The nematode Caenorhabditis elegans and the terrestrial isopod Porcellio scaber likely interact opportunistically.

Phoresy is a behavior in which an organism, the phoront, travels from one location to another by 'hitching a ride' on the body of a host as it disperses. Some phoronts are generalists, taking advantage of any available host. Others are specialists and travel only when specific hosts are located using chemical cues to identify and move (chemotax) toward the preferred host. Free-living nematodes, like Caenorhabditis elegans, are often found in natural environments that contain terrestrial isopods and other invertebrates. Additionally, the C. elegans wild strain PB306 was isolated associated with the isopod Porcellio scaber. However, it is currently unclear if C. elegans is a phoront of terrestrial isopods, and if so, whether it is a specialist, generalist, or developmental stage-specific combination of both strategies. Because the relevant chemical stimuli might be secreted compounds or volatile odorants, we used different types of chemotaxis assays across diverse extractions of compounds or odorants to test whether C. elegans is attracted to P. scaber. We show that two different strains-the wild isolate PB306 and the laboratory-adapted strain N2 -are not attracted to P. scaber during either the dauer or adult life stages. Our results indicate that C. elegans was not attracted to chemical compounds or volatile odorants from P. scaber, providing valuable empirical evidence to suggest that any associations between these two species are likely opportunistic rather than specific phoresy.

Design and applications of a novel fluorescent probe for detecting glutathione in biological samples.

This study aimed to develop a novel and practical fluorescent method for GSH detection in complex biological samples. To this end, a series of coumarin-based fluorescent probes was designed and synthesized using various aliphatic halogens as the sensing group. By using a new evaluation method of GSH/Cys/Hcy coexisting conditions, the probe with chloropropionate (CBF3) showed a high selectivity, excellent sensitivity, good stability for GSH detection. The reaction mechanism is proposed as nucleophilic substitution/cyclization and intramolecular charge transfer (ICT), which was confirmed by LC-MS and NMR analysis, as well as density functional theory calculations. In addition, CBF3 was demonstrated to be competent not only for the quantitative detection of GSH in real serum samples, but also for sensing GSH changes in different oxidative stress models in living cells and nematodes. This study showed a practical strategy for constructing GSH-specific fluorescent probes, and provided a sensitive tool for real-time sensing of GSH in real biological samples. The findings would greatly facilitate further investigations on GSH-associated clinical diagnosis and biomedical studies.

Deep sampling of Hawaiian Caenorhabditis elegans reveals high genetic diversity and admixture with global populations.

Hawaiian isolates of the nematode species Caenorhabditis elegans have long been known to harbor genetic diversity greater than the rest of the worldwide population, but this observation was supported by only a small number of wild strains. To better characterize the niche and genetic diversity of Hawaiian C. elegans and other Caenorhabditis species, we sampled different substrates and niches across the Hawaiian islands. We identified hundreds of new Caenorhabditis strains from known species and a new species, Caenorhabditis oiwi. Hawaiian C. elegans are found in cooler climates at high elevations but are not associated with any specific substrate, as compared to other Caenorhabditis species. Surprisingly, admixture analysis revealed evidence of shared ancestry between some Hawaiian and non-Hawaiian C. elegans strains. We suggest that the deep diversity we observed in Hawaii might represent patterns of ancestral genetic diversity in the C. elegans species before human influence.

Biocompatible fluorescent carbon dots derived from roast duck for in vitro cellular and in vivo C. elegans bio-imaging.

Biocompatible fluorescent carbon dots (CDs) were prepared via a simple and green route using duck breasts as a natural carbon source. The CDs from duck breasts were well dispersed, and their mean particle size decreased from 2.59 to 1.95 nm when the roasting temperature increased from 200 to 300 °C. Abundant functional groups such as OH, COOH, and NH2 were observed on the surface of the CDs, providing the CDs with good water solubility. These CDs emitted strong fluorescence under ultraviolet light irradiation and exhibited superior photostability. The absolute fluorescence quantum yield of CDs rose from 10.53% to 38.05% when the relative nitrogen content of CDs increased from 7.18% to 12.73%. The CDs showed low toxicity to PC12 cells for prolonged exposure. Therefore, the duck CDs were successfully developed as fluorescent probes for in vitro PC12 cells and in vivo Caenorhabditis elegans imaging. These results indicated that the CDs derived from roast duck were biocompatible and can potentially be used as probes in bio-imaging.

