Fecundity compensation is dependent on the generalized stress response in a nematode host

Abstract Background Fecundity compensation, increased offspring output following parasite exposure, is widely reported, but the underlying mechanisms remain unclear. General stress responses are linked to other indirect defenses against parasites, and therefore may be responsible. We challenged strains of Caenorhabditis elegans (wild type and mutants with compromised or strengthened stress responses) with Staphylococcus aureus. Results In all strains except the compromised stress response mutant, we saw elevated offspring production if hosts survived initial parasite exposure. Conclusion We infer that general stress responses are linked with fecundity compensation. These results may explain why trade‐offs are not always observed among parasite defense mechanisms.

mutant sek-1) stress responses to Staphylococcus aureus. We then tracked the reproductive output of surviving hosts over time.
We then examined whether the nematodes that survived initial parasite exposure produced more offspring.
Host mortality and offspring production were measured following parasite exposure. Nematodes were chunked from the same ancestral plate to start each trial. Newly transferred worms were maintained at 20°C for 3 days before being surface-sterilized (bleached) with sodium hypochlorite (Schulte, Makus, Hasert, Michiels, & Schulenburg, 2010) and synchronized in M9 overnight.
Synchronized L1 nematodes were then plated onto 9-cm petri dishes containing nematode growth medium (NGM) agar with a lawn of E. coli (80 μl grown overnight at 30°C in LB) and incubated at 20°C for 2 days. Subsequently, nematodes were removed from each plate and gravity-washed using M9 containing 0.1% Triton-X. Nematodes were then transferred to tryptic soy broth (TSB) agar cultured with lawns of S. aureus (80 μl grown overnight at 30°C in THB), and controls were also transferred to TSB agar but cultured with lawns of E. coli (80 μl grown overnight at 30°C in LB).
Thirty nematodes that survived the exposure to S. aureus (or were part of the control population) were picked using a platinum wire from each individual plate and transferred to new NGM plates seeded with E. coli after 12 hr, 24 hr, and subsequently every 24 hr. The transfer regime was selected after pilot experiments showed a peak in reproduction between 12 and 24 hr. After live nematodes were transferred, F1 progeny were washed off the plate using 2 ml of M9 with Triton-X into a sterile 15-ml falcon tube. The numbers of alive and dead nematodes were recorded prior to each transfer. The nematode mixture was then thoroughly vortexed, and the number of progeny present in each of the L1, L2, and L3 larval stages was counted in three aliquots of 5 μl, to provide replication and an accurate estimate of total number of F1 progeny. The numbers of live and dead nematodes were recorded after 24 hr. Dead nematodes were classified as those that did not respond via movement to being touched by platinum wire. The experiment continued until all of the nematodes F I G U R E 1 Experimental regime: synchronized L1 nematodes on NGM plate agar with a lawn of E. coli food (green) incubated at 20°C for 2 days. Nematodes were then washed using M9 containing 0.1% Triton-X and transferred to lawns of S. aureus (red) or E. coli (control) on TSB agar. Thirty parent nematodes that survived the exposure to S. aureus (or were part of the control population) were picked using a platinum wire from each individual plate and transferred to new NGM plates seeded with food after 12 hr, 24 hr, and subsequently every 24 hr. The experiment continued until all of the nematodes were dead. Treatments consisted of four biological replicates, and the whole experiment was replicated five times were dead. Treatments consisted of four biological replicates, and the whole experiment was replicated five times (see Figure 1).
Data were analyzed in R version 3.2.0 and RStudio version 0.98.1091 (R Core Team, 2014). Differences in host mortality among strains at 24 hr after exposure to the parasite or control were analyzed using binomial general linearized models (GLMs) followed by Tukey's multiple comparison tests using the "multcomp" package in R. Overall differences in offspring production among strains were analyzed using ANOVAs followed by pairwise t tests with FDR pvalue correction. Spearman's rank correlations were used to examine the relationship between proportion of nematodes alive after 24 hr of parasite exposure and cumulative number of offspring over the course of the experiment. One-way analysis of variance (ANOVA) tests were used to compare differences in offspring production following exposure to S. aureus or OP50 among nematode strains and specific differences analyzed using FDR-corrected pairwise t tests.     Minchella & Loverde, 1981;Schwanz, 2008;Thornhill et al., 1986;Vale & Little, 2012). Our results indicate that fecundity compensation has evolved and been conserved in the model host organism, C. elegans. We found that hosts exhibited greater fecundity compensation and enhanced their reproductive output if parasites were more harmful, thus highlighting the sensitivity of the life-history shift. We also show that the fecundity compensation is linked to host general stress response and related innate immune system pathways triggered upon interactions with parasites.

