Clock gene homologs lin-42 and kin-20 regulate circadian rhythms in C. elegans.

Circadian rhythms are endogenous oscillations in nearly all organisms, from prokaryotes to humans, allowing them to adapt to cyclical environments for close to 24 h. Circadian rhythms are regulated by a central clock, based on a transcription-translation feedback loop. One important protein in the central loop in metazoan clocks is PERIOD, which is regulated in part by Casein kinase 1ε/δ (CK1ε/δ) phosphorylation. In the nematode Caenorhabditis elegans, period and casein kinase 1ε/δ are conserved as lin-42 and kin-20, respectively. Here, we studied the involvement of lin-42 and kin-20 in the circadian rhythms of the adult nematode using a bioluminescence-based circadian transcriptional reporter. We show that mutations of lin-42 and kin-20 generate a significantly longer endogenous period, suggesting a role for both genes in the nematode circadian clock, as in other organisms. These phenotypes can be partially rescued by overexpression of either gene under their native promoter. Both proteins are expressed in neurons and epidermal seam cells, as well as in other cells. Depletion of LIN-42 and KIN-20, specifically in neuronal cells after development, was sufficient to lengthen the period of oscillating sur-5 expression. Therefore, we conclude that LIN-42 and KIN-20 are critical regulators of the adult nematode circadian clock through neuronal cells.

Lactobacillus paracasei subsp. paracasei 2004 improves health and lifespan in Caenorhabditis elegans.

Recent research has highlighted the importance of the gut microbiome in regulating aging, and probiotics are interventions that can promote gut health. In this study, we surveyed several novel lactic acid bacteria to examine their beneficial effect on organismal health and lifespan in C. elegans. We found that animals fed some lactic acid bacteria, including L. acidophilus 1244 and L. paracasei subsp. paracasei 2004, grew healthy. Supplementation with the lactic acid bacterial strains L. acidophilus 1244 or L. paracasei subsp. paracasei 2004 significantly improved health, including food consumption, motility, and resistance to oxidative stressor, hydrogen peroxide. Our RNA-seq analysis showed that supplementation with L. paracasei subsp. paracasei 2004 significantly increased the expression of daf-16, a C. elegans FoxO homolog, as well as genes related to the stress response. Furthermore, daf-16 deletion inhibited the longevity effect of L. paracasei subsp. paracasei 2004 supplementation. Our results suggest that L. paracasei subsp. paracasei 2004 improves health and lifespan in a DAF-16-dependent manner.

Behavioral evolution: No sex please, we're hermaphrodites.

Many 'hard-wired', innate animal behaviors are related to reproduction. So what happens when reproductive systems evolve? New research in nematodes has identified principles underlying the co-evolution of reproductive strategy and sexual behavior, revealing some surprises and raising intriguing new questions.

Microfluidic approach to correlate C. elegans neuronal functional aging and underlying changes of gene expression in mechanosensation.

The aging process has broad physiological impacts, including a significant decline in sensory function, which threatens both physical health and quality of life. One ideal model to study aging, neuronal function, and gene expression is the nematode Caenorhabditis elegans, which has a short lifespan and relatively simple, thoroughly mapped nervous system and genome. Previous works have identified that mechanosensory neuronal structure changes with age, but importantly, the actual age-related changes in the function and health of neurons, as well as the underlying genetic mechanisms responsible for these declines, are not fully understood. While advanced techniques such as single-cell RNA-sequencing have been developed to quantify gene expression, it is difficult to relate this information to functional changes in aging due to a lack of tools available. To address these limitations, we present a platform capable of measuring both physiological function and its associated gene expression throughout the aging process in individuals. Using our pipeline, we investigate the age-related changes in function of the mechanosensing ALM neuron in C. elegans, as well as some relevant gene expression patterns (mec-4 and mec-10). Using a series of devices for animals of different ages, we examined subtle changes in neuronal function and found that while the magnitude of neuronal response to a large stimulus declines with age, sensory capability does not significantly decline with age; further, gene expression is well maintained throughout aging. Additionally, we examine PVD, a harsh-touch mechanosensory neuron, and find that it exhibits a similar age-related decline in magnitude of neuronal response. Together, our data demonstrate that our strategy is useful for identifying genetic factors involved in the decline in neuronal health. We envision that this framework could be applied to other systems as a useful tool for discovering new biology.

