Background

Accurate measurements of fitness and changes in fitness over time are critical for determining a population’s response to natural selection. Nonetheless, fitness is notoriously difficult to measure because it incorporates an individual’s survival, fecundity, reproductive timing, and must account for ecological and environmental effects on individuals. Although no protocol for measuring fitness is optimal under all possible conditions, measures of fitness that account for survival and fecundity, while holding ecological and environmental effects constant, are likely to provide reliable overall estimates of fitness for a given scenario. Here we describe a protocol for measuring the relative fitness differences between C. elegans populations or strains and for determining the change in relative fitness over evolutionary time in the presence of a bacterial parasite. We utilized the gram-negative bacterium, Serratia marcescens, as a virulent parasite when consumed by C. elegans. Especially, S. marcescens strain SM2170 is capable of killing C. elegans hosts within 24 to 48 h of ingestion (Penley et al., 2017). This procedure makes use of Competitive Fitness Assays (CFAs) (Lenski et al., 1991; Wiser and Lenski, 2015), utilizing intraspecific competition to compare the relative fitness between different nematode populations or strains (Morran et al., 2009). Measurements of relative fitness, as determined via the CFA, incorporate survival and reproduction with intraspecific competition in a controlled environment to provide a comprehensive measure of fitness (Penley et al., 2017).

Relative fitness in the presence of the bacterial parasite is determined by competing a focal strain with an isogenic GFP-labeled tester strain over the course of one generation and measuring the reproductive success of the focal strain against the tester strain (Morran et al., 2009). Thus, this CFA accounts for survival against the parasite and host reproduction over the course of the nematode’s lifecycle. One single tester strain is used to measure the relative fitness of each focal nematode population or strain to facilitate comparisons of relative fitness between populations or strains. Importantly, the tester is marked with pharyngeal GFP to allow easy visualization of tester strain offspring versus focal population or strain offspring after one generation of competition. CFAs are initialized with a 50:50 mix of focal and tester strain individuals, and therefore, any deviation from 50:50 mix in the offspring indicates unequal competitive fitness between the focal and tester strains. An increase in the proportion of focal nematodes in the offspring indicates greater competitive fitness relative to the tester, while a decrease indicates reduced competitive fitness relative to the tester. The proportion of focal hosts in the offspring can be compared across multiple populations to measure the relative competitive fitness between focal strains or populations of interest. Importantly, competitive fitness measures are most effective when competing approximately equal numbers of individuals between two populations or strains with minor to moderate differences in competitive fitness. Uneven or variable starting ratios of strains or populations can confound measurements of relative fitness, while major differences in fitness between competing strains or populations are often difficult to accurately quantify (Wiser and Lenski, 2015).

This protocol is particularly useful for measuring evolutionary change after experimental evolution of C. elegans hosts in the presence of a bacterial parasite. First, the relative fitness of experimental host populations can be directly compared with the relative fitness of the ancestral population. Ancestral C. elegans populations can be stored at -80 °C during experimental evolution and then revived for CFAs to assess changes in the experimental population fitness over time (Gray and Cutter, 2014; Teotonio et al., 2017). Second, during experimental evolution, hosts may adapt to parasite exposure through altered life histories and/or increased levels of host defense. Therefore, measuring only survival in the presence of the parasite may not fully account for changes in host fitness. This CFA can account for changes in both life history and resistance that alter reproductive output in the presence of the parasite. Importantly, this procedure was originally developed to measure the change in C. elegans’ competitive fitness after multiple generations of evolution in presence of the bacterial parasite, Serratia marcescens (Morran et al., 2009; Morran et al., 2014; Parrish et al., 2016; Penley et al., 2017). Nonetheless, this protocol can be adapted to measure the relative competitive fitness of any two or more C. elegans populations or strains in the presence of any relevant bacterial parasite. Further, it can be used to measure the change in relative competitive fitness over the course of experimental evolution for any C. elegans populations evolved in the presence of a bacterial parasite.