SEMINAR REPORT

Caenorhabditis elegans: The Drunk Worms

SESSION 2015-16

SWAGATA BOSE

M.Sc (LS) Ed- IVth Semester

Roll No-18

Regional Institute Of Education,BBSR

 Caenorhabditis elegans : THE DRUNK WORMS

CLASSIFICATION

 Kingdom- Animalia

 Phylum- Nematoda

 Class- Chromadorea

 Order- Rhabditida

 Family- Rhabditidae

 Genus- Caenorhabditis

 Species- elegans

INTRODUCTION-

Caenorhabditis elegans , is a free living ,non-parasitic, transparent nematoda (roundworm), its

name was derived from Greekword ( Caeno means recent , rhabditis means rodlike) and also from
latinword ( elegans means elegant). Its size is about 1mm length, it is found in temperate soil
environment.

It is multicellular organisms, with unsegmented pseudocoelome . It lacks respiratory as well as

circulatory system. It importantly possess “gut granules” which emit a brilliant blue fluorescence.
It was the first multicellular organisms having whole genome sequenced, it feeds on microoganisms like
bacteria, majority of them are female hermaphrodites and minority of them are true males. It can be
most easily isolated from rotten fruits.

In the lab, it is grown by spreading a layer of E. coli onto a plate and letting the animal feed on the
bacterial lawn.

It was considered as the only organisms to have its complete “ connectome” (2012) a neuronal “wiring”
diagram.

They have a short generation about 3 days from egg-laying to adulthood. Its brood size is less than 300.

As they are transparent, so internal anatomy can be easily observed.

It was first introduced and used by Sydney Brenner (1963) to study development and neurology.

Sydney Brenner (1927) was born in South Africa

His fundamental contributions, ranging from molecular biology, developmental biology to genomics
played a crucial role in science and technology.

Key contributions made by Sydney Brenner were - Discovery of messenger mRNA and Solving genetic
code.
The new challenge in front of him was the Development of the “Nervous system of the model
organisms, Caenorhabditis elegans”, and the two vital reasons for selecting C.elegans as the model
organism was its simple nervous system consisting only 302 neurons and it was also amenable to
genetic analysis.

C . elegans as a Model Organisms-

C.elegans are considered as the model organisms as it is easy to work with and hence its genome
manipulation is very easy. It requires very considerable environment for its culture and hence it is cheap
to maintain.

It has 100 Mb genome which is fully sequenced and is equal to 19000 genes. They play a vital role in the
study of gene expression.

It has a very short life span of about 2-6 days which wholly depends on the temperature( About 50⁰C-2
days, 20⁰C-3 days, 06⁰C-5 days).

It consist of 302 neurons which is approximately equal to 7000 synapses.

It is a self – fertilize (hermaphrodite) organism, which produces 300-350 offsprings. Its fertilization
occur with true males and number of offsprings may exceed upto 1000 .

Its Post -embryonic cell lineages were determined by Sulston and Horwitz, 1976.

It shows Programmed cell death mechanism which was discovered by Horwitz, 1982.

Its complete embryonic cell lineages determined by Deppe et al, Sulston 1983.

Its complete connectivity of nervous system was established on (1986). The RNAi and miRNA discovered
in worms (1991-98) (Nobel Prize: Fire, Mello 2006).

The first use of GFP in animals(1994)was done (Nobel Prize: Chalfie, 2008)and also the first animal
whose whole genome was sequenced(97Mb, now 100.3Mb).
Similarities between C . elegans and Humans-
Both organisms , development proceeds through :

-morphogenesis that is the formation of the structure of an part

-differentiation and growth of tissues and organs during development

-growth giving rise to an adult.

Both of them are having a nervous system, muscular system which help in movements and a gut for
completion of various metabolic processes. It is capable of performing very simple behaviors.

It produces sperms and eggs and undergo reproduction, although normally as a hermaphrodite in case
of C.elegans. After their reproductive phase , the worms gradually undergo senescence where body
metabolisms slows down and the organism ultimately dies.

This is how a very simple organisms exhibits all the phases of life in a short span of time.

Consideration Regarding the C. elegans Genome

It has 100 Mb genome approx. equal to 19 000 genes and which is also equal to 5000 essential genes.

About 70% of human genes have a clear C. elegans homolog (this includes human disease genes).
Human genes can often rescue the worm mutants.

COMPARISON between 2 weeks organism with 70 years organism-

C.elegans preferably used for studying muscular system. Scientists prefer short life span organisms to
study the multiple generations in relatively short time frame.

C.elegans are well characterized organisms having biological functions similar to humans.

They are helpful for applying strategies in modern science and medicines to find out different ailments
occur to human.

Example: Treatment of Diabetes .

