Elegans Feeding Research Articles

Pharyngeal Pumping Assay for Quantifying Feeding Behavior in Caenorhabditis elegans.

C. elegans is a well-established nematode model organism, with 83% of its genes conserved in humans with translation potential. C. elegans is translucent, with clearly defined cellular organization, and robustly identifiable under a microscope, being an excellent model for studying feeding behavior. Its neuromuscular pharyngeal pump undergoes a pumping motion that can be quantified to study feeding behavior at specific treatment conditions and in genetically modified worms. Understanding the evolutionarily conserved feeding behaviors and regulatory signals is vital, as unhealthy eating habits increase the risk of associated diseases. The current protocol was developed to identify and study evolutionary conserved signals regulating feeding behavior. The protocol described here is very robust in calculating the pumping rate (pumping per minute) as it directly counts the pharyngeal pumping for 30 s. This protocol uses basic laboratory instrumentation, such as a stereomicroscope with an attached camera and a computer with a video program that can be used to count manually. The advantages of studying C. elegans feeding include understanding the genetic basis of feeding regulation, dysregulation of feeding behavior in a disease model, the influence of toxic or environmental substances in feeding behavior, and modulation of feeding behavior by pharmacological agents. Key features • Quantifies pharyngeal pumping, which can be used to study up/downstream signaling in feeding regulation. • Uses a phenotype (pharyngeal pumping) that is easy to score. • Requires only a stereomicroscope with a camera to record the pharyngeal pumping, which can be counted manually.

Bee Venom Toxic Effect on MDA-MB-231 Breast Cancer Cells and Caenorhabditis Elegans.

Bee venom has therapeutics and pharmacological properties. Further toxicological studies on animal models are necessary due to the severe allergic reactions caused by this product. Here, Caenorhabditis elegans was used as an in vivo toxicity model, while breast cancer cells were used to evaluate the pharmacological benefits. The bee venom utilized in this research was collected from Apis mellifera species found in Northeast Brazil. The cytotoxicity caused by bee venom was measured by MTT assay on MDA-MB-231 and J774 A.1 cells during 24 - 72 hours of exposure. C. elegans at the L4 larval stage were exposed for three hours to M9 buffer or bee venom. Survival, behavioral parameters, reproduction, DAF-16 transcription factor translocation, the expression of superoxide dismutase (SOD), and metabolomics were analyzed. Bee venom suppressed the growth of MDA-MB-231 cancer cells and exhibited cytotoxic effects on macrophages. Also, decreased C. elegans survival impacted its behaviors by decreasing C. elegans feeding behavior, movement, and reproduction. Bee venom did not increase the expression of SOD-3, but it enhanced DAF-16 translocation from the cytoplasm to the nucleus. C. elegans metabolites differed after bee venom exposure, primarily related to aminoacyl- tRNA biosynthesis, glycine, serine and threonine metabolism, and sphingolipid and purine metabolic pathways. Our findings indicate that exposure to bee venom resulted in harmful effects on the cells and animal models examined. Thus, due to its potential toxic effect and induction of allergic reactions, using bee venom as a therapeutic approach has been limited. The development of controlled-release drug strategies to improve this natural product's efficacy and safety should be intensified.

Bacterial vitamin B6 is required for post-embryonic development in C. elegans

Nutritional intake influences animal growth, reproductive capacity, and survival of animals. Under nutrition deficiency, animal developmental arrest occurs as an adaptive strategy to survive. However, the nutritional basis and the underlying nutrient sensing mechanism essential for animal regrowth after developmental arrest remain to be explored. In Caenorhabditis elegans, larvae undergo early developmental arrest are stress resistant, and they require certain nutrients to recover postembryonic development. Here, we investigated the developmental arrest in C. elegans feeding on Lactiplantibacillus plantarum, and the rescue of the diapause state with trace supplementation of Escherichia coli. We performed a genome-wide screen using 3983 individual gene deletion E. coli mutants and identified E. coli genes that are indispensable for C. elegans larval growth on originally not nutritionally sufficient bacteria L. plantarum. Among these crucial genes, we confirmed E. coli pdxH, and the downstream metabolite pyridoxal 5-P (PLP, Vitamin B6) as important nutritional factors for C. elegans postembryonic development. Transcriptome results suggest that bacterial pdxH affects host development by coordinating host metabolic processes and PLP binding. Additionally, the developmental arrest induced by the L. plantarum diet in worm does not depend on the activation of FoxO/DAF-16. Altogether, these results highlight the role of microbial metabolite PLP as a crucial cofactor to restore postembryonic development in C. elegans.

