Accelerated hermaphrodite maturation on male pheromones suggests a general principle of coordination between larval behavior and development.

Environment in general and social signals in particular could alter development. In Caenorhabditis elegans, male pheromones hasten development of hermaphrodite larvae. We show that this involves acceleration of growth and both somatic and germline development during the last larval stage (L4). Larvae exposed to male pheromones spend more time in L3 and less in the quiescent period between L3 and L4. This behavioral alteration improves provision in early L4, likely allowing for faster development. Larvae must be exposed to male pheromones in late L3 for behavioral and developmental effects to occur. Latter portions of other larval stages also contain periods of heightened sensitivity to environmental signals. Behavior during the early part of the larval stages is biased toward exploration, whereas later the emphasis shifts to food consumption. We argue that this organization allows assessment of the environment to identify the most suitable patch of resources, followed by acquisition of sufficient nutrition and salient information for the developmental events in the next larval stage. Evidence from other species indicates that such coordination of behavior and development may be a general feature of larval development.

A male pheromone that improves the quality of the oogenic germline.

Pheromones exchanged by conspecifics are a major class of chemical signals that can alter behavior, physiology, and development. In particular, males and females communicate with potential mating partners via sex pheromones to promote reproductive success. Physiological and developmental mechanisms by which pheromones facilitate progeny production remain largely enigmatic. Here, we describe how a Caenorhabditis elegans male pheromone, ascr#10, improves the oogenic germline. Before most signs of aging become evident, C. elegans hermaphrodites start producing lower-quality gametes characterized by abnormal morphology, increased rates of chromosomal nondisjunction, and higher penetrance of deleterious alleles. We show that exposure to the male pheromone substantially ameliorates these defects and reduces embryonic lethality. ascr#10 stimulates proliferation of germline precursor cells in adult hermaphrodites. Coupled to the greater precursor supply is increased physiological germline cell death, which is required to improve oocyte quality in older mothers. The hermaphrodite germline is sensitive to the pheromone only during a time window, comparable in duration to a larval stage, in early adulthood. During this period, prereproductive adults assess the suitability of the environment for reproduction. Our results identify developmental events that occur in the oogenic germline in response to a male pheromone. They also suggest that the opposite effects of the pheromone on gamete quality and maternal longevity arise from competition over resource allocation between soma and the germline.

Serotonergic signaling plays a deeply conserved role in improving oocyte quality.

Declining germline quality is a major cause of reproductive senescence. Potential remedies could be found by studying regulatory pathways that promote germline quality. Several lines of evidence, including a C. elegans male pheromone ascr#10 that counteracts the effects of germline aging in hermaphrodites, suggest that the nervous system plays an important role in regulating germline quality. Inspired by the fact that serotonin mediates ascr#10 signaling, here we show that serotonin reuptake inhibitors recapitulate the effects of ascr#10 on the germline and promote healthy oocyte aging in C. elegans. Surprisingly, we found that pharmacological increase of serotonin signaling stimulates several developmental processes in D. melanogaster, including improved oocyte quality, although underlying mechanisms appear to be different between worms and flies. Our results reveal a plausibly conserved role for serotonin in maintaining germline quality and identify a class of therapeutic interventions using available compounds that could efficiently forestall reproductive aging.

A Caenorhabditis elegans Male Pheromone Feminizes Germline Gene Expression in Hermaphrodites and Imposes Life-History Costs.

Sex pheromones not only improve the reproductive success of the recipients, but also impose costs, such as a reduced life span. The underlying mechanisms largely remain to be elucidated. Here, we show that even a brief exposure to physiological amounts of the dominant Caenorhabditis elegans male pheromone, ascr#10, alters the expression of thousands of genes in hermaphrodites. The most dramatic effect on the transcriptome is the upregulation of genes expressed during oogenesis and the downregulation of genes associated with male gametogenesis. This result reveals a way in which social signals help to resolve the inherent conflict between spermatogenesis and oogenesis in a simultaneous hermaphrodite, presumably to optimally align reproductive function with the presence of potential mating partners. We also found that exposure to ascr#10 increased the risk of persistent intestinal infections in hermaphrodites due to pathological pharyngeal hypertrophy. Thus, our study reveals ways in which the male pheromone can not only have beneficial effects on the recipients' reproduction, but also cause harmful consequences that reduce life span.

