Regulator of Lipid Metabolism NHR-49 Mediates Pathogen Avoidance through Precise Control of Neuronal Activity.

Precise control of neuronal activity is crucial for the proper functioning of neurons. How lipid homeostasis contributes to neuronal activity and how much of it is regulated by cells autonomously is unclear. In this study, we discovered that absence of the lipid regulator nhr-49, a functional ortholog of the peroxisome proliferator-activated receptor (PPAR) in Caenorhabditis elegans, resulted in defective pathogen avoidance behavior against Pseudomonas aeruginosa (PA14). Functional NHR-49 was required in the neurons, and more specifically, in a set of oxygen-sensing body cavity neurons, URX, AQR, and PQR. We found that lowering the neuronal activity of the body cavity neurons improved avoidance in nhr-49 mutants. Calcium imaging in URX neurons showed that nhr-49 mutants displayed longer-lasting calcium transients in response to an O2 upshift, suggesting that excess neuronal activity leads to avoidance defects. Cell-specific rescue of NHR-49 in the body cavity neurons was sufficient to improve pathogen avoidance, as well as URX neuron calcium kinetics. Supplementation with oleic acid also improved avoidance behavior and URX calcium kinetics, suggesting that the defective calcium response in the neuron is due to lipid dysfunction. These findings highlight the role of cell-autonomous lipid regulation in neuronal physiology and immune behavior.

Precise sensorimotor control impacts reproductive fitness of C. elegans in 3D environments.

The ability of animals to sense and navigate towards relevant cues in complex and elaborate habitats is paramount for their survival and reproductive success. The nematode Caenorhabditis elegans uses a simple and elegant sensorimotor program to track odors in its environments. Whether this allows the worm to effectively navigate a complex environment and increase its evolutionary success has not been tested yet. We designed an assay to test whether C. elegans can track odors in a complex 3D environment. We then used a previously established 3D cultivation system to test whether defect in tracking odors to find food in a complex environment affected their brood size. We found that wild-type worms can accurately migrate toward a variety of odors in 3D. However, mutants of the muscarinic acetylcholine receptor GAR-3 which have a sensorimotor integration defect that results in a subtle navigational defect steering towards attractive odors, display decreased chemotaxis to the odor butanone not seen in the traditional 2D assay. We also show that the decreased ability to locate appetitive stimuli in 3D leads to reduced brood size not observed in the standard 2D culture conditions. Our study shows that mutations in genes previously overlooked in 2D conditions can have a significant impact in the natural habitat, and highlights the importance of considering the evolutionary selective pressures that have shaped the behavior, as well as the underlying genes and neural circuits.

Dissecting the Neuronal Contributions of the Lipid Regulator NHR-49 Function in Lifespan and Behavior in C. elegans.

Although the importance of lipid homeostasis in neuronal function is undisputed, how they are regulated within neurons to support their unique function is an area of active study. NHR-49 is a nuclear hormone receptor functionally similar to PPARα, and a major lipid regulator in C. elegans. Although expressed in most tissues, little is known about its roles outside the intestine, the main metabolic organ of C. elegans. Here, using tissue- and neuron-type-specific transgenic strains, we examined the contribution of neuronal NHR-49 to cell-autonomous and non-autonomous nhr-49 mutant phenotypes. We examined lifespan, brood size, early egg-laying, and reduced locomotion on food. We found that lifespan and brood size could be rescued by neuronal NHR-49, and that NHR-49 in cholinergic and serotonergic neurons is sufficient to restore lifespan. For behavioral phenotypes, NHR-49 in serotonergic neurons was sufficient to control egg-laying, whereas no single tissue or neuron type was able to rescue the enhanced on-food slowing behavior. Our study shows that NHR-49 can function in single neuron types to regulate C. elegans physiology and behavior, and provides a platform to further investigate how lipid metabolism in neurons impact neuronal function and overall health of the organism.

Comparative Analysis of Muscle Atrophy During Spaceflight, Nutritional Deficiency and Disuse in the Nematode Caenorhabditis elegans.

While spaceflight is becoming more common than before, the hazards spaceflight and space microgravity pose to the human body remain relatively unexplored. Astronauts experience muscle atrophy after spaceflight, but the exact reasons for this and solutions are unknown. Here, we take advantage of the nematode C. elegans to understand the effects of space microgravity on worm body wall muscle. We found that space microgravity induces muscle atrophy in C. elegans from two independent spaceflight missions. As a comparison to spaceflight-induced muscle atrophy, we assessed the effects of acute nutritional deprivation and muscle disuse on C. elegans muscle cells. We found that these two factors also induce muscle atrophy in the nematode. Finally, we identified clp-4, which encodes a calpain protease that promotes muscle atrophy. Mutants of clp-4 suppress starvation-induced muscle atrophy. Such comparative analyses of different factors causing muscle atrophy in C. elegans could provide a way to identify novel genetic factors regulating space microgravity-induced muscle atrophy.

