Summary
Many animal and plant species respond to high or low population densities by phenotypic plasticity. To investigate if specific age classes and/or cross-generational signaling affect(s) phenotypic plasticity, we developed a dye-based method to differentiate co-occurring nematode age classes. We applied this method to Pristionchus pacificus, which develops a predatory mouth form to exploit alternative resources and kill competitors in response to high population densities. Remarkably, only adult, but not juvenile, crowding induces the predatory morph in other juveniles. Profiling of secreted metabolites throughout development with HPLC-MS combined with genetic mutants traced this result to the production of adult-specific pheromones. Specifically, the P. pacificus-specific di-ascaroside#1 that induces the predatory morph exhibits a binary induction in adults, even though mouth form is no longer plastic in adults. This cross-generational signaling between adults and juveniles may serve as an indication of rapidly increasing population size. Thus, phenotypic plasticity depends on critical age classes.
Introduction
Population density is an important ecological parameter, with higher densities corresponding to increased competition for resources (Hastings, 2013). In addition to density-dependent selection (MacArthur, 1962; Travis et al., 2013), which operates on evolutionary time scales, some organisms can respond dynamically to population density through phenotypic plasticity. For example, plants can sense crowding by detecting the ratio of red (chlorophyll absorbing) to far red (non-absorbing) light, and respond by producing higher shoots (Dudley and Schmitt, 2015). Locusts undergo solitary to swarm (i.e. gregarious) transition, and aphids can develop wings, both as results of increased physical contact (Pener and Simpson, 2009; Simpson et al., 2001; Slogget and Weisser, 2004). Intriguingly, population density can also have cross-generational effects. For example, adult crowding of the desert locust Schistocerca gregaria (Maeno and Tanaka, 2008; Simpson and Miller, 2007) and migratory locust Locusta migratoria (Chen et al., 2015; Hamouda et al.) also influences the egg size, number, and morphology of their progeny; and high population densities of red squirrels elicit hormonal regulation in mothers to influence faster-developing offspring (Ben Dantzer et al., 2013). In many species, population density and cross-generational signaling are detected through pheromones, however the precise nature, mechanisms of induction, age-specificity, and exact ecological role are not well understood.
Nematodes are a powerful model system to investigate the mechanisms of density-dependent plasticity because many small molecule pheromones that affect plastic phenotypes have been characterized (Butcher, 2017; Butcher et al., 2007; Reuss et al., 2012). For example, in the model organism Caenorhabditis elegans, high population densities induce entry into a stress-resistant dormant ‘dauer’ stage (Fielenbach and Antebi, 2008). The decision to enter dauer was revealed to be regulated by a family of small molecule nematode-derived modular metabolites (NDMMs) called ascarosides that act as pheromones (Butcher et al., 2007; 2008; Jeong et al., 2005). Ascarosides consist of an ascarylose sugar with a fatty acid side chain and modular head and terminus groups (Figure 1A). The level and composition of ascarosides were later shown to be dependent on sex (Chasnov et al., 2007; Izrayelit et al., 2012) and development (Kaplan et al., 2011), although it is thought that dauer can be induced by all developmental stages (Golden and Riddle, 1982). Subsequent studies revealed that specific NDMMs also regulate other life history traits, such as mating (Chasnov et al., 2007; Izrayelit et al., 2012), social behavior (Srinivasan et al., 2012) and developmental speed (Ludewig et al., 2017). Although NDMMs are broadly conserved (Choe et al., 2012; Dong et al., 2018; Markov et al., 2016), inter- and intraspecific competition have driven the evolution of distinct response regimes for the same phenotypes (Bose et al., 2014; Choe et al., 2012; Diaz et al., 2014; Falcke et al., 2018; Greene et al., 2016). In addition, more complex structures have been observed that affect distinct plastic phenotypes (Bose et al., 2012).