The serotonin circuit that coordinates germline proliferation and egg laying with other reproductive functions in Caenorhabditis elegans.

Behaviour and physiology are altered in reproducing animals, but neuronal circuits that regulate these changes remain largely unknown. Insights into mechanisms that regulate and possibly coordinate reproduction-related traits could be gleaned from the study of sex pheromones that can improve the reproductive success of potential mating partners. In Caenorhabditis elegans, the prominent male pheromone, ascr#10, modifies reproductive behaviour and several aspects of reproductive physiology in hermaphrodite recipients, including improving oocyte quality. Here we show that a circuit that contains serotonin-producing and serotonin-uptaking neurons plays a key role in mediating effects of ascr#10 on germline development and egg laying behaviour. We also demonstrate that increased serotonin signalling promotes proliferation of germline progenitors in adult hermaphrodites. Our results establish a role for serotonin in maintaining germline quality and highlight a simple neuronal circuit that acts as a linchpin that couples food intake, mating behaviour, reproductive output, and germline renewal and provisioning.

An excreted small molecule promotes C. elegans reproductive development and aging.

Excreted small-molecule signals can bias developmental trajectories and physiology in diverse animal species. However, the chemical identity of these signals remains largely obscure. Here we report identification of an unusual N-acylated glutamine derivative, nacq#1, that accelerates reproductive development and shortens lifespan in Caenorhabditis elegans. Produced predominantly by C. elegans males, nacq#1 hastens onset of sexual maturity in hermaphrodites by promoting exit from the larval dauer diapause and by accelerating late larval development. Even at picomolar concentrations, nacq#1 shortens hermaphrodite lifespan, suggesting a trade-off between reproductive investment and longevity. Acceleration of development by nacq#1 requires chemosensation and is dependent on three homologs of vertebrate steroid hormone receptors. Unlike ascaroside pheromones, which are restricted to nematodes, fatty acylated amino acid derivatives similar to nacq#1 have been reported from humans and invertebrates, suggesting that related compounds may serve signaling functions throughout metazoa.

Sex Pheromones of C. elegans Males Prime the Female Reproductive System and Ameliorate the Effects of Heat Stress.

Pheromones are secreted molecules that mediate animal communications. These olfactory signals can have substantial effects on physiology and likely play important roles in organismal survival in natural habitats. Here we show that a blend of two ascaroside pheromones produced by C. elegans males primes the female reproductive system in part by improving sperm guidance toward oocytes. Worms have different physiological responses to different ratios of the same two molecules, revealing an efficient mechanism for increasing coding potential of a limited repertoire of molecular signals. The endogenous function of the male sex pheromones has an important side benefit. It substantially ameliorates the detrimental effects of prolonged heat stress on hermaphrodite reproduction because it increases the effectiveness with which surviving gametes are used following stress. Hermaphroditic species are expected to lose female-specific traits in the course of evolution. Our results suggest that some of these traits could have serendipitous utility due to their ability to counter the effects of stress. We propose that this is a general mechanism by which some mating-related functions could be retained in hermaphroditic species, despite their expected decay.

The roles of TGFß and serotonin signaling in regulating proliferation of oocyte precursors and germline aging.

The decline of oocyte quality in aging but otherwise relatively healthy individuals compels a search for underlying mechanisms. Building upon a finding that exposure to male pheromone ascr#10 improves oocyte quality in C. elegans, we uncovered a regulatory cascade that promotes proliferation of oocyte precursors in adults and regulates oocyte quality. We found that the male pheromone promotes proliferation of oocyte precursors by upregulating LAG-2, a ligand of the Notch-like pathway in the germline stem cell niche. LAG-2 is upregulated by a TGFß-like ligand DAF-7 revealing similarity of regulatory mechanisms that promote germline proliferation in adults and larvae. A serotonin circuit that also regulates food search and consumption upregulates DAF-7 specifically in adults. The serotonin/DAF-7 signaling promotes germline expansion to compensate for oocyte expenditure which is increased by the male pheromone. Finally, we show that the earliest events in reproductive aging may be due to declining expression of LAG-2 and DAF-7. Our findings highlight neuronal signals that promote germline proliferation in response to the environment and argue that deteriorating oocyte quality may be due to reduced neuronal expression of key germline regulators.

Macro-level modeling of the response of C. elegans reproduction to chronic heat stress.

