Experiments With Planaria (PDF)

Experiments with Planarias by

Francisco Girbal Eiras Mastery Project Project Advisor: Ellen Johnson

ABSTRACT

Nowadays more than ever, stem cells become more and more important as the possible cure for some diseases. The problem relies on the source of the stem cells (mostly from new born human beings). Newborns are the most preferable ones because of their reduced level of complexity (the cells are less specialized). As the human body develops, we tend to get more and more specified, more, in a way, unique, becoming hard to obtain this cells that can acquire the functions of the tissues in which they are inserted (stem cells). In order to be able to evaluate other sources as possible donators of cells, we first need to evaluate the complexity/regeneration rate, in other words, what are the limits of the complexity of a living being that will allow him to be used as a stem cell donator? One of the goals of this project is to evaluate this complexity/regeneration rate in planarias. A planaria is a very simple coelenterate and invertebrate living being. The amazing ability of the coelenterates is their ability to restore their original form in a relatively short period of time (some weeks). It becomes easy to evaluate the limits of  certain parameters by exposing the planaria to the most extreme conditions through a set of pre-determined experiments. If they are able to respond in a positive way (by not altering anything about their own shape or functionality), then we can conclude that they are resistant to these conditions. By measuring these levels, we are able to determine whether they would fit in the parameters of the human body, for example, an conclude if they would or not work as good stem cells (at least to begin with, because the process of evaluating the necessary conditions would be much more complex). Some of the experiments that will be tested in planarias will be amputation, transplantation, food control (reduce or augment the quantity of food, maintaining all the other conditions the same), gap junction blockage and external application of  electric fields.

KEY WORDS

Planaria, regeneration, coelenterate, stem cells, amputation, food control, gap junction blockage, electric fields, light intensity, alcohol, salt.

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TABLE OF CONTENTS

ABSTRACT ........................................................................................................ 1 KEY WORDS ...................................................................................................... 1 INTRODU CTION ............................................................................... ................. 4 BACKGROUND RESEARCH ............................................................................... 5 REGENERATION IN COELENTERATES (INVERTEBRATES )..................................................... ............................................................. ........ 5 BASIC ANATOMY AND PHYSIOLOGY ....................................................... .................................................................................. ................................... ........ 5 REGENERATION PRIMER .................................................... ............................................................................... ...................................................... ............................... .... 6 SIGNALING MECHANISM ................................................... .............................................................................. ...................................................... ............................... .... 7 MAIN EXPERIMENTS MADE WITH PLANARIAS ...................................................... ............................................................................ ......................9 HOW REGENERATION IS INITIATED...................................................... ................................................................................. ........................................ .............9 METHODS ....................................................................................................... 11 PRE-EXPERIMENTAL DESIGN ..................................................... ................................................................................ .............................................. ................... 11

Materials ........................................................................................................................... 12 Intial Procedure ............................................................................................................... 12 Daily Procedure ............................................................................................................... 12 Qualitative Analysis of the Pre-Experimental design ............................................. 13 REPRODUCTIVE S TUDIE IN BROWN PLANAR IAS ................................................... ...................................................................... ................... 13 Materials ........................................................................................................................... 13 Procedure ......................................................................................................................... 13 Qualitative Analysis of the Reproductive studie in Brown Planarias ................. 14 1. AMPUTATION IN BLACK AND BROWN PLANARIAS .................................................. ............................................................ .......... 14 Materials ........................................................................................................................... 14 Procedure ......................................................................................................................... 14 Qualitative Analysis of the Amputation study in Black and brown planarias 15 2. RESISTANCE TO A DIFFERENT SALT CONCENTRATION................................................... ........................................................ ..... 16 Materials ........................................................................................................................... 16 Procedure ......................................................................................................................... 16 Qualitative Analysis Of the resistance to a different salt concentration experiment ....................................................................................................................... 17  3. RESISTANCE TO A DIFFERENT SALT CONCENTRATION (2).................................................. .................................................. 17 Materials ........................................................................................................................... 17  Procedure ......................................................................................................................... 18 Qualitative Analysis Of the resistance to A different salt concentration (2) experiment ....................................................................................................................... 18 4. REACTION TO CONCENTRATION OF ALCOHOL ..................................................... ................................................................... .............. 18 Materials ........................................................................................................................... 18 Procedure ......................................................................................................................... 19 Qualitative Analysis of Reaction to Concentration of Alcohol ............................ 19

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5. REACTION TO CONCENTRATION OF ALCOHOL (2) ................................................... ............................................................. .......... 19 Materials ........................................................................................................................... 19 Procedure ......................................................................................................................... 20