Microanatomy –
In C.elegans, “Gut granules”are characterized to be found in Rhabditida orders. A numerous gut
granules are present in the intestine of C.elegans. The structure of this granules is similar to the
lysosomes with an acidic interior.

It has a capacity of endocytosis , it acts as a storage organelle.

Its one of the remarkable feature is its reaction with UV light and emit an intense blue fluorescence that
is similar to lysosomes only as lysosomes also have acidic interior and capacity of endocytosis , it also act
as storage organelles.

Reproduction and Development-

In context to reproduction in C. elegans- its 2 types are-

• Hermaphrodite- self fertile female having 5 pair of autosomes and XX sex chromosome

• True males- only produce sperms ( not used as model organisms) 5 pairs of autosomes and XO
sex chromosome
The 5 autosomes represented as I-V and X (6 total). The chromosomes are non-disjunctional, with one
less chromosome, males develop . They have 79 more neurons and 25 more muscle cells than
hermaphrodites, used for mating.

Hermaphrodite produces many progeny about 300 worms. Adult hermaphrodites have 959 somatic

cells while males have 1031.

LIFE CYCLE-
Under favorable environmental condition, the hatched larvae develop through 4 stages or molts (L1 to
L4) .

Rapid life cycle is about 3 days at 20 degrees Celsius from eggs -> L1-L4 -> adults -> eggs.

Life span: 2-3 weeks.

Offspring: A hermaphrodite can produce about 300-350 offspring under self-fertilization and more if it
mates with males.
The Duaer stage is the “ Alternative life form of C. elegans “ the name was derived from German ( Duaer
- Permanent)

C. elegans enter an alternative third larval stage when, culture plate is over crowded or when food
scarcity is there.

Duaer larvae are stress resistant, thin and mouth is sealed ; hence cannot take in food. They remain
viable for few months (about 3 months). These worms can live 10 times more than their normal life
span.

C. elegans can adopt an alternative life form, called the dauer larval stage, if plates are too crowded or if
food is scarce. Dauer larvae are thin and can move but their mouths are plugged and they cannot eat.
Interestingly, dauers can remain viable for three months. They appear to be non-aging: dauer larvae can
roam around for months and then reenter the L4 stage when they encounter a food source and live
about 15 more days.

At 25ºC a generation time is about 3-4 days, where as it extends to almost a week at 16ºC. Some
mutations in C. elegans are also temperature sensitive, only showing a phenotype at either 16ºC or
25ºC.
C. elegans cell lineage
One amazing advantage of worms is that every worm has the exact same number of somatic cells.

 Hatching larva=558 cells

 Males (XO)=1031 somatic cells+ ~1000 sperm

 Hermaphrodite (XX)= 959 somatic cells ~ 2000 eggs and sperm.

Somatic cells arise by an INVARIANT cell lineage. The divisions are invariant in pattern, timing, and

orientation of each division.

From the figure-

1) Each branch indicates a cell division

2) When a cell differentiates and no longer divides, line ends.

Postembryonic cell lineage was completed first.

In the presence of food, cell divisions resume and postembryonic developmental program begins 3
hours after hatching. a first stage larva has approximately 671 cells, 113 undergo programmed death in
the course of development. About 10% of the remaining 558 cells in a newly hatched larva (51 in
hermaphrodites, 55 in the male), are blast cells that divide further.
DRUNKEN WORMS REVEAL A GENETIC BASIS OF ALCOHOL RESPONSE-
Gene responsible for causing alcohol sensitivity discovered by Andrew Davies in June 10, 2004

Subtle differences are seen between worm strains in a gene for a brain protein called NPR-1 (Natriuretic
peptide receptor 1). NPR-1 explains differences in the worms alcohol sensitivity.

Researchers chose worm as a subject as alcohol has effects on worm behavior .

EXPERIMENT carried out by Davies-

Davies and his collegues exposed two wild strains of worms-

-England strain of worms

-Hawaii strain of worms

Researchers used alcohol concentrations comparable to those that causes intoxication in humans.

On the basis of exposure to alcohol-

-Hawaiian worms showed a dramatic recovery from initial intoxication than did the English worms.

In accordance to the genetic analysis of the two strains led to the difference between them due to the
gene for NPR 1. NPR 1 act as a neuronal receptor-a molecular “socket” in brain cells.

Genetic difference caused the production of higher levels of NPR 1 protein in the alcohol-sensitive
English worms decreased their ability to recover.
Above figure depicts-

Former figure represents the England strain of worm with slower recovery and the latter one
represents the Hawaiian strain of worm with faster recovery.

Locomotion-

This figure shows one wild type (in upper left corner) and 5 locomotion defect worms. As it can be seen,
the moving pattern of mutants is different from wild type. This is to show how locomotion defect can
affect worm’s motion.