A major goal of systems biology is to understand how organism-level behavior arises from a myriad of molecular interactions. Often this involves complex sets of rules describing interactions among a large number of components. As an alternative, we have developed a simple, macro-level model to describe how chronic temperature stress affects reproduction in C. elegans. Our approach uses fundamental engineering principles, together with a limited set of experimentally derived facts, and provides quantitatively accurate predictions of performance under a range of physiologically relevant conditions. We generated detailed time-resolved experimental data to evaluate the ability of our model to describe the dynamics of C. elegans reproduction. We find considerable heterogeneity in responses of individual animals to heat stress, which can be understood as modulation of a few processes and may represent a strategy for coping with the ever-changing environment. Our experimental results and model provide quantitative insight into the breakdown of a robust biological system under stress and suggest, surprisingly, that the behavior of complex biological systems may be determined by a small number of key components.

Periods of environmental sensitivity couple larval behavior and development.

The typical life cycle in most animal phyla includes a larval period that bridges embryogenesis and adulthood1. Despite the great diversity of larval forms, all larvae grow, acquire adult morphology and function, while navigating their habitats to obtain resources necessary for development. How larval development is coordinated with behavior remains substantially unclear. Here, we describe features of the iterative organization of larval stages that serve to assess the environment and procure resources prior to costly developmental commitments. We found that male-excreted pheromones accelerate2-4 the onset of adulthood in C. elegans hermaphrodites by coordinately advancing multiple developmental events and growth during the last larval stage. The larvae are sensitive to the accelerating male pheromones only at the end of the penultimate larval stage, just before the acceleration begins. Other larval stages also contain windows of sensitivity to environmental inputs. Importantly, behaviors associated with search and consumption of food are distinct between early and late portions of larval stages. We infer that each larval stage in C. elegans is subdivided into two epochs: A) global assessment of the environment to identify the most suitable patch and B) consumption of sufficient food and acquisition of salient information for developmental events in the next stage. We predict that in larvae of other species behavior is also divided into distinct epochs optimized either for assessing the habitat or obtaining the resources. Thus, a major role of larval behavior is to coordinate the orderly progression of development in variable environments.

A C. elegans male pheromone feminizes germline gene expression in hermaphrodites and imposes life-history costs.

Sex pheromones improve reproductive success, but also impose costs. Here we show that even brief exposure to physiological amounts of the dominant C. elegans male pheromone, ascr#10, alters the expression of thousands of genes in hermaphrodites. The most dramatic effect on the transcriptome was the upregulation of genes expressed during oogenesis and downregulation of genes associated with male gametogenesis. Among the detrimental effects of ascr#10 on hermaphrodites is the increased risk of persistent infections caused by pathological pharyngeal hypertrophy. Our results reveal a way in which social signals help to resolve the inherent conflict between spermatogenesis and oogenesis in a simultaneous hermaphrodite, presumably to optimally align reproductive function to the presence of potential mating partners. They also show that the beneficial effects of the pheromone are accompanied by harmful consequences that reduce lifespan.

Sex-specificity of the C. elegans metabolome.

Recent studies of animal metabolism have revealed large numbers of novel metabolites that are involved in all aspects of organismal biology, but it is unclear to what extent metabolomes differ between sexes. Here, using untargeted comparative metabolomics for the analysis of wildtype animals and sex determination mutants, we show that C. elegans hermaphrodites and males exhibit pervasive metabolomic differences. Several hundred small molecules are produced exclusively or in much larger amounts in one sex, including a host of previously unreported metabolites that incorporate building blocks from nucleoside, carbohydrate, lipid, and amino acid metabolism. A subset of male-enriched metabolites is specifically associated with the presence of a male germline, whereas enrichment of other compounds requires a male soma. Further, we show that one of the male germline-dependent metabolites, an unusual dipeptide incorporating N,N-dimethyltryptophan, increases food consumption, reduces lifespan, and accelerates the last stage of larval development in hermaphrodites. Our results serve as a foundation for mechanistic studies of how the genetic sex of soma and germline shape the C. elegans metabolome and provide a blueprint for the discovery of sex-dependent metabolites in other animals.

ODR-1 acts in AWB neurons to determine the sexual identity of C. elegans pheromone blends.