In Pristionchus pacificus, a soil-associated nematode that is reliably found on scarab beetles (Figure 1A)(Herrmann et al., 2006; 2007; Sommer and McGaughran, 2013), an ascaroside dimer (dasc#1) that is not found in C. elegans regulates the development of a predatory mouth form (Bento et al., 2010a; Bose et al., 2012; Sommer et al., 2017). Mouth-form plasticity represents an example of a morphological novelty that results in predatory behavior to exploit additional resources and kill competitors. Specifically, adult P. pacificus exhibit either a narrow stenostomatous (St) mouth (Figure 1B), which is restricted to bacterial feeding, or a wide eurystomatous (Eu) mouth with an extra denticle (Figure 1C), which allows for feeding on bacteria and fungi (Sanghvi et al., 2016), and predation on other nematodes (Wilecki et al., 2015). This type of phenotypic plasticity is distinct from direct vs. indirect (dauer) development because it results in two alternative life history strategies in the adult (for review see Sommer & Mayer, 2015). Recent studies in P. pacificus have begun to investigate the dynamics and succession of nematodes on decomposing beetle carcasses to better understand the ecological significance of mouth-form plasticity (Meyer et al., 2017). These studies revealed that on a carcass (Figure 1D), P. pacificus exits the dauer diapause to feed on microbes, and then reenters dauer after food sources have been exhausted, displaying a ‘boom-and-bust’ ecology (Meyer et al., 2017; Sommer and McGaughran, 2013). Presumably different stages of this succession comprise different ratios of juveniles and adults, and recognizing the age-structure of a population as a juvenile could provide predictive value for adulthood. However, it is unknown whether the mouth-form decision is sensitive to crowding by different age classes. More broadly, while age classes are known to be important for population growth and density-dependent selection {Hastings:2013dn, Charlesworth:1994ww, Charlesworth:1970ks}, their role in phenotypic plasticity has thus far been largely unexplored.
While nematodes have many experimental advantages, including easy laboratory culture and advanced genetic, genomic, and the aforementioned chemical tools, their small size has made investigations at the organismal level and in experimental ecology challenging. For example, no in vivo methodologies are currently available to label distinct populations without the need for transgenics, which is only available in select model organisms such as C. elegans, P. pacificus, and some of their relatives. Here, we combine a novel dye-staining method with the first developmental pheromone profiling in P. pacificus to study potential effects of age on density-dependent plasticity. This vital-dye method allows tracking adults with juveniles, or juveniles with juveniles, and can be applied to any nematode system that can be cultured under laboratory conditions. In contrast to dauer, we found that mouth form is strongly affected by cross-generational signalling. Specifically, only adult crowding induces the predatory morph, which is controlled by adult-specific pheromones.
Results
A vital dye method for labeling nematode populations
To directly test if different age groups of nematodes influence plastic phenotypes, we required two synchronized populations to co-habit the same space, yet still be able to identify worms from different age groups. To do so, we developed a dye-staining methodology to robustly differentiate between nematode populations. After trying several vital dyes, we identified that Neutral Red (Thomas and Lana, 2008) and CellTracker Green BODIPY (Thermo) stain nematode intestines brightly and specifically to their respective channels (Figures 2A-E and S1). These dyes stain all nematodes tested including C. elegans (Figure S2) and dauer larvae (Figure S3A,B). They also last more than three days (Figure S3C-G), allowing long-term tracking of mixed nematode populations. Importantly, neither Neutral Red nor CellTracker Green staining affects viability, developmental rate, or the formation of specific morphological structures, such as P. pacificus mouth form (Figure S4). Thus, Neutral Red and CellTracker Green allow specific labeling of worm populations to study age-dependent effects on phenotypes.
Adult but not juvenile crowding induces the predatory mouth form in P. pacificus
To assess potential intra- or inter-generational influence on P. pacificus mouth form we stained juveniles of the highly St strain RSC017 with Neutral Red, and added an increasing number of CellTracker Green-stained RSC017 adults or juveniles (Figure 2F, 3A). Three days later we phenotyped red animals that had developed into adults, but showed no green staining. To ascertain potential differences between adding juveniles or adults, we performed a binomial regression on Eu count data from multiple independent biological replicates (n>3), with age and number of individuals added as fixed effects (Transparent Methods, Table S1). We observed a significant increase in Eu worms in response to adults, but not juveniles (p=2.59 x10-2; for display summed percents are shown in Figure 3B,C). Almost half (48%) of the population developed the Eu mouth form with just 500 adult animals, which is a greater than 50-fold induction compared to side-by-side controls (Figure 3B,C). We were also curious if dauers, which have a thickened cuticle and represent a distinct stage in the boom-and-bust life cycle of nematodes, could still respond to adults. Indeed, the same trend that was observed with juveniles was seen with dauers (p=2.96×10−3), albeit to a more muted extent (Figure 3D,E). Specifically, with a total of 200 dauers and 500 adults, 25.7% of dauers become Eu, whereas only 1.8% of dauers become Eu on a plate containing 700 dauers (and no adults) (Figure 3D). Collectively, these data indicate that adult crowding specifically induces the Eu mouth form.