Mass Surveilance of C. elegans-Smartphone-Based DIY Microscope and Machine-Learning-Based Approach for Worm Detection.

The nematode Caenorhabditis elegans (C. elegans) is often used as an alternative animal model due to several advantages such as morphological changes that can be seen directly under a microscope. Limitations of the model include the usage of expensive and cumbersome microscopes, and restrictions of the comprehensive use of C. elegans for toxicological trials. With the general applicability of the detection of C. elegans from microscope images via machine learning, as well as of smartphone-based microscopes, this article investigates the suitability of smartphone-based microscopy to detect C. elegans in a complete Petri dish. Thereby, the article introduces a smartphone-based microscope (including optics, lighting, and housing) for monitoring C. elegans and the corresponding classification via a trained Histogram of Oriented Gradients (HOG) feature-based Support Vector Machine for the automatic detection of C. elegans. Evaluation showed classification sensitivity of 0.90 and specificity of 0.85, and thereby confirms the general practicability of the chosen approach.

An Automated Microfluidic System for Morphological Measurement and Size-Based Sorting of C. Elegans.

This paper reports a vision-based automated microfluidic system for morphological measurement and size-based sorting of the nematode worm C. elegans. Exceeding the capabilities of conventional worm sorting microfluidic devices purely relying on passive sorting mechanisms, our system is capable of accurate measurement of the worm length/width and active sorting of worms with the desired sizes from a mixture of worms with different body sizes. This function is realized based on the combination of real-time, vision-based worm detection and sizing algorithms and automated on-chip worm manipulation. A double-layer microfluidic device with computer-controlled pneumatic valves is developed for sequential loading, trapping, vision-based sizing, and sorting of single worms. To keep the system operation robust, vision-based algorithms on detecting multi-worm loading and worm sizing failure have also been developed. We conducted sorting experiments on 319 worms and achieved an average sorting speed of 10.4 worms per minute (5.8 s/worm) with an operation success rate of 90.3%. This system will facilitate the worm biology studies where body size measurement and size-based sorting of many worms are needed.

Recent Advances and Trends in Microfluidic Platforms for C. elegans Biological Assays.

Microfluidics has proven to be a key tool in quantitative biological research. The C. elegans research community in particular has developed a variety of microfluidic platforms to investigate sensory systems, development, aging, and physiology of the nematode. Critical for the growth of this field, however, has been the implementation of concurrent advanced microscopy, hardware, and software technologies that enable the discovery of novel biology. In this review, we highlight recent innovations in microfluidic platforms used for assaying C. elegans and discuss the novel technological approaches and analytic strategies required for these systems. We conclude that platforms that provide analytical frameworks for assaying specific biological mechanisms and those that take full advantage of integrated technologies to extract high-value quantitative information from worm assays are most likely to move the field forward.

High-throughput sorting of eggs for synchronization of C. elegans in a microfluidic spiral chip.

In this study, we report the use of a high-throughput microfluidic spiral chip to screen out eggs from a mixed age nematode population, which can subsequently be cultured to a desired developmental stage. For the sorting of a mixture containing three different developmental stages, eggs, L1 and L4, we utilized a microfluidic spiral chip with a trapezoidal channel to obtain a sorting efficiency of above 97% and a sample purity (SP) of above 80% for eggs at different flow rates up to 10 mL min-1. The result demonstrated a cost-effective, simple, and highly efficient method for synchronizing C. elegans at a high throughput (∼4200 organisms per min at 6 mL min-1), while eliminating challenges such as clogging and non-reusability of membrane-based filtration. Due to its simplicity, our method can be easily adopted in the C. elegans research community.

A microfluidic diode for sorting and immobilization of Caenorhabditis elegans.

