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With chemical analysis, it is impossible to qualify and quantify the toxic potency of especially hydrophilic bioactive contaminants. In this study, we applied the nematode C. elegans as a model organism for detecting the toxic potency of whole influent wastewater samples. Gene expression in the nematode was used as bioanalytical tool to reveal the presence, type and potency of molecular pathways induced by 24-h exposure to wastewater from a hospital (H), nursing home (N), community (C), and influent (I) and treated effluent (E) from a local wastewater treatment plant. Exposure to influent water significantly altered expression of 464 genes, while only two genes were differentially expressed in nematodes treated with effluent. This indicates a significant decrease in bioactive pollutant-load after wastewater treatment. Surface water receiving the effluent did not induce any genes in exposed nematodes. A subset of 209 genes was differentially expressed in all untreated wastewaters, including cytochromes P450 and C-type lectins related to the nematode’s xenobiotic metabolism and immune response, respectively. Different subsets of genes responded to particular waste streams making them candidates to fingerprint-specific wastewater sources. This study shows that gene expression profiling in C. elegans can be used for mechanism-based identification of hydrophilic bioactive compounds and fingerprinting of specific wastewaters. More comprehensive than with chemical analysis, it can demonstrate the effective overall removal of bioactive compounds through wastewater treatment. This bioanalytical tool can also be applied in the process of identification of the bioactive compounds via a process of toxicity identification evaluation. Graphical abstract

Glutamylation is a post-translational modification found on tubulin that can alter the interaction between microtubules (MTs) and associated proteins. The molecular mechanisms regulating tubulin glutamylation in response to the environment are not well understood. Here, we show that in the sensory cilia of Caenorhabditis elegans, tubulin glutamylation is upregulated in response to various signals such as temperature, osmolality, and dietary conditions. Similarly, tubulin glutamylation is modified in mammalian photoreceptor cells following light adaptation. A tubulin glutamate ligase gene ttll-4, which is essential for tubulin glutamylation of axonemal MTs in sensory cilia, is activated by p38 MAPK. Amino acid substitution of TTLL-4 has revealed that a Thr residue (a putative MAPKphosphorylation site) is required for enhancement of tubulin glutamylation. Intraflagellar transport (IFT), a bidirectional trafficking system specifically observed along axonemal MTs, is required for the formation, maintenance, and function of sensory cilia. Measurement of the velocity of IFT particles revealed that starvation accelerates IFT, which was also dependent on the Thr residue of TTLL-4. Similarly, starvation-induced attenuation of avoidance behaviour from high osmolality conditions was also dependent on ttll-4. Our data suggest that a novel evolutionarily conserved regulatory system exists for tubulin glutamylation in sensory cilia in response to the environment. National Bio-Resource Project of the MEXT, Japan National Center for Research Resources, National Institutes of Health (NIH) JSPS KAKENHI 25116712 -25293044 -25440082 -12J06986 MEXT Project for Creation of Research Platforms and Sharing of Advanced Research Infrastructure MEXT JASSO Otsuka Toshimi Foundation Takeda Science Foundation

Genome maintenance defects cause complex disease phenotypes characterized by developmental failure, cancer susceptibility and premature ageing. It remains poorly understood how DNA damage responses function during organismal development and maintain tissue functionality when DNA damage accumulates with ageing. Here we show that the FOXO transcription factor DAF-16 is activated in response to DNA damage during development, whereas the DNA damage responsiveness of DAF-16 declines with ageing. We find that in contrast to its established role in mediating starvation arrest, DAF-16 alleviates DNA-damage-induced developmental arrest and even in the absence of DNA repair promotes developmental growth and enhances somatic tissue functionality. We demonstrate that the GATA transcription factor EGL-27 co-regulates DAF-16 target genes in response to DNA damage and together with DAF-16 promotes developmental growth. We propose that EGL-27/GATA activity specifies DAF-16-mediated DNA damage responses to enable developmental progression and to prolong tissue functioning when DNA damage persists.