Qualitative Analysis of Reaction to Concentration of Alcohol (2) ...................... 20 6. LIGHT INTENSITY IN BLACK AND BROWN PLANARIA .................................................... ......................................................... ..... 20 Materials ........................................................................................................................... 20 Procedure ......................................................................................................................... 21 Quantitative Analysis of Light Intensity Experiment in Black and Brown Planaria ..................................................... .......................................................... .............. 21 7. LIGHT INTENSITY IN BLACK AND BROWN PLANARIA (USING K INESIS ............................. 22 INESIS) ............................. Materials ........................................................................................................................... 22 Procedure ......................................................................................................................... 22 Quantitative Analysis of Light Intensity Experiment in Black and Brown Planaria (Using Kinesis)................................................................................................. Kinesis)................................................................................................. 23 CALCULATIONS/EXPERIMENTAL DATA HANDELING............. .................... ............. ............. .........24 1ST TRIAL ...................................................... .................................................................................. ....................................................... .................................................. ....................... 24 2ND TRIAL ..................................................... ................................................................................. ....................................................... .................................................. ....................... 24 RD 3  TRIAL ...................................................... .................................................................................. ....................................................... .................................................. ....................... 25 GRAPHING OF THE TRIAL RESULTS ...................................................... ................................................................................. ..................................... .......... 25 CONCLUSION ................................................................................................. 27 REFERENCES ................................................................................................... 30 ANNEX ............................................................................................................ 31

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INTRODUCTION

In the topic of reproduction, the use of stem cells and their respective sources, the problem “Are planarias a possible source of stem cells?” came up. This problem requires a great amount of research that would be almost impossible to perform in a high school lab, because of the lack of equipment that it is found here. However, a more theoretical approach can be taken and results can be drawn from here (even if  they don’t have the same strong conclusion). As a basic premise for this trial, we are assuming that if a cell supports a determined environment, it will, therefore, support other similar environments and consequentially, be a good substitute for another cell in the other environments. With this in mind, the hypothesis formulated was “If the planarias resist a series of trials under the conditions that human cells normally resist (if they have the same ‘comfort’ zone), then, and only then, they should be considered as a possible source of stem cells to be used in humans.” Since this is a very discussed topic in science nowadays, the relevance is almost immediate. There is, however, a whole lot of research to be done in this topic. Despite this, the background research revealed itself very important in order to understand and test the limits of these cells, as well as the different exams that could be executed using these cells.

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BACKGROUND RESEARCH

REGENERATION IN COELENTERATES (INVERTEBRATES)  The vast v ast majority of research r esearch on coelenterates has been focused on hydras and some of the colonial hydroids. hydroids. If a hydra is cut in half, the head end reconstitutes a new foot,, while the basal portion regenerates a new hydranth with mouth and tentacles. foot  This seemingly straightforward process is deceptively simple. simple . From tiny fragments of  the organism whole animals can be reconstituted. Even if an hydra is minced and the pieces scrambled, scrambled , the fragments grow together and reorganize themselves into a complete whole. The indestructibility of the hydra may well be attributed to the fact that even the intact animal is constantly regenerating itself. Just below the mouth is a growth zone from which cells migrate into the tentacles and to the foot where they eventually die. Hence, the hydra is in a ceaseless state of turnover, with the loss of cells at the foot and at the tips of the tentacles being balanced by the production of new ones in the growth zone. If such an animal is X-rayed , the proliferation of new cells is inhibited and the hydra gradually shrinks and eventually dies owing to the inexorable demise of cells and the inability to replace them. 6

BASIC ANATOMY AND PHYSIOLOGY Several species are a re used for research; Figure 1 summarizes the basic bas ic anatomy anat omy of Schmidtea of Schmidtea mediterranea planaria and outlines their major anatomical axes. axes . Planaria possess an intestine (gastrovascular tract), tract ), a body-wall musculature, musculature , a well-differentiated nervous system (including brain) brain) with most of the same neurotransmitters as humans, three tissue layers (endoderm, ectoderm, and mesoderm), and bilateral symmetry. symmetry . The gastrovascular tract consists of a highly branched gut spread throughout the entire body, with a single ventral opening from which a long muscular tube (the pharynx) pharynx) both takes in food and expels wastes.  The central nervous system is comprised of a bilobed cephalic ganglia (the brain) connected to two ventral nerve cords that run longitudinally throughout the animal and fuse in the tail. Planarias possess a diverse set of sensory Figure 1. Anatomy of a Planaria receptors that can detect light*, light*, chemical gradients, gradients , vibration, electric fields **, magnetic fields, fields, and even weak  γ–radiation. –radiation . At least 20–30 different types of planarian cells have been described, including