| D ISCUSS I ON
Our results concur with previous findings indicating a role for generalized stress responses in other nonphysiological host responses (Schulenburg & Ewbank, 2007;Schulenburg & Müller, 2004). Contrary to expectations, nematodes with higher physiological innate immunity also show stronger behavioral avoidance responses (Schulenburg & Müller, 2004). These links have led to the hypothesis that behavioral avoidance mechanisms constitute part of a larger general stress response triggered upon exposure to parasites (Schulenburg & Ewbank, 2007). Thus, it is possible that increased fecundity compensation in daf-2 mutants and decreased fecundity compensation in sek-1 mutants are also a consequence of this general stress response. Indeed, the DAF-2/DAF-16 pathway is involved in the mitigation of physical evasion and reduced oral uptake of microbial parasites (Hasshoff, Böhnisch, Tonn, Hasert, & Schulenburg, 2007), in addition to the involvement in fecundity compensation that we report here, and is also critical to general stress responses in C. elegans (Evans, Chen, & Tan, 2008;Murphy et al., 2003). The p38 MAPK pathway is critical in C. elegans for defense against gram-positive bacteria (Kim et al., 2002;Troemel et al., 2006) and, similarly to DAF-2/DAF-16, is also involved in general stress responses (Craig, Fink, Yagi, Ip, & Cagan, 2004). Furthermore, the p38 MAPK pathway is linked to the positive regulation of egg-laying behavior in C. elegans (Kim et al., 2002), providing a mechanism by which it may mediate fecundity compensation. Interestingly, although hosts in the Biomphalaria glabrata-Schistosoma mansoni trematode system do not exhibit fecundity compensation during presumably stressful drought conditions, the parasites do ramp up offspring production (Gleichsner, Cleveland, & Minchella, 2016). Such a finding suggests that the connection between general stress and fecundity compensation can be complex in natural systems and that it can nevertheless be extended beyond hosts to parasites.
Trade-offs are often predicted in evolutionary biology between indirect host responses and measures of innate immunity, a component of the generalized stress response (Schulenburg & Ewbank, 2007;Schulenburg, Kurtz, Moret, & Siva-Jothy, 2009). The idea is that host resources should be invested into either direct responses, such as innate immunity, or indirect defenses, such as fecundity compensation, behavioral avoidance (Schulenburg et al., 2009), or antimicrobial secretions (Cotter & Kilner, 2010 Our experiments are based on two extreme phenotypes: nematodes with heightened immune response compared to nematodes with a suppressed immune response. Thus, to further explore the link between the general stress response and fecundity compensation, more experiments must be carried out along the phenotypic landscape.

| CON CLUS IONS
Here, we have pinpointed a general mechanism by which fecundity compensation might operate. Future work is now needed to evaluate the specific roles of genes in the stress response pathways highlighted here. Such an approach will help us better understand the relationships between fecundity compensation and other host defenses that are triggered before and after exposure to parasites. In an evolutionary context, elucidating the mechanistic underpinning and complexity of fecundity compensation in C. elegans will ultimately yield a powerful system for directly testing how natural selection can shape host life-history traits and other indirect defenses to parasites.

ACK N OWLED G M ENTS
We would like to thank Martyna Zelek, Dylan Dahan, and Alex Betts for help in the laboratory.
F I G U R E 3 Difference in offspring production after host exposure to parasites and food. Strain with asterisk differs significantly from the wild type (N2; p < .05 pairwise t tests, FDRcorrected). Individual data points are shown within boxplots C. elegans strain Difference in cumulative number of offspring between OP50 and S. aureus exposure