Caenorhabditis Elegans

''Caenorhabditis elegans'' () is a free-living transparent nematode about 1 mm in length that lives in temperate soil environments. It is the type species of its genus. The name is a blend of the Greek ''caeno-'' (recent), ''rhabditis'' (rod-like) and Latin ''elegans'' (elegant). In 1900, Maupas initially named it ''Rhabditides elegans.'' Osche placed it in the subgenus ''Caenorhabditis'' in 1952, and in 1955, Dougherty raised ''Caenorhabditis'' to the status of genus.

''C. elegans'' is an unsegmented pseudocoelomate and lacks respiratory or circulatory systems. Most of these nematodes are hermaphrodites and a few are males. Males have specialised tails for mating that include spicules.

In 1963, Sydney Brenner proposed research into ''C. elegans,'' primarily in the area of neuronal development. In 1974, he began research into the molecular and developmental biology of ''C. elegans'', which has since been extensively used as a model organism. It was the first multicellular organism to have its whole genome sequenced, and , is the only organism to have its connectome (neuronal "wiring diagram") completed.

Anatomy

''C. elegans'' is unsegmented, vermiform, and bilaterally symmetrical. It has a cuticle (a tough outer covering, as an exoskeleton), four main epidermal cords, and a fluid-filled pseudocoelom (body cavity). It also has some of the same organ systems as larger animals. About one in a thousand individuals is male and the rest are hermaphrodites. The basic anatomy of ''C. elegans'' includes a mouth, pharynx, intestine, gonad, and collagenous cuticle. Like all nematodes, they have neither a circulatory nor a respiratory system. The four bands of muscles that run the length of the body are connected to a neural system that allows the muscles to move the animal's body only as dorsal bending or ventral bending, but not left or right, except for the head, where the four muscle quadrants are wired independently from one another. When a wave of dorsal/ventral muscle contractions proceeds from the back to the front of the animal, the animal is propelled backwards. When a wave of contractions is initiated at the front and proceeds posteriorly along the body, the animal is propelled forwards. Because of this dorsal/ventral bias in body bends, any normal living, moving individual tends to lie on either its left side or its right side when observed crossing a horizontal surface. A set of ridges on the lateral sides of the body cuticle, the alae, is believed to give the animal added traction during these bending motions.

In relation to lipid metabolism, ''C. elegans'' does not have any specialized adipose tissues, a pancreas, a liver, or even blood to deliver nutrients compared to mammals. Neutral lipids are instead stored in the intestine, epidermis, and embryos. The epidermis corresponds to the mammalian adipocytes by being the main triglyceride depot.

The pharynx is a muscular food pump in the head of ''C. elegans'', which is triangular in cross-section. This grinds food and transports it directly to the intestine. A set of "valve cells" connects the pharynx to the intestine, but how this valve operates is not understood. After digestion, the contents of the intestine are released via the rectum, as is the case with all other nematodes. No direct connection exists between the pharynx and the excretory canal, which functions in the release of liquid urine.

Males have a single-lobed gonad, a vas deferens, and a tail specialized for mating, which incorporates spicules. Hermaphrodites have two ovaries, oviducts, and spermatheca, and a single uterus.

''C. elegans'' neurons contain dendrites which extend from the cell to receive neurotransmitters, and a process that extends to the nerve ring (the "brain") for a synaptic connection between neurons. The biggest difference is that ''C. elegans'' has motor excitatory and inhibitory neurons, known as cholinergic and gabaergic neurons, which simply act as further regulation for the tiny creature. They have no influence on the nervous system besides regulating neuron impulses.

Gut granules

Numerous gut granules are present in the intestine of ''C. elegans'', the functions of which are still not fully known, as are many other aspects of this nematode, despite the many years that it has been studied. These gut granules are found in all of the Rhabditida orders. They are very similar to lysosomes in that they feature an acidic interior and the capacity for endocytosis, but they are considerably larger, reinforcing the view of their being storage organelles.
A remarkable feature of the granules is that when they are observed under ultraviolet light, they react by emitting an intense blue fluorescence. Another phenomenon seen is termed 'death fluorescence'. As the worms die, a dramatic burst of blue fluorescence is emitted. This death fluorescence typically takes place in an anterior to posterior wave that moves along the intestine, and is seen in both young and old worms, whether subjected to lethal injury or peacefully dying of old age.

