Pathogenic bacteria modulate pheromone response to promote mating.

Pathogens generate ubiquitous selective pressures and host-pathogen interactions alter social behaviours in many animals1-4. However, very little is known about the neuronal mechanisms underlying pathogen-induced changes in social behaviour. Here we show that in adult Caenorhabditis elegans hermaphrodites, exposure to a bacterial pathogen (Pseudomonas aeruginosa) modulates sensory responses to pheromones by inducing the expression of the chemoreceptor STR-44 to promote mating. Under standard conditions, C. elegans hermaphrodites avoid a mixture of ascaroside pheromones to facilitate dispersal5-13. We find that exposure to the pathogenic Pseudomonas bacteria enables pheromone responses in AWA sensory neurons, which mediate attractive chemotaxis, to suppress the avoidance. Pathogen exposure induces str-44 expression in AWA neurons, a process regulated by a transcription factor zip-5 that also displays a pathogen-induced increase in expression in AWA. STR-44 acts as a pheromone receptor and its function in AWA neurons is required for pathogen-induced AWA pheromone response and suppression of pheromone avoidance. Furthermore, we show that C. elegans hermaphrodites, which reproduce mainly through self-fertilization, increase the rate of mating with males after pathogen exposure and that this increase requires str-44 in AWA neurons. Thus, our results uncover a causal mechanism for pathogen-induced social behaviour plasticity, which can promote genetic diversity and facilitate adaptation of the host animals.

Effects of crowding stress on the hypothalamo-pituitary-interrenal axis of the self-fertilizing fish, Kryptolebias marmoratus.

We tested whether crowding stress affects the hypothalamo-pituitary-interrenal (HPI) axis of the self-fertilizing fish, Kryptolebias marmoratus, which is known to be aggressive in the laboratory conditions but sometimes found as a group from a single land crab burrow in the wild. The projection of corticotropin-releasing hormone (CRH) neurons to the adrenocorticotropic hormone (ACTH) cells in the pituitary was confirmed by dual-label immunohistochemistry; CRH-immunoreactive (ir) fibers originating from cell bodies located in the lateral tuberal nucleus (NLT) of the hypothalamus were observed to project to ACTH-ir cells in the rostral pars distalis of the pituitary. Then, fish were reared solitary or in pairs for 14 days, and the number of CRH-ir cell bodies in the NLT of the hypothalamus and cortisol levels in the body without head region were compared. The number of CRH-ir cell bodies and cortisol levels were significantly higher in paired fish. These results indicate that crowding stress affects the HPI axis in K. marmoratus which thrive in small burrows with limited water volume.

A new experimental model for the investigation of sequential hermaphroditism.

The stunning sexual transformation commonly triggered by age, size or social context in some fishes is one of the best examples of phenotypic plasticity thus far described. To date our understanding of this process is dominated by studies on a handful of subtropical and tropical teleosts, often in wild settings. Here we have established the protogynous New Zealand spotty wrasse, Notolabrus celidotus, as a temperate model for the experimental investigation of sex change. Captive fish were induced to change sex using aromatase inhibition or manipulation of social groups. Complete female-to-male transition occurred over 60 days in both cases and time-series sampling was used to quantify changes in hormone production, gene expression and gonadal cellular anatomy. Early-stage decreases in plasma 17ß-estradiol (E2) concentrations or gonadal aromatase (cyp19a1a) expression were not detected in spotty wrasse, despite these being commonly associated with the onset of sex change in subtropical and tropical protogynous (female-to-male) hermaphrodites. In contrast, expression of the masculinising factor amh (anti-Müllerian hormone) increased during early sex change, implying a potential role as a proximate trigger for masculinisation. Collectively, these data provide a foundation for the spotty wrasse as a temperate teleost model to study sex change and cell fate in vertebrates.

Distinct neuropeptide-receptor modules regulate a sex-specific behavioral response to a pheromone.