Caenorhabditis elegans (C. elegans) is a powerful model organism extensively used in studies of human aging and diseases. Despite the numerous advantages of C. elegans as a model system, two biological characteristics may introduce complexity and variability to most studies: 1. it exhibits different biological features, composition and behaviors at different developmental stages; 2. it has very high mobility. Therefore, synchronization and immobilization of worm populations are often required. Conventionally, these processes are implemented through manual and chemical methods, which can be laborious, time-consuming and of low-throughput. Here we demonstrate a microfluidic design capable of simultaneously sorting worms by size at a throughput of 97±4 worms per minute, and allowing for worm collection or immobilization for further investigations. The key component, a microfluidic diode structure, comprises a curved head and a straight tail, which facilitates worms to enter from the curved end but prevents them from translocating from the straight side. This design remarkably enhances the efficiency and accuracy of worm sorting at relatively low flow rates, and hence provides a practical approach to sort worms even with the presence of egg clusters and debris. In addition, we show that well-sorted worms could be immobilized, kept alive and identically orientated, which could facilitate many C. elegans-based studies.

Natural Genetic Variation in the Caenorhabditis elegans Response to Pseudomonas aeruginosa.

Caenorhabditis elegans responds to pathogenic microorganisms by activating its innate immune system, which consists of physical barriers, behavioral responses, and microbial killing mechanisms. We examined whether natural variation plays a role in the response of C. elegans to Pseudomonas aeruginosa using two C. elegans strains that carry the same allele of npr-1, a gene that encodes a G-protein-coupled receptor related to mammalian neuropeptide Y receptors, but that differ in their genetic backgrounds. Strains carrying an allele for the NPR-1 215F isoform have been shown to exhibit lack of pathogen avoidance behavior and deficient immune response toward P. aeruginosa relative to the wild-type (N2) strain. We found that the wild isolate from Germany RC301, which carries the allele for NPR-1 215F, shows an enhanced resistance to P. aeruginosa infection when compared with strain DA650, which also carries NPR-1 215F but in an N2 background. Using a whole-genome sequencing single-nucleotide polymorphism (WGS-SNP) mapping strategy, we determined that the resistance to P. aeruginosa infection maps to a region on chromosome V. Furthermore, we demonstrated that the mechanism for the enhanced resistance to P. aeruginosa infection relies exclusively on strong P. aeruginosa avoidance behavior, and does not involve the main immune, stress, and lifespan extension pathways in C. elegans Our findings underscore the importance of pathogen-specific behavioral immune defense in the wild, which seems to be favored over the more energy-costly mechanism of activation of physiological cellular defenses.

Balancing selection shapes density-dependent foraging behaviour.

The optimal foraging strategy in a given environment depends on the number of competing individuals and their behavioural strategies. Little is known about the genes and neural circuits that integrate social information into foraging decisions. Here we show that ascaroside pheromones, small glycolipids that signal population density, suppress exploratory foraging in Caenorhabditis elegans, and that heritable variation in this behaviour generates alternative foraging strategies. We find that natural C. elegans isolates differ in their sensitivity to the potent ascaroside icas#9 (IC-asc-C5). A quantitative trait locus (QTL) regulating icas#9 sensitivity includes srx-43, a G-protein-coupled icas#9 receptor that acts in the ASI class of sensory neurons to suppress exploration. Two ancient haplotypes associated with this QTL confer competitive growth advantages that depend on ascaroside secretion, its detection by srx-43 and the distribution of food. These results suggest that balancing selection at the srx-43 locus generates alternative density-dependent behaviours, fulfilling a prediction of foraging game theory.

Sampling and Isolation of C. elegans from the Natural Habitat.

Wild populations of the model organism C. elegans allow characterization of natural genetic variation underlying diverse phenotypic traits. Here we provide a simple protocol on how to sample and rapidly identify C. elegans wild isolates. We outline how to find suitable habitats and organic substrates, followed by describing isolation and identification of C. elegans live cultures based on easily recognizable morphological characteristics, molecular barcodes and/or mating tests. This protocol uses standard laboratory equipment and requires no prior knowledge of C. elegans biology.

Highly efficient microfluidic sorting device for synchronizing developmental stages of C. elegans based on deflecting electrotaxis.