Many theories have been posited on the functions of the gut granules, with earlier ones being eliminated by later findings. They are thought to store zinc as one of their functions. Recent chemical analysis has identified the blue fluorescent material they contain as a glycosylated form of anthranilic acid (AA). The need for the large amounts of AA the many gut granules contain is questioned. One possibility is that the AA is antibacterial and used in defense against invading pathogens. Another possibility is that the granules provide photoprotection; the bursts of AA fluorescence entail the conversion of damaging UV light to relatively harmless visible light. This is seen a possible link to the melanin–containing melanosomes.

Reproduction

The hermaphroditic worm is considered to be a specialized form of self-fertile female, as its soma is female. The hermaphroditic germline produces male gametes first, and lays eggs through its uterus after internal fertilization. Hermaphrodites produce all their sperm in the L4 stage (150 sperm cells per gonadal arm) and then produce only oocytes. The hermaphroditic gonad acts as an ovotestis with sperm cells being stored in the same area of the gonad as the oocytes until the first oocyte pushes the sperm into the spermatheca (a chamber wherein the oocytes become fertilized by the sperm).

The male can inseminate the hermaphrodite, which will preferentially use male sperm (both types of sperm are stored in the spermatheca).
The sperm of ''C. elegans'' is amoeboid, lacking flagella and acrosomes. When self-inseminated, the wild-type worm lays about 300 eggs. When inseminated by a male, the number of progeny can exceed 1,000. Hermaphrodites do not typically mate with other hermaphrodites. At 20 °C, the laboratory strain of ''C. elegans'' (N2) has an average lifespan around 2–3 weeks and a generation time of 3 to 4 days.

''C. elegans'' has five pairs of autosomes and one pair of sex chromosomes. Sex in ''C. elegans'' is based on an X0 sex-determination system. Hermaphrodites of ''C. elegans'' have a matched pair of sex chromosomes (XX); the rare males have only one sex chromosome (X0).

Sex determination

''C. elegans'' are mostly hermaphroditic organisms, producing both sperms and oocytes. Males do occur in the population in a rate of approximately 1 in 200 hermaphrodites, but the two sexes are highly differentiated. Males differ from their hermaphroditic counterparts in that they are smaller and can be identified through the shape of their tail. ''C.elegans'' reproduce through a process called androdioecy. This means that they can reproduce in two ways: either through self-fertilization in hermaphrodites or through hermaphrodites breeding with males. Males are produced through non-disjunction of the X chromosomes during meiosis. The worms that reproduce through self-fertilization are at risk for high linkage disequilibrium, which leads to lower genetic diversity in populations and an increase in accumulation of deleterious alleles. In ''C. elegans'', somatic sex determination is attributed to the '' tra-1'' gene. The ''tra-1'' is a gene within the TRA-1 transcription factor sex determination pathway that is regulated post-transcriptionally and works by promoting female development. In hermaphrodites (XX), there are high levels of ''tra-1'' activity, which produces the female reproductive system and inhibits male development. At a certain time in their life cycle, one day before adulthood, hermaphrodites can be identified through the addition of a vulva near their tail. In males (XO), there are low levels of ''tra-1'' activity, resulting in a male reproductive system. Recent research has shown that there are three other genes, ''fem-1, fem-2, and fem-3,'' that negatively regulate the TRA-1 pathway and act as the final determiner of sex in ''C. elegans''.

Evolution

The sex determination system in ''C. elegans'' is a topic that has been of interest to scientists for years. Since they are used as a model organism, any information discovered about the way their sex determination system might have evolved could further the same evolutionary biology research in other organisms. After almost 30 years of research, scientists have begun to put together the pieces in the evolution of such a system. What they have discovered is that there is a complex pathway involved that has several layers of regulation. The closely related organism '' Caenorhabditis briggsae'' has been studied extensively and its whole genome sequence has helped put together the missing pieces in the evolution of ''C. elegans'' sex determination. It has been discovered that two genes have assimilated, leading to the proteins XOL-1 and MIX-1 having an effect on sex determination in ''C. elegans'' as well. Mutations in the XOL-1 pathway leads to feminization in ''C. elegans .'' The ''mix-1'' gene is known to hypoactivate the X chromosome and regulates the morphology of the male tail in ''C. elegans.'' Looking at the nematode as a whole, the male and hermaphrodite sex likely evolved from parallel evolution. Parallel evolution is defined as similar traits evolving from an ancestor in similar conditions; simply put, the two species evolve in similar ways over time. An example of this would be marsupial and placental mammals. Scientists have also hypothesized that hermaphrodite asexual reproduction, or "selfing", could have evolved convergently by studying species similar to ''C. elegans'' Other studies on the sex determination evolution suggest that genes involving sperm evolve at the faster rate than female genes. However, sperm genes on the X chromosome have reduced evolution rates. Sperm genes have short coding sequences, high codon bias, and disproportionate representation among orphan genes. These characteristics of sperm genes may be the reason for their high rates of evolution and may also suggest how sperm genes evolved out of hermaphrodite worms. Overall, scientists have a general idea of the sex determination pathway in ''C. elegans'', however, the evolution of how this pathway came to be is not yet well defined.