Dioecious species are a hallmark of the animal kingdom, with opposing sexes responding differently to identical sensory cues. Here, we study the response of C. elegans to the small-molecule pheromone, ascr#8, which elicits opposing behavioral valences in each sex. We identify a novel neuropeptide-neuropeptide receptor (NP/NPR) module that is active in males, but not in hermaphrodites. Using a novel paradigm of neuropeptide rescue that we established, we leverage bacterial expression of individual peptides to rescue the sex-specific response to ascr#8. Concurrent biochemical studies confirmed individual FLP-3 peptides differentially activate two divergent receptors, NPR-10 and FRPR-16. Interestingly, the two of the peptides that rescued behavior in our feeding paradigm are related through a conserved threonine, suggesting that a specific NP/NPR combination sets a male state, driving the correct behavioral valence of the ascr#8 response. Receptor expression within pre-motor neurons reveals novel coordination of male-specific and core locomotory circuitries.

Photoperiod controls egg laying and caudodorsal cell hormone expression but not gonadal development in the pond snail Lymnaea stagnalis.

Photoperiod is a reliable cue to regulate growth and reproduction for seasonal adaptation. Although photoperiodism has been well studied in Chordata and Arthropoda, less is known about Mollusca. We examined photoperiodic effects on egg laying, body size, gonad-somatic index, oocyte size and relative amounts of caudodorsal cell hormone mRNA in individual rearing conditions in the pond snail Lymnaea stagnalis. Twenty-five weeks after hatching, the percentages of egg-laying snails under a photoperiod of 12 h light and 12 h darkness (12L:12D) were significantly smaller than those under longer days. The total numbers of eggs and egg masses under 12L:12D were significantly smaller than those under longer days. Significant differences between 16L:8D and 12L:12D were not observed in the soft body and ovotestis weight, and the gonad-somatic index. Photoperiodic effects were also not observed in oocyte diameters twenty-two weeks after hatching. Twenty-seven weeks after hatching amounts of caudodorsal cell hormone mRNA were significantly lower in the cerebral ganglia with commissure under 12L:12D than 16L:8D. L. stagnalis exhibited a clear photoperiodic response in egg laying and the amount of caudodorsal cell hormone mRNA, but not in gonadal development. Under 12L:12D suppression of caudodorsal cell hormone expression might suppress egg laying.

Testicular inducing steroidogenic cells trigger sex change in groupers.

Vertebrates usually exhibit gonochorism, whereby their sex is fixed throughout their lifetime. However, approximately 500 species (~ 2%) of extant teleost fishes change sex during their lifetime. Although phylogenetic and evolutionary ecological studies have recently revealed that the extant sequential hermaphroditism in teleost fish is derived from gonochorism, the evolution of this transsexual ability remains unclear. We revealed in a previous study that the tunica of the ovaries of several protogynous hermaphrodite groupers contain functional androgen-producing cells, which were previously unknown structures in the ovaries of gonochoristic fishes. Additionally, we demonstrated that these androgen-producing cells play critical roles in initiating female-to-male sex change in several grouper species. In the present study, we widened the investigation to include 7 genera and 18 species of groupers and revealed that representatives from most major clades of extant groupers commonly contain these androgen-producing cells, termed testicular-inducing steroidogenic (TIS) cells. Our findings suggest that groupers acquired TIS cells in the tunica of the gonads for successful sex change during their evolution. Thus, TIS cells trigger the evolution of sex change in groupers.

Inferences of glia-mediated control in Caenorhabditis elegans.

Astrocytes modulate synaptic transmission; yet, it remains unclear how glia influence complex behaviors. Here, we explore the effects of Caenorhabditis elegans astrocyte-like cephalic glia (CEPglia ) and the glia-specific bHLH transcription factor HLH-17 on mating behavior and the defecation motor program (DMP). In C. elegans, male mating has been explicitly described through the male tail circuit and is characterized by coordination of multiple independent behaviors to ensure that copulation is achieved. Furthermore, the sex-specific male mating circuitry shares similar components with the DMP, which is complex and rhythmic, and requires a fixed sequence of behaviors to be activated periodically. We found that loss of CEPglia reduced persistence in executing mating behaviors and hindered copulation, while males that lacked HLH-17 demonstrated repetitive prodding behavior that increased the time spent in mating but did not hinder copulation. During the DMP, we found that posterior body wall contractions (pBocs) and enteric muscle contractions (EMCs) were differentially affected by loss of HLH-17 or CEPglia in males and hermaphrodites. pBocs and EMCs required HLH-17 activity in both sexes, whereas loss of CEPglia alone did not affect DMP in males. Our data suggest that CEPglia mediate complex behaviors by signaling to the GABAergic DVB neuron, and that HLH-17 activity influences those discrete steps within those behaviors. Collectively, these data provide evidence of glia as a link in cooperative regulation of complex and rhythmic behavior that, in C. elegans links circuitry in the head and the tail.