Background Exercise exerts remarkably powerful effects on metabolism and health, with anti-disease and anti-aging outcomes. Pharmacological manipulation of exercise benefit circuits might improve the health of the sedentary and the aging populations. Still, how exercised muscle signals to induce system-wide health improvement remains poorly understood. With a long-term interest in interventions that promote animal-wide health improvement, we sought to define exercise options for Caenorhabditis elegans. Results Here, we report on the impact of single swim sessions on C. elegans physiology. We used microcalorimetry to show that C. elegans swimming has a greater energy cost than crawling. Animals that swam continuously for 90 min specifically consumed muscle fat supplies and exhibited post-swim locomotory fatigue, with both muscle fat depletion and fatigue indicators recovering within 1 hour of exercise cessation. Quantitative polymerase chain reaction (qPCR) transcript analyses also suggested an increase in fat metabolism during the swim, followed by the downregulation of specific carbohydrate metabolism transcripts in the hours post-exercise. During a 90 min swim, muscle mitochondria matrix environments became more oxidized, as visualized by a localized mitochondrial reduction-oxidation-sensitive green fluorescent protein reporter. qPCR data supported specific transcriptional changes in oxidative stress defense genes during and immediately after a swim. Consistent with potential antioxidant defense induction, we found that a single swim session sufficed to confer protection against juglone-induced oxidative stress inflicted 4 hours post-exercise. Conclusions In addition to showing that even a single swim exercise bout confers physiological changes that increase robustness, our data reveal that acute swimming-induced changes share common features with some acute exercise responses reported in humans. Overall, our data validate an easily implemented swim experience as C. elegans exercise, setting the foundation for exploiting the experimental advantages of this model to genetically or pharmacologically identify the exercise-associated molecules and signaling pathways that confer system-wide health benefits. Electronic supplementary material The online version of this article (doi:10.1186/s12915-017-0368-4) contains supplementary material, which is available to authorized users.

Many organisms have stress response pathways, components of which share homology with players in complex human disease pathways. Research on stress response in the nematode worm Caenorhabditis elegans has provided detailed insights into the genetic and molecular mechanisms underlying complex human diseases. In this review we focus on four different types of environmental stress responses – heat shock, oxidative stress, hypoxia, and osmotic stress – and on how these can be used to study the genetics of complex human diseases. All four types of responses involve the genetic machineries that underlie a number of complex human diseases such as cancer and neurodegenerative diseases, including Alzheimer's and Parkinson's. We highlight the types of stress response experiments required to detect the genes and pathways underlying human disease and suggest that studying stress biology in worms can be translated to understanding human disease and provide potential targets for drug discovery.

Increased lifespan can be associated with greater resistance to many different stressors, most notably thermal stress. Such hormetic effects have also been found in C. elegans where short-term exposure to heat lengthens the lifespan. Genetic investigations have been carried out using mutation perturbations in a single genotype, the wild type Bristol N2. Yet, induced mutations do not yield insight regarding the natural genetic variation of thermal tolerance and lifespan. We investigated the genetic variation of heat-shock recovery, i.e. hormetic effects on lifespan and associated quantitative trait loci (QTL) in C. elegans. Heat-shock resulted in an 18% lifespan increase in wild type CB4856 whereas N2 did not show a lifespan elongation. Using recombinant inbred lines (RILs) derived from a cross between wild types N2 and CB4856 we found natural variation in stress-response hormesis in lifespan. Approx. 28% of the RILs displayed a hormesis effect in lifespan. We did not find any hormesis effects for total offspring. Across the RILs there was no relation between lifespan and offspring. The ability to recover from heat-shock mapped to a significant QTL on chromosome II which overlapped with a QTL for offspring under heat-shock conditions. The QTL was confirmed by introgressing relatively small CB4856 regions into chromosome II of N2. Our observations show that there is natural variation in hormetic effects on C. elegans lifespan for heat-shock and that this variation is genetically determined.