A Smartphone-Based Imaging Method for C. elegans Lawn Avoidance Assay.

When exposed to toxic or pathogenic bacteria, the nematode Caenorhabditis elegans displays a learned lawn avoidance behavior, in which the worms gradually leave their food source and prefer to remain outside the bacterial lawn. The assay is an easy way to test the worms' ability to sense external or internal cues to properly respond to harmful conditions. Though a simple assay, counting is time consuming, particularly with multiple samples, and assay durations that span overnight are inconvenient for researchers. An imaging system that can image many plates over a long period is useful but costly. Here, we describe a smartphone-based imaging method to record lawn avoidance in C. elegans. The method requires only a smartphone and a light emitting diode (LED) light box, to serve as a transmitted light source. Using free time-lapse camera applications, each phone can image up to six plates, with sufficient sharpness and contrast to manually count worms outside the lawn. The resulting movies are processed into 10 s audio video interleave (AVI) files for every hourly time point, then cropped to show each single plate to make them more amenable for counting. This method is a cost-effective way for those looking to examine avoidance defects and can potentially be extended to other C. elegans assays.

Making "Sense" of Ecology from a Genetic Perspective: Caenorhabditis elegans, Microbes and Behavior.

Our knowledge of animal and behavior in the natural ecology is based on over a century's worth of valuable field studies. In this post-genome era, however, we recognize that genes are the underpinning of ecological interactions between two organisms. Understanding how genes contribute to animal ecology, which is essentially the intersection of two genomes, is a tremendous challenge. The bacterivorous nematode Caenorhabditis elegans, one of the most well-known genetic animal model experimental systems, experiences a complex microbial world in its natural habitat, providing us with a window into the interplay of genes and molecules that result in an animal-microbial ecology. In this review, we will discuss C. elegans natural ecology, how the worm uses its sensory system to detect the microbes and metabolites that it encounters, and then discuss some of the fascinating ecological dances, including behaviors, that have evolved between the nematode and the microbes in its environment.

Comparison of Life Traits in Two Bacterivorous Nematodes Suggest Different Ecological Strategies to Exploit Similar Habitats.

Environments can be in states of dynamic change as well as persistent stability. These different states are a result of outside external conditions, but also the constant flux of living organisms in that ecological fauna. Nematodes are tremendously diverse, and many types can reside in the same soil microenvironments at the same time. To examine how so many nematodes can thrive and exploit a single environment, we identified two bacterivorous nematodes, Caenorhabditis elegans and Acrobeloides tricornis, that can inhabit rotting apple and soil environments. We cultured both nematodes in the laboratory and compared their life traits. We found that whereas C. elegans develops and reproduces extremely quickly, A. tricornis reaches sexual maturity much later and lays eggs at a slower rate but remains fertile for a longer time. In addition, A. tricornis displays a slower feeding behavior than C. elegans. Finally, A. tricornis has a significantly longer lifespan than C. elegans. These differences in development, physiology and behavior between the two nematodes hint at different ecological strategies to exploit the same habitat over different time periods, C. elegans as a colonizer-type nematode, and A. tricornis as more of a persister.

Biotechnological Activities and Applications of Bacterial Pigments Violacein and Prodigiosin.

In this review, we discuss violacein and prodigiosin, two chromogenic bacterial secondary metabolites that have diverse biological activities. Although both compounds were "discovered" more than seven decades ago, interest into their biological applications has grown in the last two decades, particularly driven by their antimicrobial and anticancer properties. These topics will be discussed in the first half of this review. The latter half delves into the current efforts of groups to produce these two compounds. This includes in both their native bacterial hosts and heterogeneously in other bacterial hosts, including discussing some of the caveats related to the yields reported in the literature, and some of the synthetic biology techniques employed in this pursuit.

Muscle and epidermal contributions of the structural protein ß-spectrin promote hypergravity-induced motor neuron axon defects in C. elegans.