Sexual conflict in protandrous flowers and the evolution of gynodioecy.

Sexual interference between male and female function in hermaphrodite plants is reduced by protandry. In environments with insufficient pollinator service, prolongation of male function owing to limited pollen removal could restrict the duration of female function and lower seed production. We provide evidence that this form of sexual conflict has played a role in the spread of females in gynodioecious populations of Cyananthus delavayi in the pollen-limited environments in which this subalpine species occurs. Using field experiments involving artificial pollen removal from the strongly protandrous flowers of hermaphrodites, we demonstrated a trade-off between male- and female-phase duration with no influence on overall floral longevity. Pollen removal at the beginning of anthesis resulted in hermaphrodite seed production matching that of females. In contrast, restricted pollen removal increased the duration of male function at the expense of female function lowering maternal fertility compared to females. This pattern was evident in five populations with females experiencing a twofold average seed fertility advantage compared to hermaphrodites. Gynodioecy often appears to evolve from protandrous ancestors and pollen limitation is widespread in flowering plants suggesting that sexual conflict may play an unappreciated role in the evolution of this form of sexual dimorphism.

The type II integral ER membrane protein VAP-B homolog in C. elegans is cleaved to release the N-terminal MSP domain to signal non-cell-autonomously.

VAMP/synaptobrevin-associated protein B (VAP-B) is a type II ER membrane protein, but its N-terminal MSP domain (MSPd) can be cleaved and secreted. Mutations preventing the cleavage and secretion of MSPd have been implicated in cases of human neurodegenerative diseases. The site of VAP cleavage and the tissues capable in releasing the processed MSPd are not understood. In this study, we analyze the C. elegans VAP-B homolog, VPR-1, for its processing and secretion from the intestine. We show that intestine-specific expression of an N-terminally FLAG-tagged VPR-1 rescues underdeveloped gonad and sterility defects in vpr-1 null hermaphrodites. Immunofluorescence studies reveal that the tagged intestinal expressed VPR-1 is present at the distal gonad. Mass spectrometry analysis of a smaller product of the N-terminally tagged VPR-1 identifies a specific cleavage site at Leu156. Mutation of the leucine results in loss of gonadal MSPd signal and reduced activity of the mutant VPR-1. Thus, we report for the first time the cleavage site of VPR-1 and provide direct evidence that intestinally expressed VPR-1 can be released and signal in the distal gonad. These results establish the foundation for further exploration of VAP cleavage, MSPd secretion, and non-cell-autonomous signaling in development and diseases.

Social and sexual behaviors in C. elegans: the first fifty years.

For the first 25 years after the landmark 1974 paper that launched the field, most C. elegans biologists were content to think of their subjects as solitary creatures. C. elegans presented no shortage of fascinating biological problems, but some of the features that led Brenner to settle on this species-in particular, its free-living, self-fertilizing lifestyle-also seemed to reduce its potential for interesting social behavior. That perspective soon changed, with the last two decades bringing remarkable progress in identifying and understanding the complex interactions between worms. The growing appreciation that C. elegans behavior can only be meaningfully understood in the context of its ecology and evolution ensures that the coming years will see similarly exciting progress.

Seed germination of gynodioecious species: theoretical considerations and a comparison of females and hermaphrodites.