Valence of animal pheromone blends can vary due to differences in relative abundance of individual components. For example, in C. elegans, whether a pheromone blend is perceived as "male" or "hermaphrodite" is determined by the ratio of concentrations of ascr#10 and ascr#3. The neuronal mechanisms that evaluate this ratio are not currently understood. We present data that suggest that the function of guanylyl cyclase ODR-1 in AWB neurons is required for the effect of ascr#3 that counteracts the activity of ascr#10. This finding defines a new module in the neuronal mechanism that determines the sexual identity of C. elegans pheromone.

The roles of several sensory neurons and the feedback from egg laying in regulating the germline response to a sex pheromone in C. elegans hermaphrodites.

Animals broadcast small molecule pheromones that can alter behavior and physiology in conspecifics. Neuronal circuits that regulate these processes remain largely unknown. In C. elegans, male-enriched ascaroside sex pheromone ascr#10, in addition to behavioral effects, expands the population of germline precursor cells in hermaphrodites. Previously, we identified several sensory neurons required for this effect. We also found that feedback from egg laying acts via serotonergic signaling to license the pheromone response in reproducing adults. Here, using newly available reagents, we confirm and extend several of our previous conclusions: a) the ADL neurons are essential for the ascr#10 response, b) phasmid neurons (PHA and PHB) are unlikely to be involved in the ascr#10 response, c) the mod-1 receptor is the main conduit of the serotonergic feedback from egg laying, and d) serotonin remains the only currently known signal of this feedback. Our findings better define the neuronal circuits that mediate the germline response to the major male pheromone.

Coordinated Behavioral and Physiological Responses to a Social Signal Are Regulated by a Shared Neuronal Circuit.

Successful reproduction in animals requires orchestration of behavior and physiological processes. Pheromones can induce both "releaser" (behavioral) and "priming" (physiological) effects [1] in vertebrates [2, 3] and invertebrates [4, 5]. Therefore, understanding the mechanisms underlying pheromone responses could reveal how reproduction-related behaviors and physiology are coordinated. Here, we describe a neuronal circuit that couples the reproductive system and behavior in adult Caenorhabditis elegans hermaphrodites. We found that the response of the oogenic germline to the male pheromone requires serotonin signal from NSM and HSN neurons that acts via the mod-1 receptor in AIY and RIF interneurons and is antagonized by pigment-dispersing factor (PDF). Surprisingly, the same neurons and pathways have been previously implicated in regulation of exploratory behavior in the absence of male-produced signals [6]. We demonstrate that male pheromone acts via this circuit in hermaphrodites to reduce exploration and decrease mating latency, thereby tuning multiple fitness-proximal processes. Our results demonstrate how a single circuit could coordinate behavioral and physiological responses to the environment, even those that unfold on different timescales. Our findings suggest the existence of a centralized regulatory mechanism that balances organismal resources between reproductive investment and somatic maintenance.

Dynamic Regulation of Adult-Specific Functions of the Nervous System by Signaling from the Reproductive System.

Unlike juveniles, adult animals engage in suites of behaviors related to the search for and selection of potential mates and mating, including appropriate responses to sex pheromones. As in other species [1], male sex pheromones modulate several behaviors and physiological processes in C. elegans hermaphrodites [2-5]. In particular, one of these small-molecule signals, an ascaroside ascr#10, causes reduced exploration, more avid mating, and improved reproductive performance (see the accompanying paper by Aprison and Ruvinsky in this issue of Current Biology) [6]. Here, we investigated the mechanism that restricts pheromone response to adult hermaphrodites. Unexpectedly, we found that attainment of developmental adulthood was not alone sufficient for the behavioral response to the pheromone. To modify exploratory behavior in response to male pheromone, adult hermaphrodites also require functional germline and egg-laying apparatus. We show that this dependence of behavior on the reproductive system is due to feedback from the vulva muscles that reports ongoing reproduction to the nervous system. Our results reveal an activity-dependent conduit by which the reproductive system continuously licenses adult behaviors, including appropriate responses to the pheromones of the opposite sex. More broadly, our results suggest that signals from peripheral organs may serve as an important component of assuring age-appropriate functions of the nervous system.

Counteracting Ascarosides Act through Distinct Neurons to Determine the Sexual Identity of C. elegans Pheromones.