Ecotoxicology research is using many methods for engineered nanomaterials (ENMs), but the collective experience from researchers has not been documented. This paper reports the practical issues for working with ENMs and suggests nano-specific modifications to protocols. The review considers generic practical issues, as well as specific issues for aquatic tests, marine grazers, soil organisms, and bioaccumulation studies. Current procedures for cleaning glassware are adequate, but electrodes are problematic. The maintenance of exposure concentration is challenging, but can be achieved with some ENMs. The need to characterize the media during experiments is identified, but rapid analytical methods are not available to do this. The use of sonication and natural/synthetic dispersants are discussed. Nano-specific biological endpoints may be developed for a tiered monitoring scheme to diagnose ENM exposure or effect. A case study of the algal growth test highlights many small deviations in current regulatory test protocols that are allowed (shaking, lighting, mixing methods), but these should be standardized for ENMs. Invertebrate (Daphnia) tests should account for mechanical toxicity of ENMs. Fish tests should consider semistatic exposure to minimize wastewater and animal husbandry. The inclusion of a benthic test is recommended for the base set of ecotoxicity tests with ENMs. The sensitivity of soil tests needs to be increased for ENMs and shortened for logistics reasons; improvements include using Caenorhabditis elegans, aquatic media, and metabolism endpoints in the plant growth tests. The existing bioaccumulation tests are conceptually flawed and require considerable modification, or a new test, to work for ENMs. Overall, most methodologies need some amendments, and recommendations are made to assist researchers.

Induced mutants in the nematode Caenorhabditis elegans are used to study genetic pathways of processes ranging from aging to behavior. The effects of such mutations are usually analyzed in a single wildtype background: N2. However, studies in other species demonstrate that the phenotype(s) of induced mutations can vary widely depending on the genetic background. Moreover, induced mutations in one genetic background do not reveal the allelic effects that segregate in natural populations and contribute to phenotypic variation. Because other wildtype Caenorhabditis spp., including C. elegans, are now available, we review how current mapping resources and methodologies within and between species support the use of Caenorhabditis spp. for studying genetic variation, with a focus on pathways associated with human disease.

This study aims to reveal whether complexity, namely, community and trophic structure, of chronically stressed soil systems is at increased risk or remains stable when confronted with a subsequent disturbance. Therefore, we focused on a grassland with a history of four centuries of patchy contamination. Nematodes were used as model organisms because they are an abundant and trophically diverse group and representative of the soil food web and ecosystem complexity. In a field survey, a relationship between contaminants and community structures was established. Following, two groups of soil mesocosms from the field that differed in contamination level were exposed to different disturbance regimes, namely, to the contaminant zinc and a heat shock. The zinc treatment revealed that community structure is stable, irrespective of soil contamination levels. This implies that centuries of exposure to contamination led to adaptation of the soil nematode community irrespective of the patchy distribution of contaminants. In contrast, the heat shock had adverse effects on species richness in the highly contaminated soils only. The total nematode biomass was lower in the highly contaminated field samples; however, the biomass was not affected by zinc and heat treatments of the mesocosms. This means that density compensation occurred rapidly, i.e., tolerant species quickly replaced sensitive species. Our results support the hypothesis that the history of contamination and the type of disturbance determine the response of communities. Despite that ecosystems may be exposed for centuries to contamination and communities show adaptation, biodiversity in highly contaminated sites is at increased risk when exposed to a different disturbance regime. We discuss how the loss of higher trophic levels from the entire system, such as represented by carnivorous nematodes after the heat shock, accompanied by local biodiversity loss at highly contaminated sites, may result in detrimental effects on ecosystem functions. © 2007 by the Ecological Society of America.