Biology is adapted to Earth's gravity force, and the long-term effects of varying gravity on the development of animals is unclear. Previously, we reported that high gravity, called hypergravity, increases defects in the development of motor neuron axons in the nematode Caenorhabditis elegans. Here, we show that a mutation in the unc-70 gene that encodes the cytoskeletal ß-spectrin protein suppresses hypergravity-induced axon defects. UNC-70 expression is required in both muscle and epidermis to promote the axon defects in high gravity. We reveal that the location of axon defects is correlated to the size of the muscle cell that the axon traverses. We also show that mutations that compromise key proteins of hemidesmosomal structures suppress hypergravity-induced axon defects. These hemidesmosomal structures play a crucial role in coupling mechanical force between the muscle, epidermis and the external cuticle. We speculate a model in which the rigid organization of muscle, epidermal and cuticular layers under high gravity pressure compresses the narrow axon migration pathways in the extracellular matrix hindering proper axon pathfinding of motor neurons.

Consumption of Oleic Acid During Matriphagy in Free-Living Nematodes Alleviates the Toxic Effects of the Bacterial Metabolite Violacein.

Maternal behaviors benefit the survival of young, contributing directly to the mother's reproductive fitness. An extreme form of this is seen in matriphagy, when a mother performs the ultimate sacrifice and offers her body as a meal for her young. Whether matriphagy offers only a single energy-rich meal or another possible benefit to the young is unknown. Here, we characterized the toxicity of a bacterial secondary metabolite, namely, violacein, in Caenorhabditis elegans and found it is not only toxic towards adults, but also arrests growth and development of C. elegans larvae. To counteract this, C. elegans resorted to matriphagy, with the mothers holding their eggs within their bodies and hatching the young larvae internally, which eventually led to the mothers' death. This violacein-induced matriphagy alleviated some of the toxic effects of violacein, allowing a portion of the internally-hatched young to bypass developmental arrest. Using genetic and pharmacological experiments, we found the consumption of oleate, a monounsaturated fatty acid produced by the mother, during matriphagy is partially responsible. As such, our study provides experimental evidence of why such a drastic and peculiar maternal behavior may have arisen in nematode natural habitats.

Odor-dependent temporal dynamics in Caenorhabitis elegans adaptation and aversive learning behavior.

Animals sense an enormous number of cues in their environments, and, over time, can form learned associations and memories with some of these. The nervous system remarkably maintains the specificity of learning and memory to each of the cues. Here we asked whether the nematode Caenorhabditis elegans adjusts the temporal dynamics of adaptation and aversive learning depending on the specific odor sensed. C. elegans senses a multitude of odors, and adaptation and learned associations to many of these odors requires activity of the cGMP-dependent protein kinase EGL-4 in the AWC sensory neuron. We identified a panel of 17 attractive odors, some of which have not been tested before, and determined that the majority of these odors require the AWC primary sensory neuron for sensation. We then devised a novel assay to assess odor behavior over time for a single population of animals. We used this assay to evaluate the temporal dynamics of adaptation and aversive learning to 13 odors and find that behavior change occurs early in some odors and later in others. We then examined EGL-4 localization in early-trending and late-trending odors over time. We found that the timing of these behavior changes correlated with the timing of nuclear accumulation of EGL-4 in the AWC neuron suggesting that temporal changes in behavior may be mediated by aversive learning mechanisms. We demonstrate that temporal dynamics of adaptation and aversive learning in C. elegans can be used as a model to study the timing of memory formation to different sensory cues.

Hypergravity hinders axonal development of motor neurons in Caenorhabditis elegans.

As space flight becomes more accessible in the future, humans will be exposed to gravity conditions other than our 1G environment on Earth. Our bodies and physiology, however, are adapted for life at 1G gravity. Altering gravity can have profound effects on the body, particularly the development of muscles, but the reasons and biology behind gravity's effect are not fully known. We asked whether increasing gravity had effects on the development of motor neurons that innervate and control muscle, a relatively unexplored area of gravity biology. Using the nematode model organism Caenorhabditis elegans, we examined changes in response to hypergravity in the development of the 19 GABAergic DD/VD motor neurons that innervate body muscle. We found that a high gravity force above 10G significantly increases the number of animals with defects in the development of axonal projections from the DD/VD neurons. We showed that a critical period of hypergravity exposure during the embryonic/early larval stage was sufficient to induce defects. While characterizing the nature of the axonal defects, we found that in normal 1G gravity conditions, DD/VD axonal defects occasionally occurred, with the majority of defects occurring on the dorsal side of the animal and in the mid-body region, and a significantly higher rate of error in the 13 VD axons than the 6 DD axons. Hypergravity exposure increased the rate of DD/VD axonal defects, but did not change the distribution or the characteristics of the defects. Our study demonstrates that altering gravity can impact motor neuron development.

