Table 1: Experimental procedure for the evolution experiment. Columns
indicate the number of experimental host generations (1- 20), while rows
show the eight treatments. Host generations were infected with S.
aureus (purple) or given food (green), while constantly coevolving withE. faecalis. Two controls for lab effects on host evolution (dark
brown, No Protective Microbe, NPM) and E. faecalis evolution
(light brown, No Host Control, NHC) were also included, where the NPM
treatment was only ever exposed to food alone. Each evolutionary
treatment consisted of six independent evolutionary replicates.
Experimental evolution - Culturing and passaging
methods
At the start of each generation, worms were bleached as described
previously and left in M9 buffer overnight for larvae to hatch
(Stiernagle, 2006). Simultaneously, E. faecalis clones were
cultured overnight in Todd-Hewitt Broth (THB) in 600µl at 30ºC, while
food was cultured overnight in LB broth. Subsequently, 9cm NGM plates
were inoculated with 300µl of each overnight culture. Plates with
freshly inoculated bacteria were dried at room temperature before
approximately 1000 L1 worms were added to each NGM plate. After these
plates dried at room temperature, they were transferred to a 20ºC
incubator and left for 48h. Simultaneously, a liquid culture of S.
aureus was grown in THB from frozen stock, while a liquid culture of
food was grown in LB, and both were incubated under shaking conditions
at 30ºC. The following day, 100µl of each overnight culture were spread
on 9cm plates, S. aureus on Tryptone Soy Broth agar (TSB) plates
and food on NGM plates and incubated at 30ºC overnight. To transfer
worms to the pathogen or food plates, nematodes were washed off theE. faecalis plates with M9 buffer and washed three times over
small-pore filters to remove all externally attached bacteria, as
previously described (Jansen et al., 2015; Papkou et al., 2019;
Rafaluk-Mohr et al., 2018). Worms were infected with either S.
aureus or exposed to food (Table 1) and left at 25ºC for 24h. After
this time, worms were then washed off the plates with M9 buffer once
more to plate them on NGM plates seeded with food for laying eggs.
Roughly 10% of these worms were crushed and plated on E.
faecalis selective medium (TSB + 100mg/ml Rifampicin). The remaining
worms were left on food plates for 48h to allow for egg laying.
To passage E. faecalis , roughly 100 E. faecalis colonies
were picked and grown up shaking overnight in 600µl THB at 30ºC, while
worms were bleached and left to hatch overnight. This cycle was repeated
for 20 experimental host generations.
All passaged worms and E. faecalis samples were cryopreserved at
-80 ºC. A proportion of the offspring of surviving worms were frozen in
40% DMSO, and 100µl of E. faecalis liquid culture was mixed with
100µl of glycerol before cryopreservation.
Host survival and fecundity assays
All assays were conducted at the end of the evolution experiment on
archived samples. Plates were randomized and fully encoded during each
experiment to ensure the experimenter was blind to different treatments
whilst collecting data.
Basic procedures were adopted from the experimental evolution, but with
the following alterations to keep the assays feasible with higher
accuracy when scoring dead and alive worms: 400 L1 worms were exposed to
200µl of food and E. faecalis on 6cm NGM plates, while 60µlS. aureus overnight culture was used to inoculate 6cm TSB plates.
To assess microbe-mediated protection of different combinations of worms
and E. faecalis , 400 L1s were exposed to 50:50 mixtures ofE. faecalis and food for 48h. Worms were then washed off these
plates as described above and infected with S. aureus for 24h at
25°C. Survival in form of counting dead and alive worms was then scored.
To assess any long-term fitness consequences after protective microbe
exposure and pathogen infection, long-term survival and fecundity were
measured. Worms were exposed as described for the survival assays.
Subsequently, five females and five males were picked onto 3cm food
seeded NGM plates at 25°C and then transferred to new plates every 36h
to avoid any confusion between offspring produced and original adults.
At each time point, survival was scored. To measure fecundity, the
number of worm eggs on the plates at 120h since bleaching were counted.
Statistical Analysis
Statistical analyses were carried out with RStudio (Version 1.1.463 for
Mac), graphs created with the ggplot2 package (Version 2.1.0) and edited
with Inkscape (Version 0.91). All host survival and fecundity data were
analysed with nested binomial mixed effects models (R package lme4),
followed by a Tukey multiple-comparison tests (R package multcomp).
Life-span data were analysed with Kaplan Meier Log Rank test with FDR
correction for multiple testing.