Versatile multiple object tracking in sparse 2D/3D videos via deformable image registration.

Tracking body parts in behaving animals, extracting fluorescence signals from cells embedded in deforming tissue, and analyzing cell migration patterns during development all require tracking objects with partially correlated motion. As dataset sizes increase, manual tracking of objects becomes prohibitively inefficient and slow, necessitating automated and semi-automated computational tools. Unfortunately, existing methods for multiple object tracking (MOT) are either developed for specific datasets and hence do not generalize well to other datasets, or require large amounts of training data that are not readily available. This is further exacerbated when tracking fluorescent sources in moving and deforming tissues, where the lack of unique features and sparsely populated images create a challenging environment, especially for modern deep learning techniques. By leveraging technology recently developed for spatial transformer networks, we propose ZephIR, an image registration framework for semi-supervised MOT in 2D and 3D videos. ZephIR can generalize to a wide range of biological systems by incorporating adjustable parameters that encode spatial (sparsity, texture, rigidity) and temporal priors of a given data class. We demonstrate the accuracy and versatility of our approach in a variety of applications, including tracking the body parts of a behaving mouse and neurons in the brain of a freely moving C. elegans. We provide an open-source package along with a web-based graphical user interface that allows users to provide small numbers of annotations to interactively improve tracking results.

GABA attenuates neurotoxicity of zinc oxide nanoparticles due to oxidative stress via DAF-16/FoxO and SKN-1/Nrf2 pathways.

Zinc oxide nanoparticles (ZnO-NPs) are one of the most widely used metal oxide nanomaterials. The increased use of ZnO-NPs has exacerbated environmental pollution and raised the risk of neurological disorders in organisms through food chains, and it is urgent to look for detoxification strategies. γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter that has been shown to have anxiolytic, anti-aging and inhibitory effects on nervous system excitability. However, there are few reports on the prevention and control of the toxicity of nano-metal ions by GABA. In zebrafish, ZnO-NPs exposure led to increased mortality and behavioral abnormalities of larva, which could be moderated by GABA intervention. Similar results were investigated in Caenorhabditis elegans, showing lifespan extension, abnormal locomotor frequency and behavior recovery when worms fed with GABA under ZnO-NPs exposure. Moreover, GABA enhanced antioxidant enzyme activities by upregulating the expression of antioxidant-related genes and thus scavenged excessive O2-. In the case of ZnO-NPs exposure, inhibition of nuclear translocation of DAF-16 and SKN-1 was restored by GABA. Meanwhile, the protective effect of GABA was blocked in daf-16 (-) and skn-1 (-) mutant, suggesting that DAF-16/FoxO and SKN-1/Nrf2 pathways is the key targets of GABA. This study provides a new solution for the application of GABA and mitigation of metal nanoparticle neurotoxicity.

A hyperpolarizing neuron recruits undocked innexin hemichannels to transmit neural information in Caenorhabditis elegans.

While depolarization of the neuronal membrane is known to evoke the neurotransmitter release from synaptic vesicles, hyperpolarization is regarded as a resting state of chemical neurotransmission. Here, we report that hyperpolarizing neurons can actively signal neural information by employing undocked hemichannels. We show that UNC-7, a member of the innexin family in Caenorhabditis elegans, functions as a hemichannel in thermosensory neurons and transmits temperature information from the thermosensory neurons to their postsynaptic interneurons. By monitoring neural activities in freely behaving animals, we find that hyperpolarizing thermosensory neurons inhibit the activity of the interneurons and that UNC-7 hemichannels regulate this process. UNC-7 is required to control thermotaxis behavior and functions independently of synaptic vesicle exocytosis. Our findings suggest that innexin hemichannels mediate neurotransmission from hyperpolarizing neurons in a manner that is distinct from the synaptic transmission, expanding the way of neural circuitry operations.