Natural Variation in NPR-1 gene modifies Ethanol Responses of wild strains of C. elegans-

Allelic variation that alters the functional level of NPR-1, a neuropeptide Y (NPY) receptor like protein,
can account for natural variation in the acute response to ethanol in wild strains of C. elegans

NPR-1 negatively regulates the development of acute tolerance to ethanol.

In C. elegans, including NPR-1 most of the proteins has been postulated to play a role in mediating
ethanol responses. Same tissue concentrations of ethanol that produce intoxication in humans have
neurodepressive effects on multiple behaviors of C.elegans. Level of response to ethanol-acute
adaptations that occur shortly after ethanol administration.

Acute or Seasonal tolerance : If blood or tissue concentration of ethanol kept constant-decreased

intoxication observed .

The NPR-1 gene encodes a predicted G-protein coupled receptor in the neuropeptide Y (NPY) receptor
family. Variation in the acute response to ethanol between genetic basis for differences in
human populations that individuals has a significant impact on determining have different
ethanol sensitivity and susceptibility to alcoholism.
Here we show that allelic variation that alters the func- variation in ethanol responsiveness have
yet to be retional level of NPR-1, a neuropeptide Y (NPY) receptor-. like protein, can account for
natural variation in the C. elegans is an ideal organism in which to analyze
acute response to ethanol in wild strains of Caeno- natural variations in behavioral responses
due to the C. elegans.
NPR-1 negatively regulates the powerful genetic tools available and the existence of
development of acute tolerance to ethanol, a neuroadaptive- multiple wild isolates obtained from
different process that compensates for effects of ethanol. Furthermore, dynamic changes in the
NPR-1 pathway neuronal proteins have orthologs in C. elegans.
Downregulation of NPR -1

Downregulation of NPR-1 Pathway during Acute Tolerance

NPR-1 negatively regulates the rate at which acute tolerance develops. Ethanol
triggers“downregulation” of NPR-1 pathway, reduced functioning of NPR-1-results to behavioral
adaptations.

Eg : Aggregation of animals with one another in the presence of food (Clumping)

Interestingly, clumping associated with ethanol exposure is only observed when removed from ethanol

Activating effect of ethanol removed from physiological environment leads to downregulation of NPR 1
pathway exposed. Animal with reduced or no NPR-1 pathway function demonstrate faster rate of acute
tolerance development .

Ethanol Exposure-
Behavioral Responses of C. elegans to Ethanol in the graph is shown below.

Ethanol-induced decrease in body-bend amplitude during locomotion.

- Ethanol (200 mM) causes a small decrease in the amplitude of body bends.
 - Complete flattening of the body bends can occur at 400 and 500 mM ethanol.

- The effect is most pronounced on the posterior of the body (left).

The Effects of Radiation

The worms showed that they could normally repair their radiation damaged cells in spaceflight by using
apoptosis, or the programmed cell death which can also be triggered by unusual amounts of damage.
Researchers say that this makes them ideal "living dosimeters" to track accumulated radiation damage
over time.

Mechanism of BK CHANNEL (SLO-1)-

Big Potassium (BK channel), which is also called SLO-1 or Maxi-K.

Characterized by their large conductance for potassium ion (K⁺) through cell membrane (leakage
channel). These channels are activated or opened when-

- changes in membrane potential

- increase in concentration of intracellular calcium ions (Ca⁺⁺)

Opening of BK channel allow K⁺ to flow passively through the channel,down the electrochemical
gradient.
Efflux of K⁺ from the cell, leads to cell membrane ( hyperpolarization ) an increase in the electrical
potential across the cell membrane.

It also results in decrease in cell excitability.

BK channels are essential for the regulation of physiological processes-

-smooth muscle tone

-neuronal excitability

It remains to be determined if BK channels contribute to intoxication in humans.

BK CHANNEL-tetramer
BK channel is a tetrameric structure.

Each monomer of the channel forming α–subunit product of KCNMA1 gene, modulatory β–subunits
( KCNMB1, KCNMB2, KCNMB3 or KCNMB4 ) can associate with tetrameric channel .

STRUCTURE OF BK
A unique transmembrane domain ( SO ) that precedes the 6 transmembrane domains (S1-S6).

It is a conserved sequence in all voltage-dependent K⁺ channels

 A voltage-sensing domain ( S1-S4 )

 K⁺ channel pore domain ( S5-selectivity filter, S6 ) channels.

A cytoplasmic C terminal domain ( CTD ) consisting of a pair of RCK ( Regulator of Conductance of K⁺ ).

CTD contains four primary binding sites for Calcium (Ca⁺⁺) called “Calcium Bowls”.
C.elegans : Identification of BK as the major mediator of depressive behavioral
effects of Ethanol
Its approach for identification is “Forward genetic screen”.

