Natural variation in Pristionchus pacificus dauer formation reveals cross-preference rather than self-preference of nematode dauer pheromones

Many free-living nematodes, including the laboratory model organisms Caenorhabditis elegans and Pristionchus pacificus, have a choice between direct and indirect development, representing an important case of phenotypic plasticity. Under harsh environmental conditions, these nematodes form dauer larvae, which arrest development, show high resistance to environmental stress and constitute a dispersal stage. Pristionchus pacificus occurs in a strong association with scarab beetles in the wild and remains in the dauer stage on the living beetle. Here, we explored the circumstances under which P. pacificus enters and exits the dauer stage by using a natural variation approach. The analysis of survival, recovery and fitness after dauer exit of eight P. pacificus strains revealed that dauer larvae can survive for up to 1 year under experimental conditions. In a second experiment, we isolated dauer pheromones from 16 P. pacificus strains, and tested for natural variation in pheromone production and sensitivity in cross-reactivity assays. Surprisingly, 13 of the 16 strains produce a pheromone that induces the highest dauer formation in individuals of other genotypes. These results argue against a simple adaptation model for natural variation in dauer formation and suggest that strains may have evolved to induce dauer formation precociously in other strains in order to reduce the fitness of these strains. We therefore discuss intraspecific competition among genotypes as a previously unconsidered aspect of dauer formation.     

Hormone signaling and phenotypic plasticity in nematode development and evolution

Phenotypic plasticity refers to the ability of an organism to adopt different phenotypes depending on environmental conditions. In animals and plants, the progression of juvenile development and the formation of dormant stages are often associated with phenotypic plasticity, indicating the importance of phenotypic plasticity for life-history theory. Phenotypic plasticity has long been emphasized as?a crucial principle in ecology and as facilitator of phenotypic evolution. In nematodes, several examples of phenotypic plasticity have been studied at the genetic and developmental level. In addition, the influence of different environmental factors has been investigated under laboratory conditions. These studies have provided detailed insight into the molecular basis of phenotypic plasticity and its?ecological and evolutionary implications. Here, we review recent studies on the formation of dauer larvae in Caenorhabditis elegans, the evolution of nematode parasitism and the generation of a novel feeding trait in Pristionchus pacificus. These examples reveal a conserved and co-opted role of an endocrine signaling module involving the steroid hormone dafachronic acid. We will discuss how hormone signaling might facilitate life-history and morphological evolution.     

Genetic mapping of variation in dauer larvae development in growing populations of Caenorhabditis elegans

In the nematode Caenorhabditis elegans, the appropriate induction of dauer larvae development within growing populations is likely to be a primary determinant of genotypic fitness. The underlying genetic architecture of natural genetic variation in dauer formation has, however, not been thoroughly investigated. Here, we report extensive natural genetic variation in dauer larvae development within growing populations across multiple wild isolates. Moreover, bin mapping of introgression lines (ILs) derived from the genetically divergent isolates N2 and CB4856 reveals 10 quantitative trait loci (QTLs) affecting dauer formation. Comparison of individual ILs to N2 identifies an additional eight QTLs, and sequential IL analysis reveals six more QTLs. Our results also show that a behavioural, laboratory-derived, mutation controlled by the neuropeptide Y receptor homolog npr-1 can affect dauer larvae development in growing populations. These findings illustrate the complex genetic architecture of variation in dauer larvae formation in C. elegans and may help to understand how the control of variation in dauer larvae development has evolved.     

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.     

The evolution of plasticity of dauer larva developmental arrest in the nematode Caenorhabditis elegans

Organisms can end up in unfavourable conditions and to survive this they have evolved various strategies. Some organisms, including nematodes, survive unfavourable conditions by undergoing developmental arrest. The model nematode Caenorhabditis elegans has a developmental choice between two larval forms, and it chooses to develop into the arrested dauer larva form in unfavourable conditions (specifically, a lack of food and high population density, indicated by the concentration of a pheromone). Wild C. elegans isolates vary extensively in their dauer larva arrest phenotypes, and this prompts the question of what selective pressures maintain such phenotypic diversity? To investigate this we grew C. elegans in four different environments, consisting of different combinations of cues that can induce dauer larva development: two combinations of food concentration (high and low) in the presence or absence of a dauer larva-inducing pheromone. Five generations of artificial selection of dauer larvae resulted in an overall increase in dauer larva formation in most selection regimes. The presence of pheromone in the environment selected for twice the number of dauer larvae, compared with environments not containing pheromone. Further, only a high food concentration environment containing pheromone increased the plasticity of dauer larva formation. These evolutionary responses also affected the timing of the worms’ reproduction. Overall, these results give an insight into the environments that can select for different plasticities of C. elegans dauer larva arrest phenotypes, suggesting that different combinations of environmental cues can select for the diversity of phenotypically plastic responses seen in C. elegans.     