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broadly distributed pluripotent adult stem cells called neoblasts that constitute 20%– 30% of the total planarian cell population. Neoblasts are the only proliferative cells in the body, with the ability to differentiate into any other planarian cell type , and are a key component of the planarian's ability to regenerate. 5 * -  The ocellar potential (OP) of planaria was recorded using microelectrode techniques. The action spectrum and spectral sensitivity of the OP are described. Maximum OP sensitivity was found with 508 nm light. A moderate increase in sensitivity to blue light was observed. This is typical of many invertebrate photoreceptors and was shown, by selective chromatic adaptation, (in not to indicate the presence of a second pigment. http://jgp.rupress.org/content/51/2/255.abstract)

** -  The planarian ocellar potential (OP), an action potential evoked from the planarian p lanarian ocellus by a light flash, was recorded with microelectrodes. OP amplitude, latency, and peak delay varied as a function of stimulus intensity and state of adaptation in a manner similar to the responses of other photoreceptors. Changes in the OP that occurred with different directions of  incident light are described and attributed to screening effects of the ocellar pigment cells. The temperature coefficient (Q (Q10) of OP latency was 1.5; latency decreased continuously as temperature was increased to destructive levels. The energy of activation of the rate of OP formation was calculated to approximate 10 kcal. These findings suggest dependence of OP latency on ionic diffusion and of OP formation on a biocatalytic process. (in http://jgp.rupress.org/content/51/2/237.abstract)

REGENERATION PRIMER Planarias have the remarkable ability to regenerate an entire worm from a fragment that may lack any brain, brain , central nerve cords, or pharynx. Regeneration is completed through (1) closure of the wound within the first 30–45 min , (2) formation of a mass of new cells (called the blastema blastema)) at the injury site, which is visible by 48–72 h , and (3) re-patterning of both the old and the new tissues over the next 1–2 weeks. weeks . These processes together restore the normal morphology of the worm. Wound closure is facilitated by muscle contraction, contraction , but the molecular trigger for this reaction is unknown. However, migration of planarian epithelial cells to the wound site is known to be an essential component for wound closure, as is the juxtaposition of dorsal and ventral tissues. Wound closure is followed by an initial body-wide peak  of cell division (mitosis) from the neoblasts within 6h after injury; if tissue was lost, this is accompanied by the migration of neoblasts of  neoblasts towards the wound by 18h and a second, local mitotic peak at the injury site around 48–72 h after injury to promote the formation of the blastema (the mass of new cells). Conversely, these proliferative spikes are counterbalanced by an initial local increase of cell death (apoptosis) at the wound site within 1–4 h after injury, followed by a second, systemic apoptotic increase throughout the body that peaks at about 72h after injury as old tissues are remodeled.  Thus, normal morphology is restored by a tightly regulated combination com bination of new tissue growth and selective loss of old tissues, producing a new worm that has all its parts in the correct proportion for its now smaller size.

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Planarias also use this extraordinary regenerative ability to reproduce asexually .  Through the process of transverse fissioning, planarias anchor their tails and essentially pull themselves apart, resulting in two fragments (one head and one tail) that will regenerate into two genetically identical worms. 5

SIGNALING MECHANISM Regeneration in planarias has been shown to be the result of carefully orchestrated communication both within the blastema and between the newly regenerating tissues and the old existing ones . However, the exact nature of this communication is not well understood and is the focus of a majority of planarian experiments, which together support four main types of signaling mechanisms: (1) cell signaling pathways, (2) gap junctional communication, (3) ion fluxes, and (4) nervous system signals. (1) In planarias, as in other multi-cellular organisms, classical cell signaling pathways exist that allows cells to communicate with each other. Diffusing chemical factors act as messenger molecules, molecules , triggering a specific cascade of events either in the same cell or, more often, in neighboring cells. cells .  Typically, developmental signaling pathways involve the secretion of a molecule (morphogen ( morphogen)) from one cell that binds to its specific receptor on a second cell (which can be located quite distant from the originating cell). Sometimes, the same morphogen is dispersed throughout the animal in a concentration gradient, where different concentration ranges of the same morphogen produce different outcomes. This type of signaling is widespread and used in both short-range and long-range cell communication for basic cellular activities such as rearranging the cytoskeleton, cytoskeleton , changing gene expression, and global tissue patterning during embryogenesis. (2) Alternatively, cells can communicate with their immediate neighbors through the direct exchange of cytoplasm in a process known as gap junctional communication (GJC).. Gap junctions are membrane channels that dynamically allow for the direct (GJC) transmission of small molecules and ions between cells; gap junctions are passive channels that can control the amount and type of small molecules that pass through. Hence, GJC enables regulated quick bursts of communication, permitting synchronization among nearby cells, while inhibition of GJC can create regions of  isolation often needed for morphogenesis. Electrophoretic forces can drive the movement of charged substances through GJC-connected cells. Furthermore, voltage gradients can be transmitted and altered through gap junctions, such that cells are able to sense the membrane potential of neighboring cells. In planarian, about a dozen gap junction genes (the innexin family) have been found, and various combinations create junctions that selectively allow for different degrees of  communication among cells, such as that between neoblasts and their differentiated neighbors. (3) While ion flux is more commonly associated with nerve conduction, conduction , all cells