Even though we did not detect a mouth-form switch in large populations of J2s or dauers, and food was still visible on plates containing the most animals (500 adults and 200 juveniles), we could not completely rule out the possible effect of food availability on mouth form. As a proxy for starvation, we conducted assays with greatly increased numbers of juveniles from 1,000 to 10,000 that would rapidly deplete bacterial food. We noticed a stark cliff in the fraction of juveniles that reach adulthood at 4,000-5,000 animals, arguing that food is a limiting resource at this population density (Figure 3F). Importantly however, in these plates we still did not see a shift in mouth form (Figure 3G) (p=0.99, binomial regression, Table S1). With an overwhelming 10,000 worms on a plate, 5.8% were Eu, compared to 48% in the presence of only 500 adults. While longer-term starvation may yet have an impact on mouth form, under our experimental conditions it appears to be negligible.
Adult but not juvenile secretions induce the Eu mouth form
As mouth-form plasticity in P. pacificus is regulated by nematode-derived modular metabolites (NDMMs)(Bose et al., 2012), we wondered if the difference between adults and juveniles resulted from differences in secreted NDMMs. To test this hypothesis we added secretions from adult or juvenile worms to RSC017 (highly St) juveniles. We found that adult secretions from both the laboratory stain RS2333 (highly Eu) and RSC017 led to a significant increase in the Eu morph relative to juvenile secretions (p=5.27×10−06, 1.33×10−3, respectively, Fisher’s exact test)(Figure 4). To confirm the effect was caused by ascaroside pheromones, we exposed RSC017 juveniles to supernatant from a daf-22.1;22.2 double mutant, which exhibits virtually no ascaroside production in both C. elegans and P. pacificus (Golden and Riddle, 1985; Markov et al., 2016). Again, juvenile secretion had no impact on Eu frequency, but in contrast to wild-type supernatants, we observed no significant increase in Eu frequency with adult secretions (p=0.8324, Fisher’s exact test, Figure 4). Thus, adult-specific NDMMs induce development of the Eu mouth form.
Developmental-staged NDMM profiles reveal adult-specific synthesis of dasc#1
Next, we investigated whether the difference between adult and juvenile pheromones is one of dosage, or of identity. To answer this question and verify age-specific differences in pheromones, we profiled P. pacificus NDMM levels in two strains and at three time points throughout development. We used RS2333 and RSC017 and measured the exo-metabolomes of juvenile stage 2 (J2s, 24 hrs), J3s (48 hrs) and J4/adults (72 hrs) from a constant culture with excess OP50 bacterial food (Figures 5A,B, S5, Materials and methods). To assess potential differences in pheromone levels we performed a linear regression with the area under the curve for each NDMM (aoc) (Figure S5) as the response variable. Stage and strain were modeled as fixed effects, and because we performed separate regression analyses for each pheromone, we adjusted the resulting p values for multiple testing using false discovery rate (FDR)(see Table S2 for p and FDR values between stage and strain). We observed that there was a significant affect of developmental stage on the levels of ascr#9, ascr#12, npar#1, and dasc#1, and that ubas#1 and #2 are strain and stage specific (FDR<0.05). Interestingly, dasc#1 is the most potent known Eu-inducing compound when tested as a single synthesized compound, while npar#1 is both Eu- and dauer-inducing (Figure 5C,D,F-I) (Bose et al., 2012). Closer inspection revealed dasc#1, npar#1, and ascr#9 increase throughout development in both strains, and dasc#1 peaks in adults in RS2333 (p<0.05, student’s two-tailed t-test between 72 hrs and 24 hrs for each NDMM in both strains, and also 72 hrs and 48 hrs for dasc#1 in RS233, Table S3). Intriguingly, the trajectory of dasc#1 appeared binary in both strains (Figure 5F,G). In fact our statistical model for dasc#1 fits better if we assume cubic rather than linear growth (ΔAIC=3.958). In contrast, ascr#9, which was also statistically up-regulated but does not affect known plastic phenotypes (Bose et al., 2012), displays a more gradual increase in both strains (Figure 5E,J,K), and the model fits better with linear growth (AIClinear – AICcubic= −1.208). Meanwhile, the trajectory of npar#1 appears strain-specific (Figure 5H,I). Hence the mode of induction is NDMM-specific, and the kinetics of production may be related to their roles in phenotypic plasticity.