In effect assessment the comparability and applicability of LCx and ECx values, which are calculated at single points in time during exposure, relies on the ability to perform a valid extrapolation to other time points of interest. The behavior of LCx in time has been extensively studied, and the behavior of ECx in time is expected to follow similar dynamics, as it is considered that the LCx is just a specific case of ECxs. However, most models have focused on validating the dynamics of LCx, and hardly anything is known about the time dependence of ECx for other endpoints or whether it is comparable to that of LCxs. We have created four scenarios where we study the dynamics of the ECx for different endpoints and how it is affected by the characteristics of two different compounds (carbendazim and pentachlorobenzene) and of two different life history strategies (hermaphroditic and sexually reproducing strains of Caenorhabditis elegans). The observed patterns of behavior in time of the ECx for body size and for reproduction showed unexpected dynamics that deviate considerably from that of the LCx. It was demonstrated that the temporal dynamics of ECx were very different for each particular endpoint. The shape of the ECx-time curves depends on the intrinsic characteristics of the endpoint of study, as well as on the characteristics of the compound and life history strategy of the organism. This makes extrapolation in time or between endpoints difficult and hampers the comparability of results based on this summary statistic. The interpretation of the results from toxicity tests can be improved through process-based modeling, as demonstrated on the current data set. © 2006 American Chemical Society.

β-Catenin is a multifunctional protein that mediates Wnt signaling by binding to members of the T cell factor (TCF) family of transcription factors. Here, we report an evolutionarily conserved interaction of β-catenin with FOXO transcription factors, which are regulated by insulin and oxidative stress signaling. β-Catenin binds directly to FOXO and enhances FOXO transcriptional activity in mammalian cells. In Caenorhabditis elegans , loss of the β-catenin BAR-1 reduces the activity of the FOXO ortholog DAF-16 in dauer formation and life span. Association of β-catenin with FOXO was enhanced in cells exposed to oxidative stress. Furthermore, BAR-1 was required for the oxidative stress–induced expression of the DAF-16 target gene sod-3 and for resistance to oxidative damage. These results demonstrate a role for β-catenin in regulating FOXO function that is particularly important under conditions of oxidative stress.

The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity, and metabolism in Caenorhabditis elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including the expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass spectrometry (LC-MS)-based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2);daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the upregulation of many core intermediary metabolic pathways. These include glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid beta-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complexes I, II, III, and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative of spatiotemporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. This restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves and possibly also shunting metabolites through alternative energy-generating pathways to sustain longevity.

Death of the nematode Caenorhabditis elegans involves a conserved necrotic cell death cascade which generates endogenous blue anthranilate fluorescence, allowing death to be visualized., For cells the passage from life to death can involve a regulated, programmed transition. In contrast to cell death, the mechanisms of systemic collapse underlying organismal death remain poorly understood. Here we present evidence of a cascade of cell death involving the calpain-cathepsin necrosis pathway that can drive organismal death in Caenorhabditis elegans. We report that organismal death is accompanied by a burst of intense blue fluorescence, generated within intestinal cells by the necrotic cell death pathway. Such death fluorescence marks an anterior to posterior wave of intestinal cell death that is accompanied by cytosolic acidosis. This wave is propagated via the innexin INX-16, likely by calcium influx. Notably, inhibition of systemic necrosis can delay stress-induced death. We also identify the source of the blue fluorescence, initially present in intestinal lysosome-related organelles (gut granules), as anthranilic acid glucosyl esters—not, as previously surmised, the damage product lipofuscin. Anthranilic acid is derived from tryptophan by action of the kynurenine pathway. These findings reveal a central mechanism of organismal death in C. elegans that is related to necrotic propagation in mammals—e.g., in excitotoxicity and ischemia-induced neurodegeneration. Endogenous anthranilate fluorescence renders visible the spatio-temporal dynamics of C. elegans organismal death., Author Summary In the nematode Caenorhabditis elegans, intestinal lysosome-related organelles (or “gut granules”) contain a bright blue fluorescent substance of unknown identity. This has similar spectral properties to lipofuscin, a product of oxidative damage known to accumulate with age in postmitotic mammalian cells. Blue fluorescence seems to increase in aging worm populations, and lipofuscin has been proposed to be the source. To analyze this further, we measure fluorescence levels after exposure to oxidative stress and during aging in individually tracked worms. Surprisingly, neither of these conditions increases fluorescence levels; instead blue fluorescence increases in a striking and rapid burst at death. Such death fluorescence (DF) also appears in young worms when killed, irrespective of age or cause of death. We chemically identify DF as anthranilic acid glucosyl esters derived from tryptophan, and not lipofuscin. In addition, we show that DF generation in the intestine is dependent upon the necrotic cell death cascade, previously characterized as a driver of neurodegeneration. We find that necrosis spreads in a rapid wave along the intestine by calcium influx via innexin ion channels, accompanied by cytosolic acidosis. Inhibition of necrosis pathway components can delay stress-induced death, supporting its role as a driver of organismal death. This necrotic cascade provides a model system to study neurodegeneration and organismal death.