Electrophysiology performed over intact C.elegans neurons

-ethanol increases BK channels currents in wild strains

-ethanol fails to activate BK in null-mutant neurons

BK channel plays a role in the development of acute functional tolerance (AFT) to ethanol. Loss of
functioning of SLO-1 results in inability in developing AFT.

GENETIC EVIDENCE between LIPID and BK channel-

TAG ( Triacylglycerol ) lipase plays a significant role in regulating the rate of development of AFT.

TAG Lipase regulates to develop AFT. Manipulation occurs in level of TAGS( TAG lipase, lips-7 ).

By mutation : TAGS changes the basal behavior of phenotypes results to Hyperactivation of SLO-1 gene
development of AFT. This result provides genetic evidence for a link between lipid environment and BK
channel.
How long has NASA been studying C. elegans?
The first experiment utilizing C. elegans in space was conducted on STS (Space Transportation System)-
42, which launched in January 1992. The experiment was designed to study the biological effect of
exposure to cosmic rays. This mission contributed to helping scientists understand how to protect space
travelers on long missions.

The principle of biological universality-

“One point that emerges from the studies of programmed cell death in C. elegans and other organisms
is the striking similarity of genes and gene pathways among organisms that are as superficially distinct
as worms and humans...

It underlies my strong conviction that the rigorous, detailed and analytic study of the biology of any
organism is likely to lead to findings of importance in the understanding of other organisms.”

ADVANTAGES of C.elegans as a Research Tool-

-It is a simple organisms which is;

 Easy to grow

 Short life cycle

 Powerful genetic studies can be conducted

  Small and transparent

 Fully described anatomy and development

 Completely sequenced genome

 Stocks can be frozen and preserved

 Cheap to maintain

 Present no biohazard

 Large size of progeny

 Developmental biology and cell biology-

-Due to its Transparency- easy to view anatomy and development

-Easy to view Specific cells, cellular architecture which can be examined like GFP

- Programmed cell death can also be visualized.

- Individual cell lineages can be easily traced and directly inspected by DIC light microscopy.
Specific cells, cellular architecture can be directly examined using reporters like Green fluorescent
protein(GFP)* .
 Ilys genes(lysozyme) exhibit distinct patterns of pharyngeal expression.

GFP= GREEN FLUORESCENT PROTEIN .Small protein derived from luminous jelly fish and can be
introduced as a transgenic tag to mark any protein in the animal.

2. Neurobiology

• Act as a model for neuronal development and function.

• No brain per say, but have sophisticated nervous system(302neurons/959 cells).

• C. elegans responds to chemo attractants/repellants.

• Laser beams(selective cutting of neurons) and electrophysiology studies can

be conducted.

• Connectome (the complete wiring diagram) have been established.

3. Aging

• Short life span helps to conduct genetic screens to find longevity genes.

4. Human disease studies

• ~75% of human disease genes have potential C. elegans homologs.

• ~40-50% have a C. elegans ortholog.

Biochemical and genetic characterization of chromatin organizer protein Dve-1 and its role in C.
elegans development and aging.

The present study seeks to elucidate the mechanism by which chromatin organization impacts
organismal aging by studying C. elegans protein (Defective proventriculus) Dve-1.

• Identify genetic and physical interactors of C. elegans Dve1.

• Generate C. elegans strains mutated in Dve1 and characterize the function of Dve1 during C.
elegans development and aging.

C. elegans as Model in Biomedical Research

Validated C. elegans disease models are-

Central Nervous System

• Depression, Psychosis

• Parkinson’s, Alzheimer’s

• Pain .

Metabolic

• Type II diabetes

• Obesity .

Other

• Cardiovascular (arrhythmia)

• Oncology

• Muscle disease .
C. elegans in the Drug Development Line

Hence, Caenorhabditis elegans is considered as the model organism with enormous biological
applications.
 REFERENCES

 C. elegans, research community http://www.wormbook.org

 Article info: -C. elegans as a model organisms

-Natural variation in NPR1/NPY receptor

-Drunken worms reveal a genetic basis of alcohol

response

-Role of BK channel in ethanol response behavior

 Bargmann, C.I.(1998) Neurobiology of C. elegans genome

 Discussion paper on Ethanol resistance

 Experiment procedures on behavioral assay

 Genetic screens for ethanol- resistancy on C. elegans

 CONTENTS

 Classification
 Introduction
 C.elegans as a model organism
 Similarities between C.elegans and Human
 Comparison between 2 weeks organism and 70 years organism
 Microanatomy
 Reproduction and development
 Life cycle
 Drunken worm reveals a genetic basis of alcohol response
 Natural variation in NPR-1 gene
 Downregulation of NPR-1 pathway
 Ethanol exposure
 BK Channels and its Structure
 Genetic evidence between BK channel and Lipid
 Advantages of C.elegans as a research tool
 Application in biological research areas
 References