Combinatorial effects of odorants on mouse behavior.

The mechanisms by which odors induce instinctive behaviors are largely unknown. Odor detection in the mouse nose is mediated by >1, 000 different odorant receptors (ORs) and trace amine-associated receptors (TAARs). Odor perceptions are encoded combinatorially by ORs and can be altered by slight changes in the combination of activated receptors. However, the stereotyped nature of instinctive odor responses suggests the involvement of specific receptors and genetically programmed neural circuits relatively immune to extraneous odor stimuli and receptor inputs. Here, we report that, contrary to expectation, innate odor-induced behaviors can be context-dependent. First, different ligands for a given TAAR can vary in behavioral effect. Second, when combined, some attractive and aversive odorants neutralize one another's behavioral effects. Both a TAAR ligand and a common odorant block aversion to a predator odor, indicating that this ability is not unique to TAARs and can extend to an aversive response of potential importance to survival. In vitro testing of single receptors with binary odorant mixtures indicates that behavioral blocking can occur without receptor antagonism in the nose. Moreover, genetic ablation of a single receptor prevents its cognate ligand from blocking predator odor aversion, indicating that the blocking requires sensory input from the receptor. Together, these findings indicate that innate odor-induced behaviors can depend on context, that signals from a single receptor can block innate odor aversion, and that instinctive behavioral responses to odors can be modulated by interactions in the brain among signals derived from different receptors.

NGT-3D: a simple nematode cultivation system to study Caenorhabditis elegans biology in 3D.

The nematodeCaenorhabditiselegansis one of the premier experimental model organisms today. In the laboratory, they display characteristic development, fertility, and behaviors in a two dimensional habitat. In nature, however,C. elegansis found in three dimensional environments such as rotting fruit. To investigate the biology ofC. elegansin a 3D controlled environment we designed a nematode cultivation habitat which we term the nematode growth tube or NGT-3D. NGT-3D allows for the growth of both nematodes and the bacteria they consume. Worms show comparable rates of growth, reproduction and lifespan when bacterial colonies in the 3D matrix are abundant. However, when bacteria are sparse, growth and brood size fail to reach levels observed in standard 2D plates. Using NGT-3D we observe drastic deficits in fertility in a sensory mutant in 3D compared to 2D, and this defect was likely due to an inability to locate bacteria. Overall, NGT-3D will sharpen our understanding of nematode biology and allow scientists to investigate questions of nematode ecology and evolutionary fitness in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory.

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

A natural odor attraction between lactic acid bacteria and the nematode Caenorhabditis elegans.

Animal predators can track prey using their keen sense of smell. The bacteriovorous nematode Caenorhabditis elegans employs sensitive olfactory sensory neurons that express vertebrate-like odor receptors to locate bacteria. C. elegans displays odor-related behaviors such as attraction, aversion and adaptation, but the ecological significance of these behaviors is not known. Using a combination of food microbiology and genetics, we elucidate a possible predator-prey relationship between C. elegans and lactic acid bacteria (LAB) in rotting citrus fruit. LAB produces the volatile odor diacetyl as an oxidized by-product of fermentation in the presence of citrate. We show that C. elegans is attracted to LAB when grown on citrate media or Citrus medica L, commonly known as yuzu, a citrus fruit native to East Asia, and this attraction is mediated by the diacetyl odor receptor, ODR-10. We isolated a wild LAB strain and a wild C. elegans-related nematode from rotten yuzu, and demonstrate that the wild nematode was attracted to the diacetyl produced by LAB. These results not only identify an ecological function for a C. elegans olfactory behavior, but contribute to the growing understanding of ecological relationships between the microbial and metazoan worlds.

Violacein: Properties and Production of a Versatile Bacterial Pigment.

Violacein-producing bacteria, with their striking purple hues, have undoubtedly piqued the curiosity of scientists since their first discovery. The bisindole violacein is formed by the condensation of two tryptophan molecules through the action of five proteins. The genes required for its production, vioABCDE, and the regulatory mechanisms employed have been studied within a small number of violacein-producing strains. As a compound, violacein is known to have diverse biological activities, including being an anticancer agent and being an antibiotic against Staphylococcus aureus and other Gram-positive pathogens. Identifying the biological roles of this pigmented molecule is of particular interest, and understanding violacein's function and mechanism of action has relevance to those unmasking any of its commercial or therapeutic benefits. Unfortunately, the production of violacein and its related derivatives is not easy and so various groups are also seeking to improve the fermentative yields of violacein through genetic engineering and synthetic biology. This review discusses the recent trends in the research and production of violacein by both natural and genetically modified bacterial strains.