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generate and receive bioelectrical signals through channels and pumps within their membranes, producing ion currents, currents , which, in turn, create voltage gradients. gradients . Recent experiments have begun to identify these long-term, steady-state membrane voltage gradients and ion flows, the genetic networks that regulate them, and the mechanisms that transduce electrical signals into changes in patterning and morphogenesis. Thus, bioelectric signals (often transmitted by GJC) and signaling pathways interact, and together they regulate regenerative outgrowth, cell proliferation, differentiation, and migration. Recent work has begun to elucidate the bioelectric signaling that carries patterning information during planarian

Figure 2. Diagrams of the main planarian regeneration experiments found in the literature.

regeneration, including membrane voltage gradients, and fluxes of hydrogen, potassium, and calcium. calcium . Investigations in multiple model species have shown that several cellular mechanisms can transduce such electrical signals into intracellular responses, including: control of Ca2+ flux and calcium-mediated genetic regulation, phosphorylation of key regulatory enzymes induced by electric fields, subcellular

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translocation of transcription factors due to depolarization, modulation of voltagesensitive transporters, redistribution of membrane receptors, electrophoresis of  signaling molecules such as morphogens, and activation of signals by voltage-induced conformational changes in membrane proteins. (4) Finally, the nervous system itself may mediate instructive signaling during planarian regeneration. Recently, it has been shown that the planarian's ventral nerve cords have the capacity to transmit long-range information to a wound site regarding the presence or absence of anterior tissues in a fragment following amputation. It is clear that a comprehensive picture of regenerative regulation will include the integration and coordination of all of these overlapping signaling mechanisms.

MAIN EXPERIMENTS MADE WITH PLANARIAS (A) Cutting experiments amputate part of the planarian body (shadowed); normally, a complete regenerated worm results within 1–2 weeks. (B) Transplantation of diverse parts also regenerates into a complete worm. (C) Planarias degrowth when starved; they restore their original size upon feeding. (D) Octanol blocks gap junction communication between the worm cells; a trunk  fragment treated with octanol regenerates into a double-headed worm. (E) A post-pharyngeal fragment treated with octanol and with the nerve cords partially amputated regenerates into a quadruple-headed worm. (F) An external electric field applied to a trunk fragment disturbs AP polarity during regeneration when the anode is located in the head wound; low-intensity currents cause double-headed worms, whereas high-intensity currents cause reversed-polarity worms. (G) The drugs Ivermectin (IVM IVM)) and SCH-28080 (SCH ) disturb the ion pumps in the worm cells, altering their membrane voltage. IVM causes cell depolarization (more positive) and trunk fragments to regenerate into double-headed worms; SCH  causes cell hyperpolarization (more negative) and trunk fragments to regenerate into worms with no heads. 5

HOW REGENERATION IS INITIATED Following injury, a cascade of signaling events is triggered that initiates the regeneration process. Experiments using markers for both neoblasts and their mitotic activity have provided evidence for the existence of two different signaling events, which distinguish between simple wounding (resulting in wound healing alone) and the loss of tissue (requiring regeneration). After wounding, an increase in

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neoblast mitoses occurs throughout the animal; however, only tissue loss results in neoblast migration to the wound site and a second mitotic peak at the wound resulting in blastema formation. The signal that causes neoblast cells to stop proliferating and start differentiating within the blastema requires the activation of the extracellular signal-related kinase (ERK), since pharmacological experiments blocking ERK result in blastema formation without neoblast differentiation. Finally, c-Jun Nterminal kinase (JNK) signaling is required for blastema formation, while epidermal growth factor (EGF) receptors have been shown to regulate the differentiation of  several cell types during regeneration. 5

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METHODS

Planarias are very demanding living beings with some necessities that have to be satisfied. In order to avoid any error for lack of care, a pre-experiment environment was prepared.