This review paper reports the consensus of a technical workshop hosted by the European network, NanoImpactNet (NIN). The workshop aimed to review the collective experience of working at the bench with manufactured nanomaterials (MNMs), and to recommend modifications to existing experimental methods and OECD protocols. Current procedures for cleaning glassware are appropriate for most MNMs, although interference with electrodes may occur. Maintaining exposure is more difficult with MNMs compared to conventional chemicals. A metal salt control is recommended for experiments with metallic MNMs that may release free metal ions. Dispersing agents should be avoided, but if they must be used, then natural or synthetic dispersing agents are possible, and dispersion controls essential. Time constraints and technology gaps indicate that full characterisation of test media during ecotoxicity tests is currently not practical. Details of electron microscopy, dark-field microscopy, a range of spectroscopic methods (EDX, XRD, XANES, EXAFS), light scattering techniques (DLS, SLS) and chromatography are discussed. The development of user-friendly software to predict particle behaviour in test media according to DLVO theory is in progress, and simple optical methods are available to estimate the settling behaviour of suspensions during experiments. However, for soil matrices such simple approaches may not be applicable. Alternatively, a Critical Body Residue approach may be taken in which body concentrations in organisms are related to effects, and toxicity thresholds derived. For microbial assays, the cell wall is a formidable barrier to MNMs and end points that rely on the test substance penetrating the cell may be insensitive. Instead assays based on the cell envelope should be developed for MNMs. In algal growth tests, the abiotic factors that promote particle aggregation in the media (e.g. ionic strength) are also important in providing nutrients, and manipulation of the media to control the dispersion may also inhibit growth. Controls to quantify shading effects, and precise details of lighting regimes, shaking or mixing should be reported in algal tests. Photosynthesis may be more sensitive than traditional growth end points for algae and plants. Tests with invertebrates should consider non-chemical toxicity from particle adherence to the organisms. The use of semi-static exposure methods with fish can reduce the logistical issues of waste water disposal and facilitate aspects of animal husbandry relevant to MMNs. There are concerns that the existing bioaccumulation tests are conceptually flawed for MNMs and that new test(s) are required. In vitro testing strategies, as exemplified by genotoxicity assays, can be modified for MNMs, but the risk of false negatives in some assays is highlighted. In conclusion, most protocols will require some modifications and recommendations are made to aid the researcher at the bench.

Ecotoxicology research is using many methods for engineered nanomaterials (ENMs), but the collective experience from researchers has not been documented. This paper reports the practical issues for working with ENMs and suggests nano-specific modifications to protocols. The review considers generic practical issues, as well as specific issues for aquatic tests, marine grazers, soil organisms, and bioaccumulation studies. Current procedures for cleaning glassware are adequate, but electrodes are problematic. The maintenance of exposure concentration is challenging, but can be achieved with some ENMs. The need to characterize the media during experiments is identified, but rapid analytical methods are not available to do this. The use of sonication and natural/synthetic dispersants are discussed. Nano-specific biological endpoints may be developed for a tiered monitoring scheme to diagnose ENM exposure or effect. A case study of the algal growth test highlights many small deviations in current regulatory test protocols that are allowed (shaking, lighting, mixing methods), but these should be standardized for ENMs. Invertebrate (Daphnia) tests should account for mechanical toxicity of ENMs. Fish tests should consider semistatic exposure to minimize wastewater and animal husbandry. The inclusion of a benthic test is recommended for the base set of ecotoxicity tests with ENMs. The sensitivity of soil tests needs to be increased for ENMs and shortened for logistics reasons; improvements include using Caenorhabditis elegans, aquatic media, and metabolism endpoints in the plant growth tests. The existing bioaccumulation tests are conceptually flawed and require considerable modification, or a new test, to work for ENMs. Overall, most methodologies need some amendments, and recommendations are made to assist researchers.