Development

Embryonic development

The fertilized zygote undergoes rotational holoblastic cleavage.

Sperm entry into the oocyte commences formation of an anterior-posterior axis. The sperm microtubule organizing center directs the movement of the sperm pronucleus to the future posterior pole of the embryo, while also inciting the movement of PAR proteins, a group of cytoplasmic determination factors, to their proper respective locations. As a result of the difference in PAR protein distribution, the first cell division is highly asymmetric. ''C. elegans'' embryogenesis is among the best understood examples of asymmetric cell division.

All cells of the germline arise from a single primordial germ cell, called the ''P4'' cell, established early in embryogenesis. This primordial cell divides to generate two germline precursors that do not divide further until after hatching.

Axis formation

The resulting daughter cells of the first cell division are called the AB cell (containing PAR-6 and PAR-3) and the P1 cell (containing PAR-1 and PAR-2). A second cell division produces the ABp and ABa cells from the AB cell, and the EMS and P2 cells from the P1 cell. This division establishes the dorsal-ventral axis, with the ABp cell forming the dorsal side and the EMS cell marking the ventral side. Through Wnt signaling, the P2 cell instructs the EMS cell to divide along the anterior-posterior axis. Through Notch signaling, the P2 cell differentially specifies the ABp and ABa cells, which further defines the dorsal-ventral axis. The left-right axis also becomes apparent early in embryogenesis, although it is unclear exactly when specifically the axis is determined. However, most theories of the L-R axis development involve some kind of differences in cells derived from the AB cell.

Gastrulation

Gastrulation occurs after the embryo reaches the 24-cell stage. C. elegans are a species of protostomes, so the blastopore eventually forms the mouth. Involution into the blastopore begins with movement of the endoderm cells and subsequent formation of the gut, followed by the P4 germline precursor, and finally the mesoderm cells, including the cells that eventually form the pharynx. Gastrulation ends when epiboly of the hypoblasts closes the blastopore.

Post-embryonic development

Under environmental conditions favourable for reproduction, hatched larvae develop through four larval stages - L1, L2, L3, and L4 - in just 3 days at 20 °C. When conditions are stressed, as in food insufficiency, excessive population density or high temperature, ''C. elegans'' can enter an alternative third larval stage, L2d, called the dauer stage (''Dauer'' is German for permanent). A specific dauer pheromone regulates entry into the dauer state. This pheromone is composed of similar derivatives of the 3,6-dideoxy sugar, ascarylose. Ascarosides, named after the ascarylose base, are involved in many sex-specific and social behaviors. In this way, they constitute a chemical language that ''C. elegans'' uses to modulate various phenotypes. Dauer larvae are stress-resistant; they are thin and their mouths are sealed with a characteristic dauer cuticle and cannot take in food. They can remain in this stage for a few months. The stage ends when conditions improve favour further growth of the larva, now moulting into the L4 stage, even though the gonad development is arrested at the L2 stage.

Each stage transition is punctuated by a molt of the worm's transparent cuticle. Transitions through these stages are controlled by genes of the heterochronic pathway, an evolutionarily conserved set of regulatory factors. Many heterochronic genes code for microRNAs, which repress the expression of heterochronic transcription factors and other heterochronic miRNAs. miRNAs were originally discovered in ''C. elegans.'' Important developmental events controlled by heterochronic genes include the division and eventual syncitial fusion of the hypodermic seam cells, and their subsequent secretion of the alae in young adults. It is believed that the heterochronic pathway represents an evolutionarily conserved predecessor to circadian clocks.

Some nematodes have a fixed, genetically determined number of cells, a phenomenon known as eutely. The adult ''C. elegans'' hermaphrodite has 959 somatic cells and the male has 1033 cells, although it has been suggested that the number of their intestinal cells can increase by one to three in response to gut microbes experienced by mothers. Much of the literature describes the cell number in males as 1031, but the discovery of a pair of left and right MCM neurons increased the number by two in 2015. The number of cells does not change after cell division ceases at the end of the larval period, and subsequent growth is due solely to an increase in the size of individual cells.