In principle, the increase in abundance of certain pheromones could be a result of a concomitant increase in body mass, however several observations indicate more targeted regulation. First, no other compounds were significantly different in our linear model. Second, an analysis of previously published RNA-seq data (Baskaran et al., 2015) reveals the increase in NDMM abundance corresponds to an increase in transcription of the thiolase Ppa-daf-22.1 (Figure S6), the most downstream enzyme in the β-oxidation pathway of ascaroside synthesis. Third, pasc#9 and pasc#12 actually exhibit a peak in abundance at the 48 hr/J3 time point, rather than in 72 hrs/adults. Finally, we profiled the endo-metabolome of eggs, and found appreciable amounts of ascr#1, #9, #12, and pasc#9, but little to no traces of other ascaroside derivatives (Figure S5C), suggesting age-specific synthesis rather than release. Together, these results suggest that the observed increase in ubas#1 and #2, ascr#9, npar#1, and dasc#1 over time corresponds to age-specific production. The observation that dasc#1 is produced specifically during the juvenile-to-adult transition is especially intriguing because adults are no longer able to switch mouth forms, hinting at cross-generational signaling.
Discussion
Here, we introduce a novel dye-based method that allowed us to assess cross-generational influence on mouth form. Our results demonstrate adult crowding induces the Eu predatory morph, and that this effect is a result of age-specific pheromones. In doing so, we provide the first multi-stage time series of pheromone production in P. pacificus, which shows that dasc#1 exhibits a surprising ‘off-on’ switch-like induction pattern. Collectively, our results argue that adults represent a critical age group (Charlesworth, 1972) in nematode populations.
Our developmental profiling revealed an increase in two NDMMs that affect plastic phenotypes. The observation that this trend mirrors the transcriptional regulation of enzymes involved in NDMM synthesis argues that the stage-dependent increase is not simply a result of an increase in body mass, but rather that these molecules are programmed for stage-specific induction. The binary ‘off-on’ kinetics might reflect a population level feedback loop, such that the production of density-sensing pheromones is based on a threshold level of previously produced pheromones. It is also worth noting that while npar#1 is the major dauer-inducing pheromone in P. pacificus (Bose et al., 2012), we did not observe dauers in any experimental setup described herein. Thus, it seems that mouth-form phenotype is the first-level plastic response to population density. Presumably higher concentrations are required for dauer induction, reflecting a calculated response strategy depending on the level of crowding or duration of starvation. Interestingly, the effect of adult supernatants was noticeably less (23%-26% Eu) than of adult worms (up to 48% with only 500 adults). It is difficult to compare the amount of pheromone concentrations between experiments, but presumably worms in the vital-dye assay experienced a greater local concentration as they were in direct physical contact with each other, compared to worms in the supernatant assay.
Among the many environmental influences on mouth form (Werner et al., 2017), population density and starvation are perhaps the most ecologically relevant. However, teasing apart these two factors has proven difficult (Bento et al., 2010b). Here, we demonstrate that while a strong shift is observed with adult-specific pheromones, no such effect was seen under limited resource conditions. Thus, age-specific crowding is sufficient to induce the Eu mouth form. Nevertheless, this does not preclude that long-term starvation could also have an effect. Determining the relative contributions of these factors to mouth form will be important to better understand the sophisticated ecological response strategies of P. pacificus, nematodes, and phenotypic plasticity in general.