C. elegans as a powerful model organism has been widely used in fundamental biological studies. Many of these studies frequently need a large number of different stage-synchronized worms due to the stage-specific features of C. elegans among 4 distinct larval stages and the adult stage. In this work, we present an interesting and cost-effective microfluidic approach to realize simultaneous sorting of C. elegans of different developmental stages by deflecting electrotaxis. The microfluidic device was fabricated using PDMS consisting of symmetric sorting channels with specific angles, which was further hybridized to an agarose plate. While applying an electric field, different stages of C. elegans would crawl to the negative pore with different angles due to their deflecting electrotaxis. Thus, the worms were separated and synchronized by stages. lon-2 mutant was further used to study this electrotactic response and the results indicated that the body size plays a key role in determining the deflecting angle in matured adult worms. In addition to discriminating wild-type hermaphrodites, it could also be employed to sort mutants with abnormal development sizes and males. Therefore, our device provided a versatile and highly efficient platform for sorting C. elegans to meet the requirement of large numbers of different stage-synchronized worms. It can also be further used to investigate the neuronal basis of deflecting electrotaxis in worms.

Hydrogel-droplet microfluidic platform for high-resolution imaging and sorting of early larval Caenorhabditis elegans.

The nematode Caenorhabditis elegans is an important model organism in research on neuroscience and development because of its stereotyped anatomy, relevance to human biology, and ease of culture and genetic manipulation. The first larval stage (L1) is of particular interest in many biological problems, including post-embryonic developmental processes and developmental decision-making, such as dauer formation. However, L1's small size and high mobility make it difficult to manipulate; particularly in microfluidic chips, which have been used to great advantage in handling larger larvae and adult animals, small features are difficult to fabricate and these structures often get clogged easily, making the devices less robust. We have developed a microfluidic device to overcome these challenges and enable high-resolution imaging and sorting of early larval stage C. elegans via encapsulation in droplets of a thermosensitive hydrogel. To achieve precise handling of early larval stage worms, we demonstrated on-chip production, storage, and sorting of hydrogel droplets. We also demonstrated temporary immobilization of the worms within the droplets, allowing high-resolution imaging with minimal physiological perturbations. Because of the ability to array hydrogel droplets for handling a large number of L1 worms in a robust way, we envision that this platform will be widely applicable to screening in various developmental studies.

A highly efficient nonchemical method for isolating live nematodes (Caenorhabditis elegans) from soil during toxicity assays.

The success of soil toxicity tests using Caenorhabditis elegans may depend in large part on recovering the organisms from the soil. However, it can be difficult to learn the International Organization for Standardization/ASTM International recovery process that uses the colloidal silica flotation method. The present study determined that a soil-agar isolation method provides a highly efficient and less technically demanding alternative to the colloidal silica flotation method. Test soil containing C. elegans was arranged on an agar plate in a donut shape, a linear shape, or a C curve; and microbial food was placed outside the soil to encourage the nematodes to leave the soil. The effects of ventilation and the presence of food on nematode recovery were tested to determine the optimal conditions for recovery. A linear arrangement of soil on an agar plate that was sprinkled with microbial food produced nearly 83% and 90% recovery of live nematodes over a 3-h and a 24-h period, respectively, without subjecting the nematodes to chemical stress. The method was tested using copper (II) chloride dihydrate, and the resulting recovery rate was comparable to that obtained using colloidal silica flotation. The soil-agar isolation method portrayed in the present study enables live nematodes to be isolated with minimal additional physicochemical stress, making it a valuable option for use in subsequent sublethal tests where live nematodes are required.

A continuous-flow C. elegans sorting system with integrated optical fiber detection and laminar flow switching.

We present a high-throughput continuous-flow C. elegans sorting device that works based on integrated optical fiber detection and laminar flow switching. Two types of genetically engineered nematodes are allowed to flow into the device and their genotypes are detected based on their fluorescence, without the need for immobilization, by integrated optical fibers. A novel dynamic fluidic switch sorts the nematodes to desired outlets. By changing input pressures of the control inlets, the laminar flow path is altered to steer the nematodes to appropriate outlets. Compared to previously reported microfluidic C. elegans sorting devices, sorting in this system is conducted in a continuous flow environment without any immobilization technique or need for multilayer mechanical valves to open and close the outlets. The continuous flow sorter not only increases the throughput but also avoids any kind of invasive or possibly damaging mechanical or chemical stimulus. We have characterized both the detection and the switching accuracy of the sorting device at different flow rates, and efficiencies approaching 100% can be achieved with a high throughput of about one nematode per second. To confirm that there was no significant damage to C. elegans following sorting, we recovered the sorted worms, finding no deaths and no differences in behavior and propagation compared to control.

Isolation of C. elegans and related nematodes.