All living organisms react to signals from their environment such as light, temperature, sound and chemical substances in order to survive and adapt to their surroundings. In multicellular organisms communication between cells is necessary to regulate growth and differentiation [3]. Neighbouring cells can communicate with each other by direct cell-cell contact through plasma-membrane-bound signalling molecules or through the formation of gap junctions [176]. Cells are able to communicate with other cells some distance away through secretion of chemical signals. The secreted signals can be soluble molecules such as most hormones and neurotransmitters; they bind to receptors on the cell surface of target cells. Hydrophobic signal molecules such as the steroid and thyroid hormones are able to pass the lipid bilayer of the plasma membrane and bind to specific proteins inside the cell [76].

Organophosphorus pesticides (OPs) were originally designed to affect the nervous system by inhibiting the enzyme acetylcholinesterase, an important regulator of the neurotransmitter acetylcholine. Over the past years evidence is mounting that these compounds affect many other processes. Little is known, however, about gene expression responses against OPs in the nematode Caenorhabditis elegans. This is surprising because C. elegans is extensively used as a model species in toxicity studies. To address this question we performed a microarray study in C. elegans which was exposed for 72 hrs to two widely used Ops, chlorpyrifos and diazinon, and a low dose mixture of these two compounds. Our analysis revealed transcriptional responses related to detoxification, stress, innate immunity, and transport and metabolism of lipids in all treatments. We found that for both compounds as well as in the mixture, these processes were regulated by different gene transcripts. Our results illustrate intense, and unexpected crosstalk between gene pathways in response to chlorpyrifos and diazinon in C. elegans.

Background The C. elegans gene folt-1 is an ortholog of the human reduced folate carrier gene. The FOLT-1 protein has been shown to transport folate and to be involved in uptake of exogenous folate by worms. A knockout mutation of the gene, folt-1(ok1460), was shown to cause sterility, and here we investigate the source of the sterility and the effect of the folt-1 knockout on somatic function. Results Our results show that folt-1(ok1460) knockout hermaphrodites have a substantially reduced germline, generate a small number of functional sperm, and only rarely produce a functional oocyte. We found no evidence of increased apoptosis in the germline of folt-1 knockout mutants, suggesting that germline proliferation is defective. While folt-1 knockout males are fertile, their rate of spermatogenesis was severely diminished, and the males were very poor maters. The mating defect is likely due to compromised metabolism and/or other somatic functions, as folt-1 knockout hermaphrodites displayed a shortened lifespan and elongated defecation intervals. Conclusions The FOLT-1 protein function affects both the soma and the germline. folt-1(ok1460) hermaphrodites suffer severely diminished lifespan and germline defects that result in sterility. Germline defects associated with folate deficiency appear widespread in animals, being found in humans, mice, fruit flies, and here, nematodes.

More than 70 years after its initial report, caloric restriction stands strong as the most consistent non- pharmacological intervention increasing lifespan and protecting against metabolic disease. Among the different mechanisms by which caloric restriction might act, Sir2/SIRT1 (Silent information regulator 2/Silent information regulator T1) has been the focus of much attention because of its ability to integrate sensing of the metabolic status with adaptive transcriptional outputs. This review focuses on gathered evidence suggesting that Sir2/SIRT1 is a key mediator of the beneficial effects of caloric restriction and addresses the main questions that still need to be answered to consolidate this hypothesis.