Aveneu Park, Starling, Australia

Planarians of the head. They have two ventrolateral

Planarians (Dugesia) are eukaryotic
flatworms that form the class Turbellarians in the Phylum Platyhelminthes (1).
They are related to other flatworms, e.g. tape and fluke however they differ by
one thing which is that they are non-parasitic. They are invertebrates and are one of the most basal
triploblastic organisms, with derivatives of all three germ layers; ectoderm,
mesoderm, and endoderm (3). They show bilateral symmetry and are one of the
simplest animals with mesodermic layers and their tissues are organised into
organ and organ systems (1).  As shown in diagram 1,
Planarians are formed various components that allow it to survive and work well
in its environment. The eyespots (ocelli) on their head are not actually
eyes but light sensitive receptors that direct the planarians away from the
light (negative phototaxis). They also use chemoreceptors on ciliated auricles, the
ear-like extensions of the head. They have two ventrolateral and many
transverse nerve cords that detect external stimuli (3).  They have a Pharynx located on the under part
of their body which is used to feed, however it is also used for excretion. Planarians
do not have a skeletal, circulatory or respiratory system and are known as
acoelomate as they do not have the principal body cavity that most animals have

Planarians are known as freshwater worms (as they are
sensitive to pollution), found at the bottom of ponds, streams or under rocks
(3). Planarians are predators and scavengers and
eat live or dead animals using their muscular retractable pharynx which can
extend out of the mouth opening on the ventral side up to half of their body
length (1).    

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Planarians contain cilia which enables them to glide across surfaces.
The cilia is located on the surface on the planarian and is attached to the
flame cell within the excretory canal.

Cilia are the oldest known cellular organelle, discovered
in 1675 by Anthony Van Leeuwenhoek (5). Cilia
is functionally and structurally similar to eukaryotic flagella as they both
contain a central bundle of microtubules called the axoneme which is nine outer
doublet microtubules surrounding a central pair of singlet microtubules in the
(7). Cilia can be divided into two
general types, primary and motile. Primary cilia are sensory organelles that
contain a variety of receptors, including G protein-coupled receptors. Cells
typically sprout a single primary cilium with a characteristic 9+0 cytoskeletal
structure (axoneme). In contrast, motile cilia can occur in the tens and
hundreds on epithelial cells, they have a typical 9+2 axoneme, and they serve a
mechanical role (9). The ‘9+2’ formation of
microtubules is seen through an electron microscope at the cross section of axoneme.
As seen in diagram 2, the surrounding doublets contain two different tubules, A
and B. Tubule A is a complete microtubule that contains 13 protofilaments while
tubule B only contains 10 protofilaments (7). The axoneme is held together by
three sets of protein cross-links. The central pair of singlet microtubules are
connected by periodic bridges surrounded by a fibrous structure given the name
inner sheath (7). The linkage between microtubule doublets is by a protein
called Nexin and the linkage between the doublets and the singlets at the
centre of cilia is done by a multi-unit protein called Radial spoke. The inner
and outer dynein arms are permanently attached to tubule A and these reach out
to the tubule B ahead of them. These components are surround by the plasma
membrane.

The point at which the axoneme attaches to the cell, it
connects with the basal body as shown in diagram 3. Basal bodies are like
centrioles as they have a cylindrical structure containing nine triplet
microtubules. Each triplet contains one complete 13-protofilament microtubule
(tubule A), which is fused to an incomplete tubule B, which is turn is fused to
an incomplete C tubule. Tubules A and B fill the whole axonemal shaft while C
stops within the transition zone, between the basal body (Kinetosome) and
axonemal shaft (7).

For bending to occur, dynein rows must be activated and
inactivated in a synchronous alternating pattern. Individual dynein arms have
been shown to behave as oscillators. In addition to the bending movements of
cilia, the ciliary membrane also undergoes an active longitudinal sliding
movement (9).

