Body stiffness is a mechanical property that facilitates contact-mediated mate recognition in Caenorhabditis elegans.

Physical contact is prevalent in the animal kingdom to recognize suitable mates by decoding information about sex, species, and maturity. Although chemical cues for mate recognition have been extensively studied, the role of mechanical cues remains elusive. Here, we show that C. elegans males recognize conspecific and reproductive mates through short-range cues, and that the attractiveness of potential mates depends on the sex and developmental stages of the hypodermis. We find that a particular group of cuticular collagens is required for mate attractiveness. These collagens maintain body stiffness to sustain mate attractiveness but do not affect the surface properties that evoke the initial step of mate recognition, suggesting that males utilize multiple sensory mechanisms to recognize suitable mates. Manipulations of body stiffness via physical interventions, chemical treatments, and 3D-printed bionic worms indicate that body stiffness is a mechanical property for mate recognition and increases mating efficiency. Our study thus extends the repertoire of sensory cues of mate recognition in C. elegans and provides a paradigm to study the important roles of mechanosensory cues in social behaviors.

Pathogen metabolite checkpoint: NHR on guard.

How can beneficial microorganisms be distinguished from pathogenic ones? In this issue of Immunity, Peterson et al. discovered that a specific phenazine, which is part of a family of toxic metabolites expressed by pathogenic bacteria, is detected by Caenorhabditis elegans by directly binding to a nuclear hormone receptor, promoting the expression of detoxifying enzymes and immunity-related genes, thus protecting the worm.

Meisosomes, folded membrane microdomains between the apical extracellular matrix and epidermis.

Apical extracellular matrices (aECMs) form a physical barrier to the environment. In Caenorhabditis elegans, the epidermal aECM, the cuticle, is composed mainly of different types of collagen, associated in circumferential ridges separated by furrows. Here, we show that in mutants lacking furrows, the normal intimate connection between the epidermis and the cuticle is lost, specifically at the lateral epidermis, where, in contrast to the dorsal and ventral epidermis, there are no hemidesmosomes. At the ultrastructural level, there is a profound alteration of structures that we term 'meisosomes,' in reference to eisosomes in yeast. We show that meisosomes are composed of stacked parallel folds of the epidermal plasma membrane, alternately filled with cuticle. We propose that just as hemidesmosomes connect the dorsal and ventral epidermis, above the muscles, to the cuticle, meisosomes connect the lateral epidermis to it. Moreover, furrow mutants present marked modifications of the biomechanical properties of their skin and exhibit a constitutive damage response in the epidermis. As meisosomes co-localise to macrodomains enriched in phosphatidylinositol (4,5) bisphosphate, they could conceivably act, like eisosomes, as signalling platforms, to relay tensile information from the aECM to the underlying epidermis, as part of an integrated stress response to damage.

ATFS-1 plays no repressive role in the regulation of epidermal immune response.

Fungal infection triggers the induction of antimicrobial peptide (AMP) genes in the epidermis (Pujol et al, 2008). We previously showed that this effect is suppressed by the mitochondrial unfolded protein response (UPRmt), which can be activated by knockdown of select genes including the mitochondrial metalloprotease spg-7 (Zugasti et al, 2016). Here, we confirm that RNAi against spg-7 triggers the UPRmt and blocks AMP induction during infection, whereas infection itself does not trigger the UPRmt. ATFS-1 is a key factor in the UPRmt, mediating much of the associated transcriptional response. We find that, surprisingly, ATFS-1 is not required for the suppression of AMP induction provoked by spg-7(RNAi). These data show that the mitochondrial dysfunction that blocks the immune response upon infection or wounding is independent of ATFS-1.