PRE-EXPERIMENTAL DESIGN In the pre-experimental the main concern was to create a perfect environment for the planarias to grow. With the help of the Carolina Protozoa and Invertebrates Manual , the necessities and cares of the planarias are described in a very detailed way.

PLANARIA “(…) As planarias foul their water rapidly, you should  transfer them to fresh spring water immediately upon their  arrival. Use care to avoid damaging the animals. animals. Move them individually with a glass pipet or, after carefully   pouring out the shipping water, use your finger to dislodge the planarias from the walls and bottom of the jar. Do not  use deep containers. Planarias keep best for extended   periods in a large, larg e, shallow, enameled enam eled or stainless steel tray. (…) Maintain the temperature between 21 and 23ºC for  most species shipped from North Carolina. Keep the water clean and change it daily. Use only  chemical-free spring water or unpolluted water. Planarias in the laboratory thrive on fresh beef liver, hardboiled egg yolk, Lumbriculus worms, pieces of earthworm, crushed aquarium snails, etc. (…) The amount of food  depends upon the number of animals and the size of the culture vessel. For example, for 50 planarias in an 8-inch culture dish half filled with spring water, feed a pea-sized   portion (…)”  In CarolinaTM Protozoa and Invertebrates Manual 

Following the instructions, the protocol presented below was designed and the environment created.

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MATERIALS  The materials used during this phase of the methods were: •

2 x Culture dishes (about 5-inches);

•

1 x Beaker of 500 mL;

•

2 x Pipet of 3 mL;

•

2 x Beaker with cap of 40 mL;

•

Spring water (no chemicals added);

•

Beef liver;

•

Planaria (black and brown);

•

Lumbriculus worm.

INTIAL PROCEDURE 1. 2.

Fill two culture dishes half way with fresh spring water. Using a pipet, transfer the brown planarias and the Black planarias to different dishes. In case of doubt, microscopic observation might help telling the difference between one and the other. If the doubt persists, transfer that planaria to a third culture dish (with a small portion of water) that should follow the same procedure as the other 2. 3. Feed the culture according to the reference lines for the different cultures (depending on the number of planarias and quantity of water) 1. 4.  Transfer the Lumbriculus worm to the 500 mL beaker filled with water half  way (250 mL) due to the size of the culture2. 5. According to the same procedure, feed the culture of Lumbriculus of Lumbriculus worms with 2 the appropriate quantity of beef liver . Again, due to the strict needs of the planarias, the necessity of a daily procedure came up. This procedure needed to be simple and fast, but also effective, for which the following instructions were created, using the same material.

DAILY PROCEDURE 1.

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If the planarias are in the culture dishes, go to step 1.a; otherwise go to step 1.b. a. Using a pipet transfer the living beings from the culture dishes to a previously half-filled with spring water beaker with cap. Feed them a reasonable amount of beef liver (according to the number of  planarias and quantity of water) 1. Afterwards don’t forget to wash the culture dishes using only water! b. Using a pipet transfer the living beings from the beaker with a cap to a previously half-filled with spring water culture dishes. Feed them a reasonable amount of beef liver (according to the number of  planarias and quantity of water) 1. Afterwards don’t forget to wash the beakers using only water!

2.

Verify that the temperature of the room is between 21 and 23ºC.

 This procedure should be repeated everyday during 7 days in order to create a wellsustained culture.

QUALITATIVE ANALYSIS OF THE PRE-EXPERIMENTAL DESIGN  The procedure was fo llowed as mentioned for most of the t he points.  The initial procedure was realized within 24 hours of the arrival of the planarias, although it wasn’t quick due to the inexperience in these matters. The distinction between brown and Black planaria seemed, at first, rather easy to be done, but as they grew some inconsistencies appeared, for which some planarias had to be moved from one to the other.  The temperature was checked to be between 21 and 23ºC everyday. The changing occurred everyday except for 2 days (a weekend). The population didn’t increase or decreased during this time. After the 4th day, it was observable a significant increase of the number of planarias (both brown as black). At the 5 th day, a planaria in a reproduction was found. A significant number of brown planarias died, for which it was necessary to come up with a small procedure that would give the answer for the problem “What is the reason why the brown planarias are dying?”

REPRODUCTIVE STUDIE IN BROWN PLANARIAS One of the options for this was the amount of food given to the planarias. Perhaps the food given to the planarias was not a reasonable quantity. In order to test this hypothesis, the following procedure was designed and created.

MATERIALS  The materials used during this phase of the methods were: •

2 x Culture dishes (about 5-inches);

•

14 x Brown Planarias;

•

Spring Water;

PROCEDURE 1. 2. 3.