The nanotoxicity assessment of cube-like iron nitride magnetic nanoparticles at the organismal level of nematode Caenorhabditis elegans

Magnetic nanoparticles (NPs) are suitable candidates for various medical and biological applications, despite some concerns that they may have negative impacts on human health. In this study, the toxicity effects of magnetic NPs consisting of α”-Fe16N2 captured and bioaccumulated by the nematode Caenorhabditis elegans (C. elegans) in the early larval stage are evaluated. The choice of α”-Fe16N2 NPs is based on their good structural stability when stored in saline solution and high magnetic performance. The uptake and bioaccumulation of α”-Fe16N2 NPs in intestinal cells of C. elegans was evidenced by transmission electron microscopy. After exposure to NPs up to 40 mg mL−1, C. elegans larval development, survival, feeding behavior, defecation cycles, movement and reproduction were monitored. C. elegans survival and other monitored behavioral evolutions do not show significant changes, except for a slight statistical reduction in the reproductive profile. Therefore, the present results are promising and very encouraging for investigations of applications of α”-Fe16N2 NPs in the biomedical area.

<i>In Vivo</i> Toxicity Assessment of Nickel Zinc Carbon Nanotubes in a Living Host Model

Nickel zinc carbon nanotubes (NiZnCNT) has gained substantial interest among researchers lately due to its wide application in the field of biomedical fields. Nickel zinc (NiZn) is well known for its corrosion-resistant, microware and magnetic properties. On the other hand, carbon nanotube (CNT) is known for its application as biosensor and tissue engineering scaffolds. Despite the excellent properties and diverse applications, the toxicity profile of NiZnCNT remains poorly understood. In this study, the potential toxic effect of NiZnCNT to living organism was evaluated using a nematode Caenorhabditis elegans (C. elegans) model. Adult worms were exposed to NiZnCNT at 50 and 200 μg/mL, followed by the assessment of three physiological parameters, including the effect NiZnCNT on C. elegans feeding behavior, reproductive ability and the overall lifespan of the worms. No significant difference was noted between the feeding rate of worms treated with NiZnCNT at both concentrations and the control population (p>0.05). Furthermore, there was no reduction in the production of progenies in NiZnCNT-treated worms, proposing that the nanoparticles tested does not negatively affect the reproductive system of animal. The mean lifespan of worms for untreated control, 50 μg/mL and 200 μg/mL of NiZnCNT was 10.4, 9.7 and 8.9 days respectively and no significant difference was observed in statistical analysis (p>0.05). In conclusion, this study demonstrated in the context of whole organism that NiZnCNT did not possess harmful toxic effect to living system, at concentration up to 200 μg/mL. The results further support the use of this nanoparticle in the field of biomedicine.

Measuring Caenorhabditis elegans Spatial Foraging and Food Intake Using Bioluminescent Bacteria.

For most animals, feeding includes two behaviors: foraging to find a food patch and food intake once a patch is found. The nematode Caenorhabditis elegans is a useful model for studying the genetics of both behaviors. However, most methods of measuring feeding in worms quantify either foraging behavior or food intake, but not both. Imaging the depletion of fluorescently labeled bacteria provides information on both the distribution and amount of consumption, but even after patch exhaustion a prominent background signal remains, which complicates quantification. Here, we used a bioluminescent Escherichia coli strain to quantify C. elegans feeding. With light emission tightly coupled to active metabolism, only living bacteria are capable of bioluminescence, so the signal is lost upon ingestion. We quantified the loss of bioluminescence using N2 reference worms and eat-2 mutants, and found a nearly 100-fold increase in signal-to-background ratio and lower background compared to loss of fluorescence. We also quantified feeding using aggregating npr-1 mutant worms. We found that groups of npr-1 mutants first clear bacteria from within the cluster before foraging collectively for more food; similarly, during large population swarming, only worms at the migrating front are in contact with bacteria. These results demonstrate the usefulness of bioluminescent bacteria for quantifying feeding and generating insights into the spatial pattern of food consumption.