Ecology

The different '' Caenorhabditis'' species occupy various nutrient- and bacteria-rich environments. They feed on the bacteria that develop in decaying organic matter ( microbivory). Soil lacks enough organic matter to support self-sustaining populations. ''C. elegans'' can survive on a diet of a variety of bacteria, but its wild ecology is largely unknown. Most laboratory strains were taken from artificial environments such as gardens and compost piles. More recently, ''C. elegans'' has been found to thrive in other kinds of organic matter, particularly rotting fruit.

''C. elegans'' can also use different species of yeast, including '' Cryptococcus laurentii'' and '' C. kuetzingii'', as sole sources of food. Although a bacterivore, ''C. elegans'' can be killed by a number of pathogenic bacteria, including human pathogens such as ''Staphylococcus aureus'', '' Pseudomonas aeruginosa'', '' Salmonella enterica'' or '' Enterococcus faecalis''. Pathogenic bacteria can also form biofilms, whose sticky exopolymer matrix could impede ''C. elegans'' motility and cloaks bacterial quorum sensing chemoattractants from predator detection.

Invertebrates such as millipedes, insects, isopods, and gastropods can transport dauer larvae to various suitable locations. The larvae have also been seen to feed on their hosts when they die.

Nematodes can survive desiccation, and in ''C. elegans'', the mechanism for this capability has been demonstrated to be late embryogenesis abundant proteins.

''C. elegans'', as other nematodes, can be eaten by predator nematodes and other omnivores, including some insects.

The Orsay virus is a virus that affects ''C. elegans'', as well as the Caenorhabditis elegans Cer1 virus and the Caenorhabditis elegans Cer13 virus.

; Interactions with fungi
Wild isolates of ''Caenorhabditis elegans'' are regularly found with infections by Microsporidia fungi. One such species, ''Nematocida parisii'', replicates in the intestines of ''C. elegans''.

'' Arthrobotrys oligospora'' is the model organism for interactions between fungi and nematodes. It is the most common and widespread nematode capturing fungus.

Use as a model organism

In 1963, Sydney Brenner proposed using ''C. elegans'' as a model organism for the investigation primarily of neural development in animals. It is one of the simplest organisms with a nervous system. The neurons do not fire action potentials, and do not express any voltage-gated sodium channels. In the hermaphrodite, this system comprises 302 neurons the pattern of which has been comprehensively mapped, in what is known as a connectome, and shown to be a small-world network.

Research has explored the neural and molecular mechanisms that control several behaviors of ''C. elegans'', including chemotaxis, thermotaxis, mechanotransduction, learning, memory, and mating behaviour. In 2019 the connectome of the male was published using a technique distinct from that used for the hermaphrodite. The same paper used the new technique to redo the hermaphrodite connectome, finding 1,500 new synapses.

It has been used as a model organism to study molecular mechanisms in metabolic diseases.
Brenner also chose it as it is easy to grow in bulk populations, and convenient for genetic analysis. It is a multicellular eukaryotic organism, yet simple enough to be studied in great detail. The transparency of ''C. elegans'' facilitates the study of cellular differentiation and other developmental processes in the intact organism. The spicules in the male clearly distinguish males from females. Strains are cheap to breed and can be frozen. When subsequently thawed, they remain viable, allowing long-term storage. Maintenance is easy when compared to other multicellular model organisms. A few hundred nematodes can be kept on a single agar plate and suitable growth medium. Brenner described the use of a mutant of ''E. coli'' – OP50. OP50 is a uracil-requiring organism and its deficiency in the plate prevents the overgrowth of bacteria which would obscure the worms. The use of OP50 does not demand any major laboratory safety measures, since it is non-pathogenic and easily grown in Luria-Bertani (LB) media overnight.

Cell lineage mapping

The developmental fate of every single somatic cell (959 in the adult hermaphrodite; 1031 in the adult male) has been mapped. These patterns of cell lineage are largely invariant between individuals, whereas in mammals, cell development is more dependent on cellular cues from the embryo.

As mentioned previously, the first cell divisions of early embryogenesis in ''C. elegans'' are among the best understood examples of asymmetric cell divisions, and the worm is a very popular model system for studying developmental biology.

Programmed cell death

Programmed cell death ( apoptosis) eliminates many additional cells (131 in the hermaphrodite, most of which would otherwise become neurons); this "apoptotic predictability" has contributed to the elucidation of some apoptotic genes. Cell death-promoting genes and a single cell-death inhibitor have been identified.

RNA interference and gene silencing

RNA interference (RNAi) is a relatively straightforward method of disrupting the function of specific genes. Silencing

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