus lead to
increased synchronisation for each worm population. Every bleach
solution was plated out on a TSA plate to control for carried over
contaminations. 100 colonies of E. faecalis were picked from
the plates that were grown on E. faecalis selective medium for
48 hours at 30°C. These colonies were picked into 600μl of THB and
then grown up over-night. Simultaneously, a single colony ofSalmonella food was picked and added to 25ml of LB broth to be
grown under shaking conditions over night.
- On the second day of each generation, the overnight shaken and hatched
worms were exposed to 600μl of a 50:50 mixture of E. faecalisand food. At this step the population size was adjusted to only
contain 1000 individuals. Worms remained on these plates with E.
faecalis for 48 hours.
- On the third day of each generation, TSA plates to which worms were
going to be exposed on day 4 were inoculated with 100μl of eitherS. aureus or food and were incubated at 30°C overnight.
- On the fourth day of each generation, worms were washed off the plates
seeded withE. faecalis by filter tip washing. For this purpose, worms were
washed off the plates with twice 1.5ml of M9 buffer +1% Triton X100,
as previously described(Jansen et al., 2015; Papkou et al., 2019;
Rafaluk-Mohr et al., 2018).This worm and bacteria suspension was spun
down for 1 minute, 1 ml of the supernatant was discarded and the rest
of the pellet was pipetted on the top of a filter of a filter tip and
spun down for 3 min. Worms were left on the top of the filter were
washed with 400μl of M9 three times, before being re-suspended in
100μl of M9 to bring onto plate. During this method, most of the
externally attached bacteria are washed off the worms to ensure that
worm survival can be attributed to gut colonization of E.
faecalis and not external attachment of the protective microbe.Enterococcus faecalis remains in the worm’s intestine and will
establish a protective effect. After worms were transferred to the
plates containing either S. aureus or food, all plates were
moved to 25°C.
- On the fifth day of each generation, worms were washed off the plates
again by filter tip washing (as described for day 4 and previously
(Jansen et al., 2015; Papkou et al., 2019; Rafaluk-Mohr et al.,
2018)). Worms were left on plates seeded with food at 20°C for 48
hours to lay eggs. The amount of transferred bacteria (either S.
aureus or food can be neglected to have any influence on the further
development of the worms. These plates were then used for bleaching on
day 1 of the following generation. 10% of the worm mixture was
separated and used to isolate E. faecalis . For this purpose,
the suspension of worms was crushed and then plated on TSA plates with
Rifampicin, as the E. faecalis strain carries a Rifampicin
resistance. The plated gut content was allowed to grow at 30°C for 48
hours.
Statistical results:
Table A1: All statistical results summarized, including the statistical
test, the specifics associated with each test, the relevant degrees of
freedom and p-values.