Why do adults and not juveniles affect mouth form? Given that St animals can develop faster (Serobyan et al., 2013), there may be a ‘race’ to sexual maturation in emergent populations at low densities. But as the nematode population increases, there will likely be a commensurate decrease in bacterial populations. When faced with competition from other nematodes, P. pacificus has a particular advantage in developing the Eu morph; their expanded dietary range includes their competition. Indeed, when nematode prey is the only available food source, the Eu morph provides longer life spans and more progeny than the St morph (Serobyan et al., 2014). When resources become depleted as population size increases, C. elegans and other monomorphic nematodes may enter dauer and disperse (Frézal and Félix, 2015). But in St-biased dimorphic strains of P. pacificus, juveniles may switch to the Eu morph in response to adults as a first-level indication of rapidly increasing population size (Figure 6). Then, after prolonged starvation and crowding, worms will presumably enter dauer. By analogy to economic models of population growth (Malthus et al., 1992; Trewavas, 2002) mouth-form plasticity is a ‘technological innovation’ to temporarily escape a Malthusian resource trap. To what extent this occurs in nature, or with P. pacificus strains that are highly Eu, remains to be determined.
The evolution of dimorphic mouth forms is one among myriad nematode ecological strategies. For example, entomopathogenic nematodes release their symbiont bacteria in insect hosts to establish their preferred food source, and some release antibiotics to kill off competing bacteria and fungi from other entomopathogenic species (Griffin, 2012). Some free-living species, like those of the genus Oscheius, refrain from combat and stealthily feed and reproduce amidst warring entomopathogenic species. Interspecific killing also occurs in gonorchoristic species, in which both mated and virgin males are killed, implying fighting not just for mates but for resources as well (O’Callaghan et al., 2014; Zenner et al., 2014). Reproductive strategies also exist, and hermaphroditic species have an advantage over gonachristic species when colonizing a new niche, such as an insect carcass (Campos-Herrera, 2015). Meanwhile insect hosts and colonizing nematodes have their own distinct pheromone-based attraction and toxicity (Cinkornpumin et al.; Renahan and Hong, 2017). Finally, the renaissance of C. elegans sampling from around the world (Cook et al., 2017; Evans et al., 2016; Félix et al., 2013; Petersen et al., 2014; Poullet and Braendle, 2015) is rapidly building a resource of wild isolates that will almost certainly have different and fascinating ecologies. We hope our method for labeling and then combining different nematode populations on the same plate will aid in studies to identify these strategies. Perhaps the time is also ripe to complement these studies with more sophisticated ecological modelling that can lead to testable hypotheses.
Although beyond the scope of this manuscript, the cross-generational communication we observed could in principle reflect an intended signal from adults to juveniles, i.e. kin selection (Bourke, 2014). However, we favor a more simplistic view that juveniles have evolved to recognize adult-produced metabolites. Regardless of these interpretations, our results argue that age classes are a critical factor in density-dependent plasticity, as has been theorized in density-dependent selection (Charlesworth, 1994).
Limitations of the Study
Given the ubiquity of certain traits in reproductive adults and their contribution to population growth, we suspect similar results will be found in other systems. However, it may depend on the phenotype and system being studied. For example, the population dynamics of nematodes (fast hermaphroditic reproduction) may be sufficiently different from other species such that our findings are not extendable in every case. In addition, our method of staining different populations, while fast and easy, is particular to nematodes.
Methods
*See Transparent Methods in Supplemental Information
Author Contributions
MSW and RJS conceived of the project. MC conducted pheromone profiling. MSW and TR designed and conducted dye-labeling experiments. TR and MC performed supernatant experiments. MD and MSW considered ecological implications. MSW and TR wrote the manuscript with input and edits from all authors.
Acknowledgements
We would like to thank all members of the Sommer lab, Dr. Talia Karasov, Dr. Hernan Burbano and Moises Exposito-Alonso for guidance with statistical analysis, and Dr. Adrian Striet (Max Planck Institute), and Dr. Cameron Weadick (University of Sussex) for thoughtful critique and discussion.