The egg-counter: a novel microfluidic platform for characterization of Caenorhabditis elegans egg-laying.

Reproduction is a fundamental process that shapes the demography of every living organism yet is often difficult to assess with high precision in animals that produce large numbers of offspring. Here, we present a novel microfluidic research platform for studying Caenorhabditis elegans' egg-laying. The platform provides higher throughput than traditional solid-media behavioral assays while providing a very high degree of temporal resolution. Additionally, the environmental control enabled by microfluidic animal husbandry allows for experimental perturbations difficult to achieve with solid-media assays. We demonstrate the platform's utility by characterizing C. elegans egg-laying behavior at two commonly used temperatures, 15 and 20 °C. As expected, we observed a delayed onset of egg-laying at 15 °C degrees, consistent with published temperature effects on development rate. Additionally, as seen in solid media studies, egg laying output was higher under the canonical 20 °C conditions. While we validated the Egg-Counter with a study of temperature effects in wild-type animals, the platform is highly adaptable to any nematode egg-laying research where throughput or environmental control needs to be maximized without sacrificing temporal resolution.

An automatic measurement method for the response of Caenorhabditis elegans to chemicals.

BACKGROUND: Caenorhabditis elegans is a widely used model animal. Chemotaxis assay is one of the experiments that study the effects of different chemicals on nematodes. It is mainly used to study the effects of different chemicals on the perception behavior of nematodes. By conducting this experiment, not only can the neurotoxicity of chemicals be reflected, but also the impact of chemicals on physiological functions regulated by the nervous system, such as nematode feeding behavior and basic motor ability. OBJECTIVE: The experiment of detecting the response of nematode to chemicals is also a common method of chemical toxicity testing based on nematode models. In the analysis of worm tendency behavior, manual operations are generally used. Manually processing a large number of worms under a microscope is very time-consuming and labor-intensive. The current quantitative methods for nematode chemotaxis experiments are not only time-consuming and labor-intensive, but also biased in experimental results due to differences in judgment standards among experimenters. The automatic and efficient quantification method for nematode chemotaxis experiments is a very important technical difficulty in the field of nematode experiments. METHODS: Here, we have designed an automatic quantification method for nematode chemotaxis experiments by incorporating image acquisition and processing techniques into the nematode experiment. RESULTS: The experimental results show that the Pearson correlation coefficient between manual and automatic counting results is 0.978. CONCLUSION: This proves the effectiveness of our method. Applying the automatic measurement method to replace manual counting by the experimenter can improve work efficiency, and reduce errors in human counting operations.

Active nuclear positioning and actomyosin contractility maintain leader cell integrity during gonadogenesis.

Proper distribution of organelles can play an important role in a moving cell's performance. During C. elegans gonad morphogenesis, the nucleus of the leading distal tip cell (DTC) is always found at the front, yet the significance of this localization is unknown. Here, we identified the molecular mechanism that keeps the nucleus at the front, despite a frictional force that pushes it backward. The Klarsicht/ANC-1/Syne homology (KASH) domain protein UNC-83 links the nucleus to the motor protein kinesin-1 that moves along a polarized acentrosomal microtubule network. Interestingly, disrupting nuclear positioning on its own did not affect gonad morphogenesis. However, reducing actomyosin contractility on top of nuclear mispositioning led to a dramatic phenotype: DTC splitting and gonad bifurcation. Long-term live imaging of the double knockdown revealed that, while the gonad attempted to perform a planned U-turn, the DTC was stretched due to the lagging nucleus until it fragmented into a nucleated cell and an enucleated cytoplast, each leading an independent gonadal arm. Remarkably, the enucleated cytoplast had polarity and invaded, but it could only temporarily support germ cell proliferation. Based on a qualitative biophysical model, we conclude that the leader cell employs two complementary mechanical approaches to preserve its integrity and ensure proper organ morphogenesis while navigating through a complex 3D environment: active nuclear positioning by microtubule motors and actomyosin-driven cortical contractility.