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Fill two culture dishes half way with fresh spring water. Using a pipet, transfer 7 brown planarias to one dish and 7 to the other. In one of the dishes, the planarias are to be fed daily with ½ the portion of  food that a culture of 14 would consume. In the other one, ¼ of the normal portion should be provided.

 The water should be changed daily in order to avoid another variable, and the temperature and lightening conditions should also remain constant.

QUALITATIVE ANALYSIS OF THE REPRODUCTIVE STUDIE IN BROWN PLANARIAS  The results show that the culture being fed with ¼ of the food grew better (more organisms at a more advanced state of development) than the one with ½ the quantity of food. Therefore, and from this point on, the full brown planaria colony should be fed ½ the original though quantity (which was indicated from the fabricant). After this, the planarias grew with these new better conditions for a week (7 days), growing at a stable rate. After these 7 days, 23 brown planarias in various states of  development were observable, as well as 35 black planarias, the majority underdeveloped. Now that the colony of planarias seems to be relatively stable, growing at a stable rate and with all the conditions to be called a “healthy” colony, it is time to start testing the planarias ability to regenerate and resistance to certain unfavorable conditions.

1. AMPUTATION IN BLACK AND BROWN PLANARIAS In order to understand the capacity of regeneration in planarias and how fast this process really happens, a procedure was developed, in which this capacity would be tested according to the state of development (to see if it has any influence in the time and how they regenerate) 3.

MATERIALS  The materials used during this phase of the methods were: •

4 x Culture dishes (about 5-inches);

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1 x Brown Planaria in a full-grown state;

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1 x Brown Planaria in an underdeveloped state;

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1 x Black planaria in a full-grown state;

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1 x Black planaria in an underdeveloped state;

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Disinfected Scalpel;

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Beef liver;

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Spring water (no chemicals added).

PROCEDURE 1. 2.

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Fill the 4 culture dishes half way with fresh spring water. Using a pipet, transfer the full-grown brown planaria and the full-grown Black  planaria to different dishes. Repeat this procedure with the undeveloped ones, so that in the end you have a 4 culture dishes with one planaria each.

3.

After this use the scalpel to cut the underdeveloped planarias (both) in half, and the full-grown ones in quarters. 4.  The same procedure as in the ‘Pre Experimental Design” should be followed and applied to these individual colonies, adjusting the amount of food, and changing the water once in two days.  The planarias were let grown for 20 days in what is called “ideal conditions”1. Observations were taken once every two days and the results are presented below.

QUALITATIVE ANALYSIS OF THE AMPUTATION STUDY IN BLACK AND BROWN PLANARIAS Day 2: 2: the full-grown pieces of the planarias seem to be alive, although weak, and their movement is slow, as well as the underdeveloped ones. Day 4: 4: there seems to be no growth in the planarias, although there is a small black  visible line around them. Day 6: 6: there seems to be almost no movement of the planarias, but they are still all alive. Day 8: 8: although the planarias grew a little bit, nothing very significant; it is still easy to identify which where each of the parts of the amputated planarias; both species are almost in the same state of development for both the underdeveloped and the fullgrown ones. Day 10: 10: almost no development from the last day in which the growth was registered. Day 12: 12: there is a certain reshape more visible in the black planarias than in the brown, however, nothing very visible for now. Day 14: 14: in the brown planarias there is a clear formation of new tissues in the section where it was amputated, for both the full-grown as for the underdeveloped; for the black ones there is some formation of tissues, but very small and hard to see without a microscope. Day 16: 16: the brown planarias are both clearly fully recovered from the amputation; the black ones are getting close, but the line of the cut is still visible. Day 18: 18: no developments on the brown ones; the black ones have achieved a state of  almost full recovery (the line has disappeared), but not totally. Day 20: 20: both species at both states have achieved a full state of regeneration and development equal to their prevenient. In 20 days the total number of planarias grew from 4 to 12 (for both species at both states) through amputation, which indicates a quick regeneration rate. However, there are a lot more trials that the planarias have to surpass in order to be possible

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candidates for donating cells, such as the Resistance to a different salt concentration test.

2. RESISTANCE TO A DIFFERENT SALT CONCENTRATION It is important to remember that the main objective of this study is to decide if  theoretically the planarias have the ability to be used as a possible source of stem cells, for which it is very important to compare the conditions that we observe in the human body. The concentration of the intracellular content is a key feature, and it is therefore very important to verify if the planaria cells can handle the concentration of the cells.