Isolating Caenorhabditis and other nematodes from the wild first requires field sampling (reviewed in Section 1). The easiest and most efficient way to recover the animals from any substrate is to place the sample onto a standard C. elegans culture plate (Section 2.1). Alternative methods used by nematologists to recover soil nematodes (Sections 2.2, 2.3, and 2.4) are in our hands more difficult to implement and only yield a fraction of the individuals in the sample. A tricky step is to recognize your species of interest out of the zoo of nematode species that comes with a typical sample (Section 3). Culture (Section 4) and freezing (Section 5) conditions are then reviewed. Finally, we briefly summarize the organization and timing of an isolation experiment (Section 6), as well as the available collections (Section 7). Bear in mind that this chapter is strongly focused towards the isolation of Caenorhabditis elegans and close relatives.

Automated separation of C. elegans variably colonized by a bacterial pathogen.

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Mitochondrial DNA variant in COX1 subunit significantly alters energy metabolism of geographically divergent wild isolates in Caenorhabditis elegans.

Mitochondrial DNA (mtDNA) sequence variation can influence the penetrance of complex diseases and climatic adaptation. While studies in geographically defined human populations suggest that mtDNA mutations become fixed when they have conferred metabolic capabilities optimally suited for a specific environment, it has been challenging to definitively assign adaptive functions to specific mtDNA sequence variants in mammals. We investigated whether mtDNA genome variation functionally influences Caenorhabditis elegans wild isolates of distinct mtDNA lineages and geographic origins. We found that, relative to N2 (England) wild-type nematodes, CB4856 wild isolates from a warmer native climate (Hawaii) had a unique p.A12S amino acid substitution in the mtDNA-encoded COX1 core catalytic subunit of mitochondrial complex IV (CIV). Relative to N2, CB4856 worms grown at 20°C had significantly increased CIV enzyme activity, mitochondrial matrix oxidant burden, and sensitivity to oxidative stress but had significantly reduced lifespan and mitochondrial membrane potential. Interestingly, mitochondrial membrane potential was significantly increased in CB4856 grown at its native temperature of 25°C. A transmitochondrial cybrid worm strain, chpIR (M, CB4856>N2), was bred as homoplasmic for the CB4856 mtDNA genome in the N2 nuclear background. The cybrid strain also displayed significantly increased CIV activity, demonstrating that this difference results from the mtDNA-encoded p.A12S variant. However, chpIR (M, CB4856>N2) worms had significantly reduced median and maximal lifespan relative to CB4856, which may relate to their nuclear-mtDNA genome mismatch. Overall, these data suggest that C. elegans wild isolates of varying geographic origins may adapt to environmental challenges through mtDNA variation to modulate critical aspects of mitochondrial energy metabolism.

The prevalence of Caenorhabditis elegans across 1.5 years in selected North German locations: the importance of substrate type, abiotic parameters, and Caenorhabditis competitors.

BACKGROUND: Although the nematode Caenorhabditis elegans is a major model organism in diverse biological areas and well studied under laboratory conditions, little is known about its ecology. Therefore, characterization of the species' natural habitats should provide a new perspective on its otherwise well-studied biology. The currently best characterized populations are in France, demonstrating that C. elegans prefers nutrient- and microorganism-rich substrates such as rotting fruits and decomposing plant matter. In order to extend these findings, we sampled C. elegans continuously across 1.5 years from rotting apples and compost heaps in three North German locations. RESULTS: C. elegans was found throughout summer and autumn in both years. It shares its habitat with the related nematode species C. remanei, which could thus represent an important competitor for a similar ecological niche. The two species were isolated from the same site, but rarely the same substrate sample. In fact, C. elegans was mainly found on compost and C. remanei on rotten apples, possibly suggesting niche separation. The occurrence of C. elegans itself was related to environmental humidity and rain, although the correlation was significant for only one sampling site each. Additional associations between nematode prevalence and abiotic parameters could not be established. CONCLUSIONS: Taken together, our findings vary from the previous results for French C. elegans populations in that the considered German populations always coexisted with the congeneric species C. remanei (rather than C. briggsae as in France) and that C. elegans prevalence can associate with humidity and rain (rather than temperature, as suggested for French populations). Consideration of additional locations and time points is thus essential for full appreciation of the nematode's natural ecology.