MAIN CONCLUSION: Better seed germination of females than of hermaphrodites is not a major contributor to the greater geometric lifetime fitness that females require to be maintained in a gynodioecious population. Gynodioecy is a sexually dimorphic breeding system in which females (F, male sterile) and hermaphrodites (H) coexist in the same population. For plants with nuclear (biparental) inheritance of male sterility, theory predicts that except when the product of selfing rate (s) and inbreeding depression (δ) in H is high (sδ > 0.50), F must compensate (female advantage) for the loss of gene transmission via pollen production by producing more or higher-quality offspring than H to be maintained in the population. For species with cytoplasmic (maternal) inheritance of male sterility, the female requires only a small compensation in seed production or some other offspring fitness trait to persist. Reallocation to seeds of resources saved by loss of pollen production is expected to increase the quantity (number) and/or quality (mass, germinability) of seeds produced by F, thus compensating for the lack of pollen production. The primary aim of our study was to compare seed germination of F and H via a literature review. Based on theoretical considerations, we hypothesized that seeds of F should germinate better or equally as well as those of H. We found that of 235 case studies for 47 species Fgerm > Hgerm in 48.1%, Fgerm = Hgerm in 38.3% and Fgerm < Hgerm in 13.6%. Our results are very similar to those of a previously published meta-analysis that included germination of F and H for 12 species. For 162 cases on seed size, F > H in 29.0%, F = H in 63.6% and F < H in 7.4%. Since [(Fgerm > Hgerm) < (Fgerm ≤ Hgerm)] and [(Fseedsize > Hseedsize) < (Fseedsize ≤ Hseedsize)], these results suggest that seed quality is not a major fitness component of female advantage.

Small molecule signals mediate social behaviors in C. elegans.

The last few decades have seen the structural and functional elucidation of small-molecule chemical signals called ascarosides in C. elegans. Ascarosides mediate several biological processes in worms, ranging from development, to behavior. These signals are modular in their design architecture, with their building blocks derived from metabolic pathways. Behavioral responses are not only concentration dependent, but also are influenced by the current physiological state of the animal. Cellular and circuit-level analyses suggest that these signals constitute a complex communication system, employing both synergistic molecular elements and sex-specific neuronal circuits governing the response. In this review, we discuss research from multiple laboratories, including our own, that detail how these chemical signals govern several different social behaviors in C. elegans. We propose that the ascaroside repertoire represents a link between diverse metabolic and neurobiological life-history traits and governs the survival of C. elegans in its natural environment.

What about the males? the C. elegans sexually dimorphic nervous system and a CRISPR-based tool to study males in a hermaphroditic species.

Sexual dimorphism is a device that supports genetic diversity while providing selective pressure against speciation. This phenomenon is at the core of sexually reproducing organisms. Caenorhabditis elegans provides a unique experimental system where males exist in a primarily hermaphroditic species. Early works of John Sulston, Robert Horvitz, and John White provided a complete map of the hermaphrodite nervous system, and recently the male nervous system was added. This addition completely realized the vision of C. elegans pioneer Sydney Brenner: a model organism with an entirely mapped nervous system. With this 'connectome' of information available, great strides have been made toward understanding concepts such as how a sex-shared nervous system (in hermaphrodites and males) can give rise to sex-specific functions, how neural plasticity plays a role in developing a dimorphic nervous system, and how a shared nervous system receives and processes external cues in a sexually-dimorphic manner to generate sex-specific behaviors. In C. elegans, the intricacies of male-mating behavior have been crucial for studying the function and circuitry of the male-specific nervous system and used as a model for studying human autosomal dominant polycystic kidney disease (ADPKD). With the emergence of CRISPR, a seemingly limitless tool for generating genomic mutations with pinpoint precision, the C. elegans model system will continue to be a useful instrument for pioneering research in the fields of behavior, reproductive biology, and neurogenetics.

Phenological variation of flower longevity and duration of sex phases in a protandrous alpine plant: potential causes and fitness significance.