Recombinant buckwheat trypsin inhibitor decreases fat accumulation via the IIS pathway in Caenorhabditis elegans.

Buckwheat trypsin inhibitor (BTI) is a low molecular weight polypeptide that can help to prevent metabolic diseases such as obesity, hyperglycemia and hyperlipidemia. Herein, the effects of recombinant BTI (rBTI) on fat accumulation in Caenorhabditis elegans were studied. rBTI prevented fat accumulation under normal and high glucose conditions, and led to significantly shorter body widths without affecting C. elegans feeding behavior. Results also indicate that rBTI altered fat breakdown, synthesis, and accumulation by altering the transcription, expression and activity of key enzymes in lipolysis and fat synthesis. In daf-2 and daf-16 mutants, rBTI did not prevent fat accumulation, indicating that rBTI activity relies on the insulin/insulin-like growth factor (IIS) pathway. Overall rBTI may regulate changes in lipolysis and fat synthesis by down-regulating the IIS pathway, which can affect fat accumulation. These findings support the application of rBTI in preventing obesity, hyperglycemia and hyperlipemia.

Similar Neural Pathways Control Foraging in Mosquitoes and Worms.

Female Aedes aegypti mosquitoes bite human hosts to obtain a blood meal and, in the process, act as vectors for many disease-causing viruses, including the dengue, chikungunya, yellow fever, and Zika viruses. After a complete meal, the female mosquitoes lose attraction to their hosts for several days. New research shows that pharmacological activation of neuropeptide Y-like receptor (NPYLR) signaling elicits host aversion in female mosquitoes. This behavior of mosquitoes shows remarkable similarities to a bacterial-aversion behavior of the nematode Caenorhabditis elegans Feeding on pathogenic bacteria causes bloating of the gut in C. elegans that leads to activation of NPYLR signaling and bacterial aversion. Several studies suggest that this newly discovered mechanism underlying foraging may be conserved across a large number of species. A better understanding of the regulation of NPYLR signaling pathways could provide molecular targets for the control of eating behaviors in different animals, including human-disease vectors.

Toxicity Evaluation of Graphene and Poly(Lactic-Acid) Using a Nematode Model

Graphene has gained tremendous attention due to its unlimited potential in various applications while poly(lactic acid) (PLA) is a biodegradable thermoplastic polyester produced from fermenting corn starch. The incorporation of graphene into PLA has been proven to exhibit excellent mechanical and thermal properties. However, there are not many reports on the potential toxic effect of these materials towards living organisms. In this study, we investigated the possible toxicity of graphene and PLA-graphene in a live animal model, the nematode Caenorhabdits elegans (C. elegans). Alive adult worms were exposed directly to graphene and PLA-graphene across a range of concentrations from 50 µg/mL to 1000 µg/mL. After certain hours of exposure, the pharyngeal pumping rate (indicative of the C. elegans feeding activity), reproductive rate and lifespan of the worms were determined and compared to the untreated worm population. At all concentrations tested, both graphene and PLA-graphene do not affect the feeding rate of the nematode. Additionally, there was no significant difference between the lifespan of worms exposed to graphene and PLA-graphene as compared to the untreated control population (p>0.05). We examined the effect of graphene on nematode’s ability to reproduce and no reduction in progenies was detected (p>0.05). Taken together, our findings suggest that graphene and PLA-graphene do not possess a negative effect on the feeding activity, reproduction and overall lifespan of the host, indicating that these materials are safe to living organism at concentration up to 1000 µg/mL.

An automated method for the analysis of food intake behaviour in Caenorhabditis elegans

The study of mechanisms that govern feeding behaviour and its related disorders is a matter of global health interest. The roundworm Caenorhabditis elegans is becoming a model organism of choice to study these conserved pathways. C. elegans feeding depends on the contraction of the pharynx (pumping). Thanks to the worm transparency, pumping can be directly observed under a stereoscope. Therefore, C. elegans feeding has been historically investigated by counting pharyngeal pumping or by other indirect approaches. However, those methods are short-term, time-consuming and unsuitable for independent measurements of sizable numbers of individuals. Although some particular devices and long-term methods have been lately reported, they fail in the automated, scalable and/or continuous aspects. Here we present an automated bioluminescence-based method for the analysis and continuous monitoring of worm feeding in a multi-well format. We validate the method using genetic, environmental and pharmacological modulators of pharyngeal pumping. This flexible methodology allows studying food intake at specific time-points or during longer periods of time, in single worms or in populations at any developmental stage. Additionally, changes in feeding rates in response to differential metabolic status or external environmental cues can be monitored in real time, allowing accurate kinetic measurements.