CONCENTRATION PROBLEMS

OF

SALTS

AND

DEFICIENCY

“(…) discovered that twelve inorganic minerals were key  constituents of cells. His theory was that a deficiency or an imbalance of these cell salts led to disease. By  supplementing these naturally occurring biochemical cell  salts, one can stimulate the cells to assimilate nutrients more efficiently. Replenishing and balancing these minerals restores the proper structure and the function of each cell, tissue, and organ. Ferrum Phosphoricum cell salt, for  example, is a homeopathic dilution of iron, making it an excellent treatment for anemia because it bolsters iron assimilation in the cells, increasing the oxygen-carrying capacity of red blood cells.”  In http://www.ecomii.com/alternative-medicine/cell-salts

It is therefore, very important that this conditions can verify. In order to test this, the following protocol was designed.

MATERIALS  The materials used during this phase of the methods were: •

0.125 mL of Salts Mixture (NaCl, Na 2SO4, KCl, NaHCO3, KBr, H3BO3, NaF, etc);

•

2 x 1L of Spring water (no chemicals added);

•

4 x Culture dishes (about 5-inches);

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2 x Brown Planaria in a full-grown state;

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2 x Brown Planaria in an underdeveloped state;

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2 x Black planaria in a full-grown state;

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2 x Black planaria in an underdeveloped state;

PROCEDURE

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1. 2. 3. 4. 5.

Insert the 0.125 mL of Salts Mixture in one of the 1L bottle of Spring Water and label it properly; Fill 2 culture dishes with the new mixture (Salt + Water), and the other 2 with plain spring water; Insert 1 full and 1 underdeveloped planaria of the Brown planarias in one culture dish with the new mixture and in the culture dish with spring water; Repeat the last step with Black planarias.  The same procedure as in the ‘Pre Experimental Design” should be followed and applied to these individual colonies, adjusting the amount of food, and changing the water once in two days.

 The planarias were let l et grown gro wn for f or 7 days in the “ideal “id eal conditions”1. The final result was the most important part of this protocol, for which it is the only thing referred in the qualitative analysis, despite the fact that they were followed everyday.

QUALITATIVE ANALYSIS OF THE RESISTANCE TO A DIFFERENT SALT CONCENTRATION EXPERIMENT After 7 days of exposure to the ideal amount of food for each specie1, and a constant concentration of either 0% (for the control group) or 0.9% (for the experimental group), it is observable that all the organisms in the higher concentration died. died. The organisms in the control group kept their growing rate. In order to evaluate how problematic could this intolerance to salts be to the hypothesis, another “Resistance to a different salt concentration” trial was made.

3. RESISTANCE TO A DIFFERENT SALT CONCENTRATION (2) Since the planarias seem to have an intolerance to high concentration of salts, one of  the problems that came up was “How intolerant to different salt concentrations are the planaria?”. It is important to respond this question, because some of the intolerance to salt can be corrected with minor genetic modifications (despite the fact that this is more common for plants) 4.The following protocol was designed to test the hypothesis that perhaps planarias can tolerate a concentration of 0.09% of salt.

MATERIALS  The materials used during this phase o f the methods were:

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•

0.0125 mL of Salts Mixture (NaCl, Na2SO4, KCl, NaHCO3, KBr, H3BO3, NaF, etc);

•

2 x 1L of Spring water (no chemicals added);

•

4 x Culture dishes (about 5-inches);

•

2 x Brown Planaria in a full-grown state;

•

2 x Brown Planaria in an underdeveloped state;

•

2 x Black planaria in a full-grown state;

•

2 x Black planaria in an underdeveloped state;

PROCEDURE 1. 2. 3. 4. 5.

Insert the 0.0125 mL of Salts Mixture in one of the 1L bottle of Spring Water and label it properly; Fill 2 culture dishes with the new mixture (Salt + Water), and the other 2 with plain spring water; Insert 1 full and 1 underdeveloped planaria of the Brown planarias in one culture dish with the new mixture and in the culture dish with spring water; Repeat the last step with Black planarias.  The same procedure as in the ‘Pre Experimental Design” should be followed and applied to these individual colonies, adjusting the amount of food, and changing the water once in two days.

 The planarias were let l et grown gro wn for f or 7 days in the “ideal “id eal conditions”1. The final result was the most important part of this protocol, for which it is the only thing referred in the qualitative analysis, despite the fact that they were followed everyday.

QUALITATIVE ANALYSIS OF THE RESISTANCE TO A DIFFERENT SALT CONCENTRATION (2) EXPERIMENT After 7 days of exposure to the ideal amount of food for each specie1, and a constant concentration of either 0% (for the control group) or 0.09% (for the experimental group), it is observable that all the organisms in the higher concentration survived, survived, as well as the ones in the control group. Both organisms kept a constant rate of  growing that also equal each other. Even if the planarias cannot support the normal concentration of salts in human cells, this can be fixed with genetic modification4.