BACKGROUND: Flower longevity plays an important role in pollen dispersal and reproductive success in plants. In dichogamous plants, the duration of anthesis as well as the time allocated to male and female functions can vary in response to intrinsic factors (e.g., flowering time and resource allocation) and pollination context along a growth season. However, the fitness consequences of phenological dynamics have rarely been examined. This study aims to unravel the potential causes driving variation in flower longevity, duration of sex phases, and phenotypic sex during a flowering season of strongly protandrous Aconitum gymnandrum, and particularly reproductive consequences of the phenological pattern. RESULTS: Population floral sex ratio shifted from completely male at the beginning to completely female at the end of the season, as is common in other protandrous plants. Phenological dynamics of the floral sex ratio and the duration of sex phases caused a shift from femaleness to maleness in the mean phenotypic sex over the whole season. Floral longevity was negatively correlated with flower size and positively affected by temperature. Early flowers within inflorescences rather than early-flowering individuals emphasized the duration of female over male phase. Owing to the dominance of male-phase flowers, early flowering for individual flowers and plants, or female-biased sex resulted in higher pollen deposition per flower and seed set. At the flower level, flower longevity positively affected female reproductive success, while the effect of flower size was negative. By contrast, plant-level female reproductive success was negatively affected by flower longevity but positively correlated to flower size. CONCLUSIONS: The major result of this study lies in elucidating the relationship between variation in phenological sex expression and floral longevity and their fitness consequences of protandrous A. gymnandrum. The contrasting results on female fitness for individual flowers and plants contribute to our current understanding of the adaptive significance of floral longevity.

Effects of two seminal fluid transcripts on post-mating behaviour in the simultaneously hermaphroditic flatworm Macrostomum lignano.

The seminal fluid proteins (SFPs) transferred to mating partners along with sperm often play crucial roles in mediating post-mating sexual selection. One way in which sperm donors can maximize their own reproductive success is by modifying the partner's (sperm recipient's) post-copulatory behaviour to prevent or delay re-mating, thereby decreasing the likelihood or intensity of sperm competition. Here, we adopted a quantitative genetic approach combining gene expression and behavioural data to identify candidates that could mediate such a response in the simultaneously hermaphroditic flatworm Macrostomum lignano. We identified two putative SFPs-Mlig-pro46 and Mlig-pro63-linked to both mating frequency and 'suck' frequency, a distinctive behaviour, in which, upon ejaculate receipt, the worm places its pharynx over its female genital opening and apparently attempts to remove the received ejaculate. We, therefore, performed a manipulative experiment using RNA interference-induced knockdown to ask how the loss of Mlig-pro46 and Mlig-pro63 expression, singly and in combination, affects mating frequency, partner suck propensity and sperm competitive ability. None of the knockdown treatments impacted strongly on the mating frequency or sperm competitive ability, but knockdown of Mlig-pro63 resulted in a significantly decreased suck propensity of mating partners. This suggests that Mlig-pro63 may normally act as a cue in the ejaculate to trigger recipient suck behaviour and-given that other proteins in the ejaculate have the opposite effect-could be one component of an ongoing arms race between donors and recipients over the control of ejaculate fate. However, the adaptive significance of Mlig-pro46 and Mlig-pro63 from a donor perspective remains enigmatic.

Three multi-allelic gene pairs are responsible for self-sterility in the ascidian Ciona intestinalis.

Many hermaphroditic organisms possess a self-incompatibility system to avoid inbreeding. Although the mechanisms of self-incompatibility in flowering plants are well known, little is known about the mechanisms of self-sterility in hermaphroditic marine invertebrates. Ascidians are hermaphroditic sessile marine invertebrates that release sperm and eggs into the surrounding seawater. Several species, including Ciona intestinalis type A (Ciona robusta), exhibit strict self-sterility. In a previous study, we found that the candidate genes responsible for self-sterility in Ciona reside in chromosome 2q (locus A) and chromosome 7q (locus B). Two pairs of multi-allelic genes, named s(sperm)-Themis-A and v(vitelline-coat)-Themis-A in locus A and s-Themis-B and v-Themis-B in locus B, are responsible for self-sterility. In this study, we identified a third multi-allelic gene pair, s-Themis-B2 and v-Themis-B2, within locus B that is also involved in this system. Genetic analysis revealed that the haplotypes of s/v-Themis-A, s/v-Themis-B and s/v-Themis-B2 play essential roles in self-sterility. When three haplotypes were matched between s-Themis and v-Themis, fertilization never occurred even in nonself crossing. Interestingly, gene targeting of either s/v-Themis-B/B2 or s/v-Themis-A by genome editing enabled self-fertilization. These results indicate that s/v-Themis-A, -B and -B2 are S-determinant genes responsible for self-sterility in the ascidian C. intestinalis type A.