Sertraline, Paroxetine, and Chlorpromazine Are Rapidly Acting Anthelmintic Drugs Capable of Clinical Repurposing

Parasitic helminths infect over 1 billion people worldwide, while current treatments rely on a limited arsenal of drugs. To expedite drug discovery, we screened a small-molecule library of compounds with histories of use in human clinical trials for anthelmintic activity against the soil nematode Caenorhabditis elegans. From this screen, we found that the neuromodulatory drugs sertraline, paroxetine, and chlorpromazine kill C. elegans at multiple life stages including embryos, developing larvae and gravid adults. These drugs act rapidly to inhibit C. elegans feeding within minutes of exposure. Sertraline, paroxetine, and chlorpromazine also decrease motility of adult Trichuris muris whipworms, prevent hatching and development of Ancylostoma caninum hookworms and kill Schistosoma mansoni flatworms, three widely divergent parasitic helminth species. C. elegans mutants with resistance to known anthelmintic drugs such as ivermectin are equally or more susceptible to these three drugs, suggesting that they may act on novel targets to kill worms. Sertraline, paroxetine, and chlorpromazine have long histories of use clinically as antidepressant or antipsychotic medicines. They may represent new classes of anthelmintic drug that could be used in combination with existing front-line drugs to boost effectiveness of anti-parasite treatment as well as offset the development of parasite drug resistance.

Editor’s Highlight: Comparative Toxicity of Organophosphate Flame Retardants and Polybrominated Diphenyl Ethers toCaenorhabditiselegans

With the phasing-out of the polybrominated diphenyl ether (PBDE) flame retardants due to concerns regarding their potential developmental toxicity, the use of replacement compounds such as organophosphate flame retardants (OPFRs) has increased. Limited toxicity data are currently available to estimate the potential adverse health effects of the OPFRs. The toxicological effects of 4 brominated flame retardants, including 3 PBDEs and 3,3',5,5'-tetrabromobisphenol A, were compared with 6 aromatic OPFRs and 2 aliphatic OPFRs. The effects of these chemicals were determined using 3 biological endpoints in the nematode Caenorhabditis elegans (feeding, larval development, and reproduction). Because C. elegans development was previously reported to be sensitive to mitochondrial function, results were compared with those from an in vitro mitochondrial membrane permeabilization (MMP) assay. Overall 11 of the 12 flame retardants were active in 1 or more C. elegans biological endpoints, with only tris(2-chloroethyl) phosphate inactive across all endpoints including the in vitro MMP assay. For 2 of the C. elegans endpoints, at least 1 OPFR had similar toxicity to the PBDEs: triphenyl phosphate (TPHP) inhibited larval development at levels comparable to the 3 PBDEs; whereas TPHP and isopropylated phenol phosphate (IPP) affected C. elegans reproduction at levels similar to the PBDE commercial mixture, DE-71. The PBDEs reduced C. elegans feeding at lower concentrations than any OPFR. In addition, 9 of the 11 chemicals that inhibited C. elegans larval development also caused significant mitochondrial toxicity. These results suggest that some of the replacement aromatic OPFRs may have levels of toxicity comparable to PBDEs.

Feeding behaviour of Caenorhabditis elegans is an indicator of Pseudomonas aeruginosa PAO1 virulence.

Caenorhabditis elegans is commonly used as an infection model for pathogenesis studies in Pseudomonas aeruginosa. The standard virulence assays rely on the slow and fast killing or paralysis of nematodes but here we developed a behaviour assay to monitor the preferred bacterial food sources of C. elegans. We monitored the food preferences of nematodes fed the wild type PAO1 and mutants in the type III secretion (T3S) system, which is a conserved mechanism to inject secreted effectors into the host cell cytosol. A ΔexsEΔpscD mutant defective for type III secretion served as a preferred food source, while an ΔexsE mutant that overexpresses the T3S effectors was avoided. Both food sources were ingested and observed in the gastrointestinal tract. Using the slow killing assay, we showed that the ΔexsEΔpscD had reduced virulence and thus confirmed that preferred food sources are less virulent than the wild type. Next we developed a high throughput feeding behaviour assay with 48 possible food colonies in order to screen a transposon mutant library and identify potential virulence genes. C. elegans identified and consumed preferred food colonies from a grid of 48 choices. The mutants identified as preferred food sources included known virulence genes, as well as novel genes not identified in previous C. elegans infection studies. Slow killing assays were performed and confirmed that several preferred food sources also showed reduced virulence. We propose that C. elegans feeding behaviour can be used as a sensitive indicator of virulence for P. aeruginosa PAO1.