Olfactory receptor genes expressed in distinct lineages are sequestered in different nuclear compartments.

The olfactory system translates a vast array of volatile chemicals into diverse odor perceptions and innate behaviors. Odor detection in the mouse nose is mediated by 1,000 different odorant receptors (ORs) and 14 trace amine-associated receptors (TAARs). ORs are used in a combinatorial manner to encode the unique identities of myriad odorants. However, some TAARs appear to be linked to innate responses, raising questions about regulatory mechanisms that might segregate OR and TAAR expression in appropriate subsets of olfactory sensory neurons (OSNs). Here, we report that OSNs that express TAARs comprise at least two subsets that are biased to express TAARs rather than ORs. The two subsets are further biased in Taar gene choice and their distribution within the sensory epithelium, with each subset preferentially expressing a subgroup of Taar genes within a particular spatial domain in the epithelium. Our studies reveal one mechanism that may regulate the segregation of Olfr (OR) and Taar expression in different OSNs: the sequestration of Olfr and Taar genes in different nuclear compartments. Although most Olfr genes colocalize near large central heterochromatin aggregates in the OSN nucleus, Taar genes are located primarily at the nuclear periphery, coincident with a thin rim of heterochromatin. Taar-expressing OSNs show a shift of one Taar allele away from the nuclear periphery. Furthermore, examination of hemizygous mice with a single Taar allele suggests that the activation of a Taar gene is accompanied by an escape from the peripheral repressive heterochromatin environment to a more permissive interior chromatin environment.

Olfactory receptor patterning in a higher primate.

The mammalian olfactory system detects a plethora of environmental chemicals that are perceived as odors or stimulate instinctive behaviors. Studies using odorant receptor (OR) genes have provided insight into the molecular and organizational strategies underlying olfaction in mice. One important unanswered question, however, is whether these strategies are conserved in primates. To explore this question, we examined the macaque, a higher primate phylogenetically close to humans. Here we report that the organization of sensory inputs in the macaque nose resembles that in mouse in some respects, but not others. As in mouse, neurons with different ORs are interspersed in the macaque nose, and there are spatial zones that differ in their complement of ORs and extend axons to different domains in the olfactory bulb of the brain. However, whereas the mouse has multiple discrete band-like zones, the macaque appears to have only two broad zones. It is unclear whether the organization of OR inputs in a rodent/primate common ancestor degenerated in primates or, alternatively became more sophisticated in rodents. The mouse nose has an additional small family of chemosensory receptors, called trace amine-associated receptors (TAARs), which may detect social cues. Here we find that TAARs are also expressed in the macaque nose, suggesting that TAARs may also play a role in human olfactory perception. We further find that one human TAAR responds to rotten fish, suggesting a possible role as a sentinel to discourage ingestion of food harboring pathogenic microorganisms.

The multiple faces of calcineurin signaling in Caenorhabditis elegans: development, behaviour and aging.

Calcineurin, a well-conserved protein phosphatase 2B (PP2B), is a Ca2+-calmodulin-dependent serine/threonine protein phosphatase that is known to be involved in a myriad of cellular processes and signal transduction pathways. The biological role of calcineurin has been extensively studied in diverse groups of organisms. Homologues of mammalian and Drosophila calcineurin subunits exist in the nematode, Caenorhabditis elegans. The C. elegans counterpart of the catalytic subunit, calcineurin A, cna-1/tax-6, and the regulatory subunit, calcineurin B, cnb-1, are known to express ubiquitously in multiple tissues including neurons. The characterization of C. elegans calcineurin mutants facilitates identification of its physiological functions and signaling pathways. Genetic interactions between cna-1/tax-6 and cnb-1 mutants with a number of mutants involved in several signaling pathways have exemplified the pivotal role of calcineurin in regulating nematode development, behaviour and lifespan (aging). The present review has been aimed to provide a succinct summary of the multiple functions of calcineurin in C. elegans relating to its development, fertility, proliferation, behaviour and lifespan. Analyses of cna-1/tax-6 and cnb-1 interacting proteins and regulators of the phosphatase in this fascinating worm model have an immense scope to identify potential drug targets in various parasitic nematodes, which cause many diseases inflicting huge economic loss; and also for many human diseases, particularly neurodegenerative and myocardial diseases.