The Evolution of Egg Trading in Simultaneous Hermaphrodites.

Egg trading-whereby simultaneous hermaphrodites exchange each other's eggs for fertilization-constitutes one of the few rigorously documented and most widely cited examples of direct reciprocity among unrelated individuals. Yet how egg trading may initially invade a population of nontrading simultaneous hermaphrodites is still unresolved. Here, we address this question with an analytical model that considers mate encounter rates and costs of egg production in a population that may include traders (who provide eggs for fertilization only if their partners also have eggs to reciprocate), providers (who provide eggs regardless of whether their partners have eggs to reciprocate), and withholders (cheaters who mate only in the male role and just use their eggs to elicit egg release from traders). Our results indicate that a combination of intermediate mate encounter rates, sufficiently high costs of egg production, and a sufficiently high probability that traders detect withholders (in which case eggs are not provided) is conducive to the evolution of egg trading. Under these conditions, traders can invade-and resist invasion from-providers and withholders alike. The prediction that egg trading evolves only under these specific conditions is consistent with the rare occurrence of this mating system among simultaneous hermaphrodites.

A perspective on C. elegans neurodevelopment: from early visionaries to a booming neuroscience research.

The formation of the nervous system and its striking complexity is a remarkable feat of development. C. elegans served as a unique model to dissect the molecular events in neurodevelopment, from its early visionaries to the current booming neuroscience community. Soon after being introduced as a model, C. elegans was mapped at the level of genes, cells, and synapses, providing the first metazoan with a complete cell lineage, sequenced genome, and connectome. Here, I summarize mechanisms underlying C. elegans neurodevelopment, from the generation and diversification of neural components to their navigation and connectivity. I point out recent noteworthy findings in the fields of glia biology, sex dimorphism and plasticity in neurodevelopment, highlighting how current research connects back to the pioneering studies by Brenner, Sulston and colleagues. Multifaceted investigations in model organisms, connecting genes to cell function and behavior, expand our mechanistic understanding of neurodevelopment while allowing us to formulate emerging questions for future discoveries.

Seminal Fluid-Mediated Manipulation of Post-mating Behavior in a Simultaneous Hermaphrodite.

Seminal fluid proteins (SFPs) are uniquely positioned to mediate post-mating sexual selection and sexual conflict [1-3]. This role may be especially important in simultaneous hermaphrodites, in which individuals will often agree to receive sperm in order to be able to donate it, shifting the arena of sexual selection to post-mating reproductive interactions [4-7]. Nevertheless, as in separate-sexed organisms, identifying individual SFPs responsible for specific post-mating effects is difficult, owing to the complexity, rapid evolution, and functional redundancy of seminal fluid [8-11]. Here, we sought to identify SFPs that influence one striking post-mating behavior of the simultaneously hermaphroditic flatworm Macrostomum lignano, the so-called "suck behavior," in which worms respond to ejaculate receipt by placing their pharynx over their female genital opening and seemingly attempt to remove sperm and/or other ejaculate components [12-14]. We hypothesized that sucking is counter to the sperm donor's interests, potentially selecting for SFPs that reduce the suck propensity of mating partners. We tested this using a combination of quantitative genetics and RNA interference (RNAi) knockdown. As predicted, we found negative genetic correlations between the expression levels of six (out of 58) seminal fluid transcripts and partner suck propensity. RNAi knockdown confirmed that two of these transcripts, designated suckless-1 and suckless-2, indeed caused mating partners to suck less often. We suggest that these proteins are male counter-adaptations to recipient suck behavior, which itself is likely a female counter-adaptation in the ongoing evolutionary conflict to (re)gain control over ejaculate fate after mating in this hermaphroditic organism.