Materials and Methods
Worm host and bacteria
system
As a bacteriovore, Caenorhabditis elegans interacts constantly
with a variety of bacteria either by feeding or hosting them (Cabreiro
and Gems, 2013; Garsin et al., 2001; Schulenburg and Ewbank, 2004).
Consequently, C. elegans is an established model for studying
innate immunity (Gravato-Nobre and Hodgkin, 2005), as it can be infected
with its natural (Jansson, 1994; Schulenburg and Ewbank, 2004) as well
as opportunistic pathogens (Garsin et al., 2001; Tan et al., 1999). Most
pathogens are taken up orally by the worm (Marsh and May, 2012), and
some can proliferate and colonize the worm gut (King et al., 2016;
Rafaluk-Mohr et al., 2018).
Naturally, C. elegans is a self-fertilising hermaphrodite
(Brenner, 1974), but in this experiment obligate outcrossing worm
populations (line EEVD00) with males and females (hermaphrodites that
carry the fog-2(q71) mutation) were used (Theologidis et al.,
2014). This lineage was generated by Henrique Teotonio (ENS Paris) and
encompasses the genetic diversity of 16 natural worm isolates
(Theologidis et al., 2014). Worms were kept on Nematode Growth Medium
(NGM), inoculated with Salmonella , hereafter referred to as food.
Worms were infected with the pathogenic S. aureus (MSSA476)
(Holden et al., 2004), which is virulent and kills worm hosts by lysing
the intestinal cells lining the gut wall (Sifri et al., 2003). Worms
were exposed to E. faecalis (OG1RF) (Garsin et al., 2001), which
was isolated from the human digestive system, but has been previously
shown to colonize and proliferate in the host gut (Ford et al., 2017;
King et al., 2016; Rafaluk-Mohr et al., 2018), where it provides
protection.
Experimental evolution -
Design
Six single clones of E. faecalis (one for each of the six
replicate populations) and a single population of C. elegans were
the ancestors (hereafter referred to as the Ancestor) for all evolving
populations. To account for potential differences in virulence, a stock
of four clones of S. aureus was used for pathogen infections.
Both C. elegans and colonising E. faecalis were allowed to
evolve in presence of each other, while S. aureus was kept
evolutionarily static. Infection withS. aureus was varied over host evolutionary time (indicated by
purple in Table 1) to represent temporal heterogeneity in pathogen
infection, including a range from always to every 2ndgeneration, every 5th generation, and never (Table 1).
Moreover, we included differences in whether pathogens were present at
the initial formation of the symbiotic interaction or later (2.1. vs.
2.2., and 5.1. vs. 5.2. in Table 1). Controls for lab adaptation were
maintained for the host
(No Protective-Microbe control, NPM in Table 1) and E. faecalis(No Host Control, NHC in Table 1).