The Geometry of Locomotive Behavioral States in C. elegans

We develop a new hidden Markov model-based method to analyze C elegans locomotive behavior and use this method to quantitatively characterize behavioral states. In agreement with previous work, we find states corresponding to roaming, dwelling, and quiescence. However, we also find evidence for a continuum of intermediate states. We suggest that roaming, dwelling, and quiescence may best be thought of as extremes which, mixed in any proportion, define the locomotive repertoire of C elegans foraging and feeding behavior.

Dissecting a central flip-flop circuit that integrates contradictory sensory cues in C. elegans feeding regulation

Feeding behaviour is modulated by both environmental cues and internal physiological states. Appetite is commonly boosted by the pleasant smell (or appearance) of food and destroyed by a bad taste. In reality, animals sense multiple environmental cues at the same time and it is not clear how these sensory inputs are integrated and a decision is made to regulate feeding behaviour accordingly. Here we show that feeding behaviour in Caenorhabditis elegans can be either facilitated by attractive odours or suppressed by repellents. By identifying mutants that are defective for sensory-mediated feeding regulation, we dissected a central flip-flop circuit that integrates two contradictory sensory inputs and generates bistable hormone output to regulate feeding behaviour. As feeding regulation is fundamental to animal survival, we speculate that the basic organizational logic identified here in C. elegans is likely convergent throughout different phyla.

Swietenia macrophylla extract promotes the ability of Caenorhabditis elegans to survive Pseudomonas aeruginosa infection

Ethnopharmacological relevanceSwietenia macrophylla or commonly known as big leaf mahogany, has been traditionally used as an antibacterial and antifungal agent. Aim of the studyThe unwanted problem of antibiotic resistance in many bacterial species advocates the need for the discovery of the new anti-infective drugs. Here, we investigated the anti-infective properties of Swietenia macrophylla with an assay involving lethal infection of Caenorhabditis elegans with the opportunistic human pathogen Pseudomonas aeruginosa. Materials and methodsUsing a slow killing assay, Caenorhabditis elegans was challenged with an infective strain of Pseudomonas aeruginosa (PA14). The ability of Swietenia macrophylla seed ethyl acetate extract to promote the survival of infected worms was assessed by comparing the percentage of survival between extract treated and non-treated worm populations. The effect of Swietenia macrophylla towards PA14 growth, Caenorhabditis elegans feeding rate and degree of PA14 colonization in the worm gut was also evaluated. Lastly, using a fluorescent transgenic Caenorhabditis elegans strain and real time PCR, the effect of Swietenia macrophylla on the expression of lys-7, an immune response gene was also investigated. ResultsOur results demonstrate the ability of Swietenia macrophylla seed ethyl acetate extract in rescuing Caenorhabditis elegans from fatal PA14 infection. Consequently, we showed that the extract promotes the survival without exhibiting any bactericidal effect or perturbation of Caenorhabditis elegans feeding rate. We also showed that Swietenia macrophylla was able to restore the initially repressed lys-7 level in PA14 infected Caenorhabditis elegans. ConclusionSwietenia macrophylla extract is able to enhance the ability of Caenorhabditis elegans to survive PA14 infection without directly killing the pathogen. We further showed that the extract boosted the expression of a gene pivotal for innate immunity in Caenorhabditis elegans. Collectively, these findings strongly suggest the presence of compounds within Swietenia macrophylla seed that either reduces Pseudomonas aeruginosa virulence and/or enhance host resistance.