Fibration symmetries and cluster synchronization in the Caenorhabditis elegans connectome.

Capturing how the Caenorhabditis elegans connectome structure gives rise to its neuron functionality remains unclear. It is through fiber symmetries found in its neuronal connectivity that synchronization of a group of neurons can be determined. To understand these we investigate graph symmetries and search for such in the symmetrized versions of the forward and backward locomotive sub-networks of the Caenorhabditi elegans worm neuron network. The use of ordinarily differential equations simulations admissible to these graphs are used to validate the predictions of these fiber symmetries and are compared to the more restrictive orbit symmetries. Additionally fibration symmetries are used to decompose these graphs into their fundamental building blocks which reveal units formed by nested loops or multilayered fibers. It is found that fiber symmetries of the connectome can accurately predict neuronal synchronization even under not idealized connectivity as long as the dynamics are within stable regimes of simulations.

A tonically active master neuron modulates mutually exclusive motor states at two timescales.

Continuity of behaviors requires animals to make smooth transitions between mutually exclusive behavioral states. Neural principles that govern these transitions are not well understood. Caenorhabditis elegans spontaneously switch between two opposite motor states, forward and backward movement, a phenomenon thought to reflect the reciprocal inhibition between interneurons AVB and AVA. Here, we report that spontaneous locomotion and their corresponding motor circuits are not separately controlled. AVA and AVB are neither functionally equivalent nor strictly reciprocally inhibitory. AVA, but not AVB, maintains a depolarized membrane potential. While AVA phasically inhibits the forward promoting interneuron AVB at a fast timescale, it maintains a tonic, extrasynaptic excitation on AVB over the longer timescale. We propose that AVA, with tonic and phasic activity of opposite polarities on different timescales, acts as a master neuron to break the symmetry between the underlying forward and backward motor circuits. This master neuron model offers a parsimonious solution for sustained locomotion consisted of mutually exclusive motor states.

The coupling between healthspan and lifespan in Caenorhabditis depends on complex interactions between compound intervention and genetic background.

Aging is characterized by declining health that results in decreased cellular resilience and neuromuscular function. The relationship between lifespan and health, and the influence of genetic background on that relationship, has important implications in the development of pharmacological anti-aging interventions. Here we assessed swimming performance as well as survival under thermal and oxidative stress across a nematode genetic diversity test panel to evaluate health effects for three compounds previously studied in the Caenorhabditis Intervention Testing Program and thought to promote longevity in different ways - NP1 (nitrophenyl piperazine-containing compound 1), propyl gallate, and resveratrol. Overall, we find the relationships among median lifespan, oxidative stress resistance, thermotolerance, and mobility vigor to be complex. We show that oxidative stress resistance and thermotolerance vary with compound intervention, genetic background, and age. The effects of tested compounds on swimming locomotion, in contrast, are largely species-specific. In this study, thermotolerance, but not oxidative stress or swimming ability, correlates with lifespan. Notably, some compounds exert strong impact on some health measures without an equally strong impact on lifespan. Our results demonstrate the importance of assessing health and lifespan across genetic backgrounds in the effort to identify reproducible anti-aging interventions, with data underscoring how personalized treatments might be required to optimize health benefits.

Gut-associated functions are favored during microbiome assembly across a major part of C. elegans life.

