Regeneration of Planaria

Aim: To study the effect of temperature on regeneration of planaria and the establishment of polarity.

Introduction:

Fig1.

• External characteristics of freshwater planarian, Schimidtea mediterranea showing dorsal and ventral view.
• Nomenclature of planarian body parts
• Amputations used to study regeneration of planaria

Scientific classification –                        Kingdom: Animalia

                                                               Subkingdom: Eumetazoa

                                                               Superphylum: Platyzoa

                                                               Phylum:          Platyhelminthes

                                                               Class:              Turbellaria

                                                               Order              Seriata

                                                               Suborder:        Tricladida

                                                               Family:            Planariidae

Planaria are non parasitic (free living) flatworms inhabiting fresh water resources. They are very simple multi cellular organisms which are of great interest due to some of their unique properties, such as, presence of advanced synaptic nervous system and encephalization in addition to regeneration by means of fission giving rise to two or more planaria. Molecular genetics of planaria has evolved into a an area of importance due to experiments on interference RNA (Alvarado, Sanchez and Newmark, 1999), in situ hybridizations (Umesono et al.1997) and characterization of complementary DNA (cDNA) of species Schimidtea mediterranea (Alvarado, Sanchez, 2002) and so on.

Anatomy- Planaria are acoelomates and contain organs derived from all three germ layers (ectoderm, mesoderm and endoderm). The nervous system is organized into bi-lobed cephalic ganglia connected to two longitudinal nerve cords (Cebria et al. 2002). Sensory structures are found at the anterior region of the head.

They reproduce asexually (transverse fission) or sexually (cross fertilizing hermaphrodites). The gonopore located in the posterior ventral surface is the opening that leads to the muscular copulatory apparatus (Hyman, 1951).

Planarian regeneration can be of two types, epimorphosis and morphallaxis. (Morgan, 1901), by which he implied that epimorphosis is cell division at wound site and morphallaxis is a phenomenon of remodeling the tissue.

Specific type of stem cells are involved in regeneration and depending on the availability of these stem cells, the regeneration capacity in each region is different.

Materials: planaria, small petri dishes, pipettes, magnifying glass and additional materials like spring water, thermometer, paper napkins etc.

Method:

 For testing the effect of temperature on regeneration-

•  Five planaria are taken and are each cut at 2 places, one below the head (pre pharyngeal) and the other at the end of trunk (post pharyngeal) just above the tail.
• The three resulting pieces are taken in three different petri dishes that are labeled as head, trunk and tail.
• Add about 10 ml of spring water to the petri dishes to preserve the specimen.
• These petri dishes are incubated at 4 deg Celsius and left overnight. The same is repeated and the petri dishes are placed in 12 deg Celsius, 20 deg Celsius and 30 deg Celsius.
• The petri dishes are observed everyday for a week and the changes are noted for wound healing, blastema formation, blastema growth and differentiation.

Observation and results:

C

Day1

Day2

Day3

Day4

Day5

Day6

Day7

Day8

Day9

Day10

Day   11

Day 12

Day 13

Day 14

Day 15

04

Wh

B

B

Dea

Dea

Dea

–

–

–

–

–

–

–

–

–

12

Wh

B

B

BG

BG

BG

BG

BG

BG

Diff

Diff

Diff

Diff

Diff

Diff

20

Wh

B

B

BG

BG

BG

BG

Diff

Diff

–

–

–

–

–

–

30

Wh

B

B

BG

BG

BG

Diff

Diff

Diff

–

–

–

–

–

–

Wh – wound healing

B – blastema

BG – blastema growth

Diff – differentiation

Dea – death

Thus based on the above observations, it can be concluded that the physical factors, in this case, temperature plays a major role in the induction of regeneration in planaria.

Discussion-

Regeneration is defined as the replacement of missing structures following injury (Morgan 1901). The stimulus for regeneration is injury (Needham, 1952), the response to which is withdrawal from the causing agent followed by release of rhabdites, specialized mucosal covering. Thus wound healing primarily is the epithelial cell spreading around the site of injury.

The resulting cell division forms an epithelial/mesenchymal bud called as regeneration blastema. This stage is followed by migration of totipotent stem cells called neoblasts which are small undifferentiated cells with large nuclei and very little cytoplasm to the site of blastema, differentiating and producing the lost anatomical parts.

Fig: 2 Electron micrographs of neoblasts at the site of amputation in Schimidtea mediterranea

It is well known that temperature affects the rate of all chemical and biochemical reactions. And there exists an optimum temperature at which differentiation is most favored. Based on the results obtained it can be concluded that maximum regeneration rate is observed at 30 deg C.

Method:

For testing gradients of regenerative capacity:

• Five planaria are cut transversely just below the head, five are cut transversely just above the tail, and five are cut longitudinally. These parts are all taken into different petri dishes and spring water is sprinkled on them and are allowed to regenerate at room temperature. The observations are noted for about a fortnight.

Observation and Results:

	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7	Day 8	Day 9
head	Wh	B	B	B	BG	BG	Diff	Diff	Wo
tail	Wh	B	B	BG	BG	BG	Diff	Diff	Wo
Long	Wh	B	B	BG	BG	BG	Diff	Diff	2o

Wo – Whole organism

2o – double headed organism

Therefore, when planaria is amputated transversely it is noticed that the anterior wound always gives rise to a head and the posterior gives rise to a tail, thereby maintaining specific polarity.

Similarly, the lateral fragment also gives rise to an anterior head, middle trunk and a posterior tail.

Discussion:

The rate of regeneration of photoreceptors in a head blastema declines the more posterior the amputation is made (Sivicks, Bronsted  and Child). The blastema of a planarian which has its head cut off (decapitated) produces a head. Likewise in some planarian species which just has the tail cut off produces a head itself, thus producing a double headed organism and proving that blastema does not necessarily replace lost parts of the body. Blastema present in the head region, when divided into two, within two days of initial cut, both the blastema give rise to two complete heads.

Method:

For testing the effect of lithium chloride on planaria-

• Planaria are cut at various sites, pre pharyngeal, pharyngeal, post pharyngeal and laterally and each cut planaria is treated with a known concentration of lithium chloride and a similarly cut planaria is just kept in petri dish containing spring water (control).
• This is observed everyday for about a week and the results are noted.

Observation and results: Cut planaria in lithium chloride-

Cut	Day1	Day 2	Day 3	Day 4	Day 5	Day 6
Into half	Wh	B	B	2o	dead	dead
Many piece	Wh	B	B	dead	dead	dead
At tail	Wh	Wh	B	dead	dead	dead
Laterally	Wh	B	B	B	B	B

The control planaria all survived and showed regeneration.

Discussion:

Lithium chloride is known for its properties in excitation of neurons and in planaria which is cut laterally produces fusion. From the results obtained it is clear that survival by fusing is possible in planaria which is cut laterally and poisons the transversely cut planaria.

Works cited

• Bronsted HV, 1955, Planarian Regeneration Biol. Rev. 30:65_126
• Hyman LH, 1951. The Invertebrates: Platyhelminthis and Rhyncocoela acoelomate bilateia. New York : Mcgraw Hill

• Levin, Michael. The Forsyth Institute.

      www.drmichaellevin.org/Planaria

• Reddien, Peter.W and Alvarado. Sanchez.A

Fundamentals of Planarian Regeneration

• Alvarado. Sanchez A. Regeneration in metazoans Bioessays 22:578_90
• www.planarians.org