Mitogen-activated pathway kinases (MAPKs) are protein
Serine-Theronine kinases that convert extracellular stimuli into a wide range
of cellular responses. MAPKs
are among the most ancient signal transduction pathways and are widely used
throughout evolution in many physiological processes. All eukaryotic cells
possess multiple MAPK pathways, which coordinately regulate gene expression,
mitosis, metabolism, motility, survival, apoptosis, and differentiation (10).  The
mammalian MAPK family consists of extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal
kinase (11). Each group of conventional MAPKs is composed of a set of three
evolutionarily conserved, sequentially acting kinases: a MAPK, a MAPK kinase
(MAPKK), and a MAPKK kinase (MAPKKK). The MAPKKKs, which are protein Ser/Thr
kinases, are often activated through phosphorylation and/or as a result of
their interaction with a small GTP-binding protein of the Ras/Rho family in
response to extracellular stimuli (10). MAPK pathways are activated either because of a series of binary
interactions between the kinase components or through the formation of a signalling
complex containing multiple kinases that is guided by a scaffold protein (11). Scaffolding
proteins also mediate MAPK cascade specificity by simultaneously binding several
components and organizing pathways in specific modules (10).

 p38 mitogen-activated protein kinase (p38 MAPK), an
intracellular signal-transducing molecule, plays an important role in the
regulation of a variety of inflammatory responses, including expression of
proinflammatory cytokines, leukocyte adhesion and chemotaxis (14). There are four p38 MAP kinases in mammals: ?, ?, ? and ?. Among all p38 MAPK isoforms, p38?
is the best characterised and is expressed in most cell types (13). The p38
MAPK subfamily can further be divided into two distinct subsets, on the one
hand p38? and p38? and on the other, p38? and p38?. Their susceptibilities
to inhibition at low concentrations by the compounds SB203580 and SB202190
(13).

The experiment performed was to see how the inhibitor SB203580, affected
the locomotion of Planarians.

Matherials
and Methods

The Dugesia were located and collected from a pond in Kingston
university. They were transferred to a tank in the lab which was kept at room
temperature (approx. 25°C). Each week the water in the tank was filtered to
remove any ions and excess waste. These flatworms were varied in shape and
sizes, where most of them were between 5-7mm.

The Dugesia were fed a small amount of chicken liver every Friday every
week. The experiments were conducted a few days after the feed. Flatworms used
were kept in the same tank before and after use to recover from the experiment.

Movement
of the planarians were recorded using a …camera and a tripod to keep the
exposure of light and position of the camera the same. As said before the
Dugesia have eyespots which are connected to the cerebral ganglia and are used to
detect and avoid sunlight (negative phototaxis) but do not detect images (1),
therefore conditions remained the same.

 

In the experiment, a 6 well plate was
used, each filled with 2ml of filtered water, followed by 2µl of DMSO (0.1%) added to 2 wells. A 20µM solution of SB203580 was made up with DMSO
(100%), 2µl of that was pipetted
into 2 different wells. Dugesia were placed in each well and left in the dark
for 60minutes, recording movements every 30 minutes for 30 seconds, to help
analyse speed, movement and behaviour. During the recording, Dugesia were moved
to the centre of the plate so they can have the same starting point and to also
help with activity. Dugesia were put back into the tank after experiments for
them to recover. The experiment was repeated using a 10-fold dilution with
SB203580 at 2µM and 0.2µM.

 

The speed and
movement of the Dugesia was analysed by transferring the video from the camera
into Microsoft PowerPoint. Here the movement was tracked by using the freeform
tool to follow the Dugesia precisely. The line was converted into a dashed line
and compared to a known length of dashed lines. This determined the distance
(mm) the Dugesia travelled in the first 20 seconds of the video, permitting
speed (mm/s) to be calculated. 

 

Behavioural movement of Dugesia was also analysed in the 30 second video
taken, creating a tally for each parameter. Parameters used to assess behaviour
were; Number of stops, number of turns made over 45°, number of abrupt changes
in movement. 

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