The microbiome expresses a variety of functions that influence host biology. The range of functions depends on the microbiome's composition, which can change during the host's lifetime due to neutral assembly processes, host-mediated selection, and environmental conditions. To date, the exact dynamics of microbiome assembly, the underlying determinants, and the effects on host-associated functions remain poorly understood. Here, we used the nematode Caenorhabditis elegans and a defined community of fully sequenced, naturally associated bacteria to study microbiome dynamics and functions across a major part of the worm's lifetime of hosts under controlled experimental conditions. Bacterial community composition initially shows strongly declining levels of stochasticity, which increases during later time points, suggesting selective effects in younger animals as opposed to more random processes in older animals. The adult microbiome is enriched in genera Ochrobactrum and Enterobacter compared to the direct substrate and a host-free control environment. Using pathway analysis, metabolic, and ecological modeling, we further find that the lifetime assembly dynamics increase competitive strategies and gut-associated functions in the host-associated microbiome, indicating that the colonizing bacteria benefit the worm. Overall, our study introduces a framework for studying microbiome assembly dynamics based on stochastic, ecological, and metabolic models, yielding new insights into the processes that determine host-associated microbiome composition and function. IMPORTANCE: The microbiome plays a crucial role in host biology. Its functions depend on the microbiome composition that can change during a host's lifetime. To date, the dynamics of microbiome assembly and the resulting functions still need to be better understood. This study introduces a new approach to characterize the functional consequences of microbiome assembly by modeling both the relevance of stochastic processes and metabolic characteristics of microbial community changes. The approach was applied to experimental time-series data obtained for the microbiome of the nematode Caenorhabditis elegans across the major part of its lifetime. Stochastic processes played a minor role, whereas beneficial bacteria as well as gut-associated functions enriched in hosts. This indicates that the host might actively shape the composition of its microbiome. Overall, this study provides a framework for studying microbiome assembly dynamics and yields new insights into C. elegans microbiome functions.

Development of equation of motion deciphering locomotion including omega turns of Caenorhabditis elegans.

Locomotion is a fundamental behavior of Caenorhabditis elegans (C. elegans). Previous works on kinetic simulations of animals helped researchers understand the physical mechanisms of locomotion and the muscle-controlling principles of neuronal circuits as an actuator part. It has yet to be understood how C. elegans utilizes the frictional forces caused by the tension of its muscles to perform sequenced locomotive behaviors. Here, we present a two-dimensional rigid body chain model for the locomotion of C. elegans by developing Newtonian equations of motion for each body segment of C. elegans. Having accounted for friction-coefficients of the surrounding environment, elastic constants of C. elegans, and its kymogram from experiments, our kinetic model (ElegansBot) reproduced various locomotion of C. elegans such as, but not limited to, forward-backward-(omega turn)-forward locomotion constituting escaping behavior and delta-turn navigation. Additionally, ElegansBot precisely quantified the forces acting on each body segment of C. elegans to allow investigation of the force distribution. This model will facilitate our understanding of the detailed mechanism of various locomotive behaviors at any given friction-coefficients of the surrounding environment. Furthermore, as the model ensures the performance of realistic behavior, it can be used to research actuator-controller interaction between muscles and neuronal circuits.

Optogenetic manipulation of lysosomal physiology and autophagy-dependent clearance of amyloid beta.

Lysosomes are degradation centers of cells and intracellular hubs of signal transduction, nutrient sensing, and autophagy regulation. Dysfunction of lysosomes contributes to a variety of diseases, such as lysosomal storage diseases (LSDs) and neurodegeneration, but the mechanisms are not well understood. Altering lysosomal activity and examining its impact on the occurrence and development of disease is an important strategy for studying lysosome-related diseases. However, methods to dynamically regulate lysosomal function in living cells or animals are still lacking. Here, we constructed lysosome-localized optogenetic actuators, named lyso-NpHR3.0, lyso-ArchT, and lyso-ChR2, to achieve optogenetic manipulation of lysosomes. These new actuators enable light-dependent control of lysosomal membrane potential, pH, hydrolase activity, degradation, and Ca2+ dynamics in living cells. Notably, lyso-ChR2 activation induces autophagy through the mTOR pathway, promotes Aß clearance in an autophagy-dependent manner in cellular models, and alleviates Aß-induced paralysis in the Caenorhabditis elegans model of Alzheimer's disease. Our lysosomal optogenetic actuators supplement the optogenetic toolbox and provide a method to dynamically regulate lysosomal physiology and function in living cells and animals.