C. elegans: out on an evolutionary limb.

The natural environment of the free-living nematode Caenorhabditis elegans is rich in pathogenic microbes. There is now ample evidence to indicate that these pathogens exert a strong selection pressure on C. elegans, and have shaped its genome, physiology, and behaviour. In this short review, we concentrate on how C. elegans stands out from other animals in terms of its immune repertoire and innate immune signalling pathways. We discuss how C. elegans often detects pathogens because of their effects on essential cellular processes, or organelle integrity, in addition to direct microbial recognition. We illustrate the extensive molecular plasticity that is characteristic of immune defences in C. elegans and highlight some remarkable instances of lineage-specific innovation in innate immune mechanisms.

Antagonistic fungal enterotoxins intersect at multiple levels with host innate immune defences.

Animals and plants need to defend themselves from pathogen attack. Their defences drive innovation in virulence mechanisms, leading to never-ending cycles of co-evolution in both hosts and pathogens. A full understanding of host immunity therefore requires examination of pathogen virulence strategies. Here, we take advantage of the well-studied innate immune system of Caenorhabditis elegans to dissect the action of two virulence factors from its natural fungal pathogen Drechmeria coniospora. We show that these two enterotoxins have strikingly different effects when expressed individually in the nematode epidermis. One is able to interfere with diverse aspects of host cell biology, altering vesicle trafficking and preventing the key STAT-like transcription factor STA-2 from activating defensive antimicrobial peptide gene expression. The second increases STA-2 levels in the nucleus, modifies the nucleolus, and, potentially as a consequence of a host surveillance mechanism, causes increased defence gene expression. Our results highlight the remarkably complex and potentially antagonistic mechanisms that come into play in the interaction between co-evolved hosts and pathogens.

ifas-1 is upregulated by fungal infection in a GPA-12 and STA-2-independent manner in the Caenorhabditis elegans epidermis.

Skin infection with the fungus Drechmeria coniospora leads to a transcriptional response in the worm epidermis. This involves an increased expression of a group of antimicrobial peptide (AMP) genes including those in the nlp-29 and cnc-2 clusters. The major pathways leading to the expression of these AMP genes have been well characterized and converge on the STAT transcription factor STA-2. We reported previously that expression in the epidermis of a constitutively active (gain of function, gf) form of the Gα protein GPA-12 (GPA-12gf) recapitulates much of the response to infection. To reveal parallel pathways activated by infection, we focus here on an effector gene that is not induced by GPA-12gf. This gene, ifas-1, encodes a protein with a fascin domain, associated with actin binding. Its induction upon fungal infection does not require sta-2. A transcriptional reporter revealed induction in the epidermis of ifas-1 by infection and wounding. Thus, ifas-1 represents part of a previously unexplored aspect of the innate immune response to infection.

Innate immunity in C. elegans.

In its natural habitat, C. elegans encounters a wide variety of microbes, including food, commensals and pathogens. To be able to survive long enough to reproduce, C. elegans has developed a complex array of responses to pathogens. These activities are coordinated on scales that range from individual organelles to the entire organism. Often, the response is triggered within cells, by detection of infection-induced damage, mainly in the intestine or epidermis. C. elegans has, however, a capacity for cell non-autonomous regulation of these responses. This frequently involves the nervous system, integrating pathogen recognition, altering host biology and governing avoidance behavior. Although there are significant differences with the immune system of mammals, some mechanisms used to limit pathogenesis show remarkable phylogenetic conservation. The past 20 years have witnessed an explosion of host-pathogen interaction studies using C. elegans as a model. This review will discuss the broad themes that have emerged and highlight areas that remain to be fully explored.

unc-119 mutants have an increased fungal spore adhesion that is not rescued by Cb-unc-119.

If the cuticle acts as a protective barrier against environmental insults, several pathogens have developed strategies that use it as a way to infect C. elegans. The fungus Drechmeria coniospora produces spores that attach to the cuticle, before hyphae invade the body. Mutants with an altered surface coat, the outermost layer of the cuticle, including bus-2, bus-4, bus-12 and bus-17 show increased adhesion of fungal spores (Rouger et al, 2014; Zugasti et al, 2016). We unexpectedly found that D. coniospora spores attach unusually densely around the mouth of unc-119 mutants. Interestingly, this phenotype is not rescued by the C. briggsae unc-119 construct that is conventionally used to rescue neuronal unc-119 phenotypes.