Stressful environments induce novel phenotypic variation: hierarchical reaction norms for sperm performance of a pervasive invader

Genetic variation for phenotypic plasticity is ubiquitous and important. However, the scale of such variation including the relative variability present in reaction norms among different hierarchies of biological organization (e.g., individuals, populations, and closely related species) is unknown. Complicating interpretation is a trade‐off in environmental scale. As plasticity can only be inferred over the range of environments tested, experiments focusing on fine tuned responses to normal or benign conditions may miss cryptic phenotypic variation expressed under novel or stressful environments. Here, we sought to discern the presence and shape of plasticity in the performance of brown trout sperm as a function of optimal to extremely stressful river pH, and demarcate if the reaction norm varies among genotypes. Our overarching goal was to determine if deteriorating environmental quality increases expressed variation among individuals. A more applied aim was to ascertain whether maintaining sperm performance over a wide pH range could help explain how brown trout are able to invade diverse river systems when transplanted outside of their native range. Individuals differed in their reaction norms of phenotypic expression of an important trait in response to environmental change. Cryptic variation was revealed under stressful conditions, evidenced through increasing among‐individual variability. Importantly, data on population averages masked this variability in plasticity. In addition, canalized reaction norms in sperm swimming velocities of many individuals over a very large range in water chemistry may help explain why brown trout are able to colonize a wide variety of habitats.     

Gαo and Gαq)regulate the expression of daf-7, a TGFβ-like gene, in Caenorhabditis elegans

Caenorhabditis elegans enter an alternate developmental stage called dauer in unfavorable conditions such as starvation, overcrowding, or high temperature. Several evolutionarily conserved signaling pathways control dauer formation. DAF-7/TGFβ and serotonin, important ligands in these signaling pathways, affect not only dauer formation, but also the expression of one another. The heterotrimeric G proteins GOA-1 (Gα(o)) and EGL-30 (Gα(q)) mediate serotonin signaling as well as serotonin biosynthesis in C. elegans. It is not known whether GOA-1 or EGL-30 also affect dauer formation and/or daf-7 expression, which are both modulated in part by serotonin. The purpose of this study is to better understand the relationship between proteins important for neuronal signaling and developmental plasticity in both C. elegans and humans. Using promoter-GFP transgenic worms, it was determined that both goa-1 and egl-30 regulate daf-7 expression during larval development. In addition, the normal daf-7 response to high temperature or starvation was altered in goa-1 and egl-30 mutants. Despite the effect of goa-1 and egl-30 mutations on daf-7 expression in various environmental conditions, there was no effect of the mutations on dauer formation. This paper provides evidence that while goa-1 and egl-30 are important for normal daf-7 expression, mutations in these genes are not sufficient to disrupt dauer formation.     

The phenotypic variance gradient – a novel concept

Evolutionary ecologists commonly use reaction norms, which show the range of phenotypes produced by a set of genotypes exposed to different environments, to quantify the degree of phenotypic variance and the magnitude of plasticity of morphometric and life‐history traits. Significant differences among the values of the slopes of the reaction norms are interpreted as significant differences in phenotypic plasticity, whereas significant differences among phenotypic variances (variance or coefficient of variation) are interpreted as differences in the degree of developmental instability or canalization. We highlight some potential problems with this approach to quantifying phenotypic variance and suggest a novel and more informative way to plot reaction norms: namely “a plot of log (variance) on the y‐axis versus log (mean) on the x‐axis, with a reference line added”. This approach gives an immediate impression of how the degree of phenotypic variance varies across an environmental gradient, taking into account the consequences of the scaling effect of the variance with the mean. The evolutionary implications of the variation in the degree of phenotypic variance, which we call a “phenotypic variance gradient”, are discussed together with its potential interactions with variation in the degree of phenotypic plasticity and canalization.     

Two Neuronal G Proteins Are Involved in Chemosensation of the Caenorhabditis Elegans Dauer-Inducing Pheromone

Caenorhabditis elegans uses chemosensation to determine its course of development. Young larvae can arrest as dauer larvae in response to increasing population density, which they measure by a nematode-excreted pheromone, and decreasing food supply. Dauer larvae can resume development in response to a decrease in pheromone and increase in food concentration. We show here that two novel G protein alpha subunits (GPA-2 and GPA-3) show promoter activity in subsets of chemosensory neurons and are involved in the decision to form dauer larvae primarily through the response to dauer pheromone. Dominant activating mutations in these G proteins result in constitutive, pheromone-independent dauer formation, whereas inactivation results in reduced sensitivity to pheromone, and, under certain conditions, an alteration in the response to food. Interactions between gpa-2, gpa-3 and other genes controlling dauer formation suggest that these G proteins may act in parallel to regulate the neuronal decision making that precedes dauer formation.     