Generation of environmentally persistent free radicals on photoaged tire wear particles and their neurotoxic effects on neurotransmission in Caenorhabditis elegans.

Tire wear particles (TWP) are a prevalent form of microplastics (MPs) extensively distributed in the environment, raising concerns about their environmental behaviors and risks. However, knowledge regarding the properties and toxicity of these particles at environmentally relevant concentrations, specifically regarding the role of environmentally persistent free radicals (EPFRs) generated during TWP photoaging, remains limited. In this study, the evolution of EPFRs on TWP under different photoaging times and their adverse effects on Caenorhabditis elegans were systematically investigated. The photoaging process primarily resulted in the formation of EPFRs and reactive oxygen species (O2•-, ⋅OH, and 1O2), altering the physicochemical properties of TWP. The exposure of nematodes to 100 µg/L of TWP-50 (TWP with a photoaging time of 50 d) led to a significant decrease in locomotory behaviors (e.g., head thrashes, body bends, and wavelength) and neurotransmitter contents (e.g., dopamine, glutamate, and serotonin). Similarly, the expression of neurotransmission-related genes was reduced in nematodes exposed to TWP-50. Furthermore, the addition of free-radical inhibitors significantly suppressed TWP-induced neurotoxicity. Notably, correlation analysis revealed a significantly negative correlation between EPFRs levels and the locomotory behaviors and neurotransmitter contents of nematodes. Thus, it was concluded that EPFRs on photoaged TWP induce neurotoxicity by affecting neurotransmission. These findings elucidate the toxicity effects and mechanisms of EPFRs, emphasizing the importance of considering their contributions when evaluating the environmental risks associated with TWP.

Comparison of intestinal toxicity in enhancing intestinal permeability and in causing ROS production of six PPD quinones in Caenorhabditis elegans.

As the derivatives of p-phenylenediamines (PPDs), PPD quinones (PPDQs) have received increasing attention due to their possible exposure risk. We compared the intestinal toxicity of six PPDQs (6-PPDQ, 77PDQ, CPPDQ, DPPDQ, DTPDQ and IPPDQ) in Caenorhabditis elegans. In the range of 0.01-10 µg/L, only 77PDQ (10 µg/L) moderately induced the lethality. All the examined PPDQs at 0.01-10 µg/L did not affect intestinal morphology. Different from this, exposure to 6-PPDQ (1-10 µg/L), 77PDQ (0.1-10 µg/L), CPPDQ (1-10 µg/L), DPPDQ (1-10 µg/L), DTPDQ (1-10 µg/L), and IPPDQ (10 µg/L) enhanced intestinal permeability to different degrees. Meanwhile, exposure to 6-PPDQ (0.1-10 µg/L), 77PDQ (0.01-10 µg/L), CPPDQ (0.1-10 µg/L), DPPDQ (0.1-10 µg/L), DTPDQ (1-10 µg/L), and IPPDQ (1-10 µg/L) resulted in intestinal reactive oxygen species (ROS) production and activation of both SOD-3::GFP and GST-4::GFP. In 6-PPDQ, 77PDQ, CPPDQ, DPPDQ, DTPDQ, and/or IPPDQ exposed nematodes, the ROS production was strengthened by RNAi of genes (acs-22, erm-1, hmp-2, and pkc-3) governing functional state of intestinal barrier. Additionally, expressions of acs-22, erm-1, hmp-2, and pkc-3 were negatively correlated with intestinal ROS production in nematodes exposed to 6-PPDQ, 77PDQ, CPPDQ, DPPDQ, DTPDQ, and/or IPPDQ. Therefore, exposure to different PPDQs differentially induced the intestinal toxicity on nematodes. Our data highlighted potential exposure risk of PPDQs at low concentrations to organisms by inducing intestinal toxicity.