Comparison of lipidome profiles of Caenorhabditis elegans-results from an inter-laboratory ring trial.

INTRODUCTION: Lipidomic profiling allows 100s if not 1000s of lipids in a sample to be detected and quantified. Modern lipidomics techniques are ultra-sensitive assays that enable the discovery of novel biomarkers in a variety of fields and provide new insight in mechanistic investigations. Despite much progress in lipidomics, there remains, as for all high throughput "omics" strategies, the need to develop strategies to standardize and integrate quality control into studies in order to enhance robustness, reproducibility, and usability of studies within specific fields and beyond. OBJECTIVES: We aimed to understand how much results from lipid profiling in the model organism Caenorhabditis elegans are influenced by different culture conditions in different laboratories. METHODS: In this work we have undertaken an inter-laboratory study, comparing the lipid profiles of N2 wild type C. elegans and daf-2(e1370) mutants lacking a functional insulin receptor. Sample were collected from worms grown in four separate laboratories under standardized growth conditions. We used an UPLC-UHR-ToF-MS system allowing chromatographic separation before MS analysis. RESULTS: We found common qualitative changes in several marker lipids in samples from the individual laboratories. On the other hand, even in this controlled experimental system, the exact fold-changes for each marker varied between laboratories. CONCLUSION: Our results thus reveal a serious limitation to the reproducibility of current lipid profiling experiments and reveal challenges to the integration of such data from different laboratories.

Innate Immunity Promotes Sleep through Epidermal Antimicrobial Peptides.

Wounding and infection trigger a protective innate immune response that includes the production of antimicrobial peptides in the affected tissue as well as increased sleep. Little is known, however, how peripheral wounds or innate immunity signal to the nervous system to increase sleep. We found that, during C. elegans larval molting, an epidermal tolloid/bone morphogenic protein (BMP)-1-like protein called NAS-38 promotes sleep. NAS-38 is negatively regulated by its thrombospondin domain and acts through its astacin protease domain to activate p38 mitogen-activated protein (MAP)/PMK-1 kinase and transforming growth factor ß (TGF-ß)-SMAD/SMA-3-dependent innate immune pathways in the epidermis that cause STAT/STA-2 and SLC6 (solute carrier)/SNF-12-dependent expression of antimicrobial peptide (AMP) genes. We show that more than a dozen epidermal AMPs act as somnogens, signaling across tissues to promote sleep through the sleep-active RIS neuron. In the adult, epidermal injury activates innate immunity and turns up AMP production to trigger sleep, a process that requires epidermal growth factor receptor (EGFR) signaling that is known to promote sleep following cellular stress. We show for one AMP, neuropeptide-like protein (NLP)-29, that it acts through the neuropeptide receptor NPR-12 in locomotion-controlling neurons that are presynaptic to RIS and that depolarize this neuron to induce sleep. Sleep in turn increases the chance of surviving injury. Thus, we found a novel mechanism by which peripheral wounds signal to the nervous system to increase protective sleep. Such a cross-tissue somnogen-signaling function of AMPs might also boost sleep in other animals, including humans.

New Strains for Tissue-Specific RNAi Studies in Caenorhabditis elegans.