A modular concept of phenotypic plasticity in plants

Based on empirical evidence from the literature we propose that, in nature, phenotypic plasticity in plants is usually expressed at a subindividual level. While reaction norms (i.e. the type and the degree of plant responses to environmental variation) are a property of genotypes, they are expressed at the level of modular subunits in most plants. We thus contend that phenotypic plasticity is not a whole-plant response, but a property of individual meristems, leaves, branches and roots, triggered by local environmental conditions. Communication and behavioural integration of interconnected modules can change the local responses in different ways: it may enhance or diminish local plastic effects, thereby increasing or decreasing the differences between integrated modules exposed to different conditions. Modular integration can also induce qualitatively different responses, which are not expressed if all modules experience the same conditions. We propose that the response of a plant to its environment is the sum of all modular responses to their local conditions plus all interaction effects that are due to integration. The local response rules to environmental variation, and the modular interaction rules may be seen as evolving traits targeted by natural selection. Following this notion, whole-plant reaction norms are an integrative by-product of modular plasticity, which has far-reaching methodological, ecological and evolutionary implications.     

Adaptive divergence in body size overrides the effects of plasticity across natural habitats in the brown trout

The evolution of life-history traits is characterized by trade-offs between different selection pressures, as well as plasticity across environmental conditions. Yet, studies on local adaptation are often performed under artificial conditions, leaving two issues unexplored: (i) how consistent are laboratory inferred local adaptations under natural conditions and (ii) how much phenotypic variation is attributed to phenotypic plasticity and to adaptive evolution, respectively, across environmental conditions? We reared fish from six locally adapted (domesticated and wild) populations of anadromous brown trout (Salmo trutta) in one semi-natural and three natural streams and recorded a key life-history trait (body size at the end of first growth season). We found that population-specific reaction norms were close to parallel across different streams and Q_ST was similar – and larger than F_ST – within all streams, indicating a consistency of local adaptation in body size across natural environments. The amount of variation explained by population origin exceeded the variation across stream environments, indicating that genetic effects derived from adaptive processes have a stronger effect on phenotypic variation than plasticity induced by environmental conditions. These results suggest that plasticity does not “swamp” the phenotypic variation, and that selection may thus be efficient in generating genetic change.     

The thioredoxin TRX-1 modulates the function of the insulin-like neuropeptide DAF-28 during dauer formation in Caenorhabditis elegans

Thioredoxins comprise a conserved family of redox regulators involved in many biological processes, including stress resistance and aging. We report that the C. elegans thioredoxin TRX-1 acts in ASJ head sensory neurons as a novel modulator of the insulin-like neuropeptide DAF-28 during dauer formation. We show that increased formation of stress-resistant, long-lived dauer larvae in mutants for the gene encoding the insulin-like neuropeptide DAF-28 requires TRX-1 acting in ASJ neurons, upstream of the insulin-like receptor DAF-2. Genetic rescue experiments demonstrate that redox-independent functions of TRX-1 specifically in ASJ neurons are needed for the dauer formation constitutive (Daf-c) phenotype of daf-28 mutants. GFP reporters of trx-1 and daf-28 show opposing expression patterns in dauers (i.e. trx-1 is up-regulated and daf-28 is down-regulated), an effect that is not observed in growing L2/L3 larvae. In addition, functional TRX-1 is required for the down-regulation of a GFP reporter of daf-28 during dauer formation, a process that is likely subject to DAF-28-mediated feedback regulation. Our findings demonstrate that TRX-1 modulates DAF-28 signaling by contributing to the down-regulation of daf-28 expression during dauer formation. We propose that TRX-1 acts as a fluctuating neuronal signaling modulator within ASJ neurons to monitor the adjustment of neuropeptide expression, including insulin-like proteins, during dauer formation in response to adverse environmental conditions.     