Sex pheromones facilitate reproduction by attracting potential mates and altering their behavior and physiology. In C. elegans, males and hermaphrodites secrete similar blends of pheromone molecules, two of which are present in different relative concentrations: ascr#3, which is more abundant in hermaphrodites, and ascr#10, which is more abundant in males. It is not currently understood how this compositional difference results in sex-specific effects, for example, the slower aging of the hermaphrodite germline in the presence of physiologically relevant concentrations of male pheromones. Here we report three key elements of the mechanism responsible for this phenomenon. First, ascr#3 counters the activity of ascr#10. This antagonism decreases the magnitude and the sensitivity of the hermaphrodite response to the male pheromone, restricting it to situations in which the presence of a male could be inferred with high confidence. Second, hermaphrodites recognize pheromone as male if the concentration of ascr#10 is higher than that of ascr#3. Third, the response to ascr#10 requires TRPV channel function in the ADL neurons and the daf-7 signaling from the ASI neurons, whereas the response to ascr#3 relies on cyclic guanosine monophosphate (cGMP)-gated channels and activity of the ASJ, AWB, and AWC neurons. These results argue that the counteracting activities of distinct neuronal circuits determine the sexual identity of the pheromone. The parallels between this mechanism and other signaling systems suggest that diverse organisms may perform particular neuronal computations using similar general principles.

Sexually Antagonistic Male Signals Manipulate Germline and Soma of C. elegans Hermaphrodites.

Males and females pursue different reproductive strategies, which often bring them into conflict-many traits exist that benefit one sex at a cost to another [1]. Decreased female survival following mating dramatically demonstrates one aspect of this phenomenon [2-5]. Particularly intriguing is the evidence that secreted compounds can shorten lifespan of members of the opposite sex in Drosophila [6] and Caenorhabditid nematodes [7] even without copulation taking place. The purpose of such signals is not clear, however. While it is possible that they could limit subsequent mating with competitors or hasten post-reproductive demise, thus decreasing competition for resources, they are also likely to harm unmated individuals. Why would a system exist that reduces the vigor of potential mates prior to mating? Addressing this question could provide insights into mechanisms and evolution of sexual conflict and reveal sensory inputs that regulate aging. Here, we describe two distinct ways in which Caenorhabditis elegans males cause faster somatic aging of hermaphrodites but also manipulate different aspects of their reproductive physiology. The first, mediated by conserved ascaroside pheromones, delays the loss of germline progenitor cells. The second accelerates development, resulting in faster sexual maturation. These signals promote male reproductive strategy and the effects harmful to hermaphrodites appear to be collateral damage rather than the goal.

Experience Modulates the Reproductive Response to Heat Stress in C. elegans via Multiple Physiological Processes.

Natural environments are considerably more variable than laboratory settings and often involve transient exposure to stressful conditions. To fully understand how organisms have evolved to respond to any given stress, prior experience must therefore be considered. We investigated the effects of individual and ancestral experience on C. elegans reproduction. We documented ways in which cultivation at 15°C or 25°C affects developmental time, lifetime fecundity, and reproductive performance after severe heat stress that exceeds the fertile range of the organism but is compatible with survival and future fecundity. We found that experience modulates multiple aspects of reproductive physiology, including the male and female germ lines and the interaction between them. These responses vary in their environmental sensitivity, suggesting the existence of complex mechanisms for coping with unpredictable and stressful environments.

Balanced trade-offs between alternative strategies shape the response of C. elegans reproduction to chronic heat stress.

To ensure long-term reproductive success organisms have to cope with harsh environmental extremes. A reproductive strategy that simply maximizes offspring production is likely to be disadvantageous because it could lead to a catastrophic loss of fecundity under unfavorable conditions. To understand how an appropriate balance is achieved, we investigated reproductive performance of C. elegans under conditions of chronic heat stress. We found that following even prolonged exposure to temperatures at which none of the offspring survive, worms could recover and resume reproduction. The likelihood of producing viable offspring falls precipitously after exposure to temperatures greater than 28°C primarily due to sperm damage. Surprisingly, we found that worms that experienced higher temperatures can recover considerably better, provided they did not initiate ovulation. Therefore mechanisms controlling this process must play a crucial role in determining the probability of recovery. We show, however, that suppressing ovulation is only beneficial under relatively long stresses, whereas it is a disadvantageous strategy under shorter stresses of the same intensity. This is because the benefit of shutting down egg laying, and thus protecting the reproductive system, is negated by the cost associated with implementing this strategy--it takes considerable time to recover and produce offspring. We interpret these balanced trade-offs as a dynamic response of the C. elegans reproductive system to stress and an adaptation to life in variable and unpredictable conditions.