RNA interference is a powerful tool for dissecting gene function. In Caenorhabditis elegans, ingestion of double stranded RNA causes strong, systemic knockdown of target genes. Further insight into gene function can be revealed by tissue-specific RNAi techniques. Currently available tissue-specific C. elegans strains rely on rescue of RNAi function in a desired tissue or cell in an otherwise RNAi deficient genetic background. We attempted to assess the contribution of specific tissues to polyunsaturated fatty acid (PUFA) synthesis using currently available tissue-specific RNAi strains. We discovered that rde-1(ne219), a commonly used RNAi-resistant mutant strain, retains considerable RNAi capacity against RNAi directed at PUFA synthesis genes. By measuring changes in the fatty acid products of the desaturase enzymes that synthesize PUFAs, we found that the before mentioned strain, rde-1(ne219) and the reported germline only RNAi strain, rrf-1(pk1417) are not appropriate genetic backgrounds for tissue-specific RNAi experiments. However, the knockout mutant rde-1(ne300) was strongly resistant to dsRNA induced RNAi, and thus is more appropriate for construction of a robust tissue-specific RNAi strains. Using newly constructed strains in the rde-1(null) background, we found considerable desaturase activity in intestinal, epidermal, and germline tissues, but not in muscle. The RNAi-specific strains reported in this study will be useful tools for C. elegans researchers studying a variety of biological processes.

Microtubule plus-end dynamics link wound repair to the innate immune response.

The skin protects animals from infection and physical damage. In Caenorhabditis elegans, wounding the epidermis triggers an immune reaction and a repair response, but it is not clear how these are coordinated. Previous work implicated the microtubule cytoskeleton in the maintenance of epidermal integrity (Chuang et al., 2016). Here, by establishing a simple wounding system, we show that wounding provokes a reorganisation of plasma membrane subdomains. This is followed by recruitment of the microtubule plus end-binding protein EB1/EBP-2 around the wound and actin ring formation, dependent on ARP2/3 branched actin polymerisation. We show that microtubule dynamics are required for the recruitment and closure of the actin ring, and for the trafficking of the key signalling protein SLC6/SNF-12 toward the injury site. Without SNF-12 recruitment, there is an abrogation of the immune response. Our results suggest that microtubule dynamics coordinate the cytoskeletal changes required for wound repair and the concomitant activation of innate immunity.

Evolutionary plasticity in the innate immune function of Akirin.

Eukaryotic gene expression requires the coordinated action of transcription factors, chromatin remodelling complexes and RNA polymerase. The conserved nuclear protein Akirin plays a central role in immune gene expression in insects and mammals, linking the SWI/SNF chromatin-remodelling complex with the transcription factor NFκB. Although nematodes lack NFκB, Akirin is also indispensable for the expression of defence genes in the epidermis of Caenorhabditis elegans following natural fungal infection. Through a combination of reverse genetics and biochemistry, we discovered that in C. elegans Akirin has conserved its role of bridging chromatin-remodellers and transcription factors, but that the identity of its functional partners is different since it forms a physical complex with NuRD proteins and the POU-class transcription factor CEH-18. In addition to providing a substantial step forward in our understanding of innate immune gene regulation in C. elegans, our results give insight into the molecular evolution of lineage-specific signalling pathways.

A Damage Sensor Associated with the Cuticle Coordinates Three Core Environmental Stress Responses in Caenorhabditis elegans.

Extracellular matrix barriers and inducible cytoprotective genes form successive lines of defense against chemical and microbial environmental stressors. The barrier in nematodes is a collagenous extracellular matrix called the cuticle. In Caenorhabditis elegans, disruption of some cuticle collagen genes activates osmolyte and antimicrobial response genes. Physical damage to the epidermis also activates antimicrobial responses. Here, we assayed the effect of knocking down genes required for cuticle and epidermal integrity on diverse cellular stress responses. We found that disruption of specific bands of collagen, called annular furrows, coactivates detoxification, hyperosmotic, and antimicrobial response genes, but not other stress responses. Disruption of other cuticle structures and epidermal integrity does not have the same effect. Several transcription factors act downstream of furrow loss. SKN-1/Nrf and ELT-3/GATA are required for detoxification, SKN-1/Nrf is partially required for the osmolyte response, and STA-2/Stat and ELT-3/GATA for antimicrobial gene expression. Our results are consistent with a cuticle-associated damage sensor that coordinates detoxification, hyperosmotic, and antimicrobial responses through overlapping, but distinct, downstream signaling.