Differences in developmental strategies between long‐settled and invasion‐front populations of the cane toad in Australia

Phenotypic plasticity can enhance a species’ ability to persist in a new and stressful environment, so that reaction norms are expected to evolve as organisms encounter novel environments. Biological invasions provide a robust system to investigate such changes. We measured the rates of early growth and development in tadpoles of invasive cane toads (Rhinella marina) in Australia, from a range of locations and at different larval densities. Populations in long‐colonized areas have had the opportunity to adapt to local conditions, whereas at the expanding range edge, the invader is likely to encounter challenges that are both novel and unpredictable. We thus expected invasion‐vanguard populations to exhibit less phenotypic plasticity than range‐core populations. Compared to clutches from long‐colonized areas, clutches from the invasion front were indeed less plastic (i.e. rates of larval growth and development were less sensitive to density). In contrast, those rates were highly variable in clutches from the invasion front, even among siblings from the same clutch under standard conditions. Clutches with highly variable rates of growth and development under constant conditions had lower phenotypic plasticity, suggesting a trade‐off between these two strategies. Although these results reveal a strong pattern, further investigation is needed to determine whether these different developmental strategies are adaptive (i.e. adaptive phenotypic plasticity vs. bet‐hedging) or instead are driven by geographic variation in genetic quality or parental effects.     

Artificial selection on the shape of reaction norms for eyespot size in the butterfly Bicyclus anynana: direct and correlated responses

The tropical butterfly Bicyclus anynana shows phenotypic plasticity in its ventral wing pattern as an adaptive response to wet‐dry seasonality. Wet season form individuals have large eyespots, whereas individuals of the dry season generation have small eyespots. In the laboratory these forms can be obtained by rearing larvae at high and low temperatures, respectively. To study the extent to which the shape of the nearly linear reaction norms for eyespot size can be changed we applied four generations of sib selection by rearing full‐sib families across three temperatures. In addition, we measured ecdysteroid titres shortly after pupation in the final generation. Although phenotypic variation in shape was present initially, the experiment yielded lines with reaction norms with similar shapes but different elevations. High, positive genetic correlation across temperatures can explain this lack of response. Differences in ecdysteroid titres did not readily relate to differences in eyespot size.     

Variable salinity tolerance in ascidian larvae is primarily a plastic response to the parental environment

Both phenotypic plasticity and local genetic adaptation may contribute to a species’ ability to inhabit different environmental conditions. While phenotypic plasticity is usually considered costly, local adaptation takes generations to respond to environmental change and may be constrained by strong gene flow. The majority of marine species have complex life-cycles with pelagic stages that might be expected to promote gene flow and plastic responses, and yet several notable examples of local adaptation have been found in species with broadcast larvae. In the ascidian, Ciona intestinalis (Linnaeus, 1767),—a common marine species with broadcast spawning and a short larval stage—previous studies have found marked differences in salinity tolerance of early life-history stages among populations from different salinity regimes. We used common-garden experiments to test whether observed differences in salinity tolerance could be explained by phenotypic plasticity. Adult ascidians from two low salinity populations [2–5 m depth, ~25 practical salinity units (PSU)], and two full salinity populations (25–27 m depth, ~31 PSU) were acclimated for 2–4 weeks at both 25 and 31 PSU. Gametes were fertilized at the acclimation salinities, and the newly formed embryos were transferred to 10 different salinities (21–39 PSU) and cultured to metamorphosis. Adult acclimation salinity had an overriding and significant effect on larval metamorphic success: tolerance norms for larvae almost fully matched the acclimation salinity of the parents, independent of parental origin (deep or shallow). However we also detected minor population differences that could be attributed to either local adaptation or persistent environmental effects. We conclude that differences in salinity tolerance of C. intestinalis larvae from different populations are driven primarily by transgenerational phenotypic plasticity, a strategy that seems particularly favourable for an organism living in coastal waters where salinity is less readily predicted than in the open oceans.     

Habitat-specific differences in thermal plasticity in natural populations of a soil arthropod

When populations experience substantial variation in environmental conditions, they may evolve phenotypic plasticity in response to these varying selection pressures. Evolutionary theory predicts differentiation in the level of phenotypic plasticity among different habitats. We evaluated temperature-induced phenotypic responses in juvenile growth rate in natural populations of the springtail Orchesella cincta , inhabiting forest and heathland. These habitats typically co-occur but differ strongly with respect to, for example, thermal regime, relative humidity, and structure. Offspring of females from the two habitats were reared at different temperatures in climate rooms and the temperature response of juvenile growth rate and egg size was measured. We found a habitat-specific difference in plasticity of juvenile growth rate. The reaction norms of the forest populations were steeper than the reaction norms for heath populations at two replicated sampling sites. Egg weight itself was demonstrated to be a plastic trait with a higher egg weight at low temperatures, but the thermal response did not differ between habitats. We conclude that these populations have diverged due to strong local natural selection. Our results support the argument that the level of phenotypic plasticity itself can be under selection and that differentiation in reaction norms can occur even in neighbouring habitats with no barrier to gene flow.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 265–271.