Serotonin Regulates C. elegans Fat and Feeding through Independent Molecular Mechanisms

We investigated serotonin signaling in C. elegans as a paradigm for neural regulation of energy balance and found that serotonergic regulation of fat is molecularly distinct from feeding regulation. Serotonergic feeding regulation is mediated by receptors whose functions are not required for fat regulation. Serotonergic fat regulation is dependent on a neurally expressed channel and a G protein-coupled receptor that initiate signaling cascades that ultimately promote lipid breakdown at peripheral sites of fat storage. In turn, intermediates of lipid metabolism generated in the periphery modulate feeding behavior. These findings suggest that, as in mammals, C. elegans feeding behavior is regulated by extrinsic and intrinsic cues. Moreover, obesity and thinness are not solely determined by feeding behavior. Rather, feeding behavior and fat metabolism are coordinated but independent responses of the nervous system to the perception of nutrient availability.

Effects of Genetic Mutations and Chemical Exposures on Caenorhabditis elegans Feeding: Evaluation of a Novel, High-Throughput Screening Assay

BackgroundGovernment agencies have defined a need to reduce, refine or replace current mammalian-based bioassays with testing methods that use alternative species. Invertebrate species, such as Caenorhabditis elegans, provide an attractive option because of their short life cycles, inexpensive maintenance, and high degree of evolutionary conservation with higher eukaryotes. The C. elegans pharynx is a favorable model for studying neuromuscular function, and the effects of chemicals on neuromuscular activity, i.e., feeding. Current feeding methodologies, however, are labor intensive and only semi-quantitative.Methodology/Principal FindingsHere a high-throughput assay is described that uses flow cytometry to measure C. elegans feeding by determining the size and intestinal fluorescence of hundreds of nematodes after exposure to fluorescent-labeled microspheres. This assay was validated by quantifying fluorescence in feeding-defective C. elegans (eat mutants), and by exposing wild-type nematodes to the neuroactive compounds, serotonin and arecoline. The eat mutations previously determined to cause slow pumping rates exhibited the lowest feeding levels with our assay. Concentration-dependent increases in feeding levels after serotonin exposures were dependent on food availability, while feeding levels decreased in arecoline-exposed nematodes regardless of the presence of food. The effects of the environmental contaminants, cadmium chloride and chlorpyrifos, on wild-type C. elegans feeding were then used to demonstrate an application of the feeding assay. Cadmium exposures above 200 µM led to a sharp drop in feeding levels. Feeding of chlorpyrifos-exposed nematodes decreased in a concentration-dependent fashion with an EC50 of 2 µM.Conclusions/SignificanceThe C. elegans fluorescence microsphere feeding assay is a rapid, reliable method for the assessment of neurotoxic effects of pharmaceutical drugs, industrial chemicals or environmental agents. This assay may also be applicable to large scale genetic or RNAi screens used to identify genes that are necessary for the development or function of the pharynx or other neuromuscular systems.

Acetylcholine

Acetylcholine is the major excitatory neurotransmitter at nematode neuromuscular junctions, and more than a third of the cells in the C. elegans nervous system release acetylcholine. Through a combination of forward genetics, drug-resistance selections, and genomic analysis, mutants have been identified for all of the steps specifically required for cholinergic function. These include two enzymes, two transporters, and a bewildering assortment of receptors. Cholinergic transmission is involved, directly or indirectly, in many C. elegans behaviors, including locomotion, egg laying, feeding, and male mating.

C. elegans feeding defective mutants have shorter body lengths and increased autophagy.

BackgroundMutations that cause feeding defects in the nematode C. elegans are known to increase life span. Here we show that feeding defective mutants also have a second general trait in common, namely that they are small.ResultsOur measurements of the body lengths of a variety of feeding defective mutants, or of a variety of double mutants affecting other pathways that regulate body length in C. elegans, i.e. the DBL-1/TGFβ, TAX-6/calcineurin and the SMA-1/βH-spectrin pathways, indicate that food uptake acts as a separate pathway regulating body length. In early stages, before eating begins, feeding defective worms have no defect in body length or, in some cases, have only slightly smaller body length compared to wild-type. A significant difference in body length is first noticeable at later larval stages, a difference that probably correlates with increasing starvation. We also show that autophagy is induced and that the quantity of fat is decreased in starved worms.ConclusionOur results indicate that the long-term starvation seen in feeding-defective C. elegans mutants activates autophagy, and leads to depletion of fat deposits, small cell size and small body size.