4. REACTION TO CONCENTRATION OF ALCOHOL Alcohol is included in most of our cells, despite the fact that normally its concentration is very low. However, the consumption of alcohol is not uncommon for some people, so it is important to know how well can these cells take a high concentration of  alcohol, and what is its effect on mechanisms such as the Gap Junction 5.

MATERIALS  The materials used during this phase of the methods were:

18

•

1 mL of Alcohol;

•

50 mL of Spring water (no chemicals added);

•

10 mL beaker;

•

4 x Culture dishes (about 5-inches);

•

2 x Brown Planaria in a full-grown state;

•

2 x Black planaria in a full-grown state;

PROCEDURE 1.

Insert the 1 mL of Alcohol in one of the 10mL beaker with 9 mL of Spring Water and label it properly; 2. Fill 2 culture dishes with the new mixture (Alcohol + Water), and the other 2 with plain spring water; 3.  The same procedure as in the ‘Pre Experimental Design” should be followed and applied to these individual colonies, adjusting the amount of food, and changing the water once in two days.  The planarias plana rias should shou ld be let grown gro wn for 7 days in the “ideal conditions” co nditions”1. The final result is the most important part of this protocol, for which it is the only thing to be referred in the qualitative analysis, despite the fact that they are to be followed everyday.

QUALITATIVE ANALYSIS OF REACTION TO CONCENTRATION OF ALCOHOL After 7 days of exposure to the ideal amount of food for each specie1, and a constant concentration of either 0% (for the control group) or 0.1% (for the experimental group), it is observable that all the organisms in the higher concentration died. died. The organisms in the control group kept their growing rate.  This is possibly problematic, since the concentration of alcohol in the cells can reach 0.1%, but it is important that another trial is made to test how much alcohol can the planarias survive. For this reason, another trial of “Resistance to concentration of  Alcohol” was designed.

5. REACTION TO CONCENTRATION OF ALCOHOL (2) It is important to test how resistant to alcohol are the planaria cells, because again, smaller problems can be corrected using a genetic modification, but not every problem. This protocol is designed to test a concentration of 0.01% of alcohol.

MATERIALS  The materials used during this phase o f the methods were:

19

•

1 mL of Alcohol;

•

100 mL of Spring water (no chemicals added);

•

100 mL beaker;

•

4 x Culture dishes (about 5-inches);

•

2 x Brown Planaria in a full-grown state;

•

2 x Black planaria in a full-grown state;

PROCEDURE 4.

Insert the 1 mL of Alcohol in one of the 100mL beaker with 99 mL of Spring Water and label it properly; 5. Fill 2 culture dishes with the new mixture (Alcohol + Water), and the other 2 with plain spring water; 6.  The same procedure as in the ‘Pre Experimental Design” should be followed and applied to these individual colonies, adjusting the amount of food, and changing the water once in two days.  The planarias plana rias should shou ld be let grown for 7 days in the “ideal conditions”1. The final result is the most important part of this protocol, for which it is the only thing to be referred in the qualitative analysis, despite the fact that they are to be followed everyday.

QUALITATIVE ANALYSIS OF REACTION TO CONCENTRATION OF ALCOHOL (2) After 7 days of exposure to the ideal amount of food for each specie1, and a constant concentration of either 0% (for the control group) or 0.01% (for the experimental group), it is observable that all the organisms in the higher concentration survived, survived, as well as the ones in the control group. The organisms in the control group kept their growing rate and this rate also equals the one of the organisms exposed to the higher alcohol concentration.

6. LIGHT INTENSITY IN BLACK AND BROWN PLANARIA It is important to know the preference of the black and brown planarias to more or less light. This experiment will allow us to take quantitative data (in a graph form), which will let us take important conclusions about the real possibilities of usage to planaria cells.

MATERIALS  The materials used during this phase of the methods were:

20

•

3 x Culture dishes (about 5-inches);

•

9 x Brown Planaria in a full-grown state;

•

9 x Black planaria in a full-grown state;

•

Spring water (no chemicals added);

•

Black duct tape;

•

24 inches high light.

PROCEDURE 1.

Insert 3 brown and 3 black planarias in each of the 3 culture dishes pre-filled with spring water; 2. Cover half of each of the culture dishes’ surface with a piece of black duct tape; 3.  The culture cu lture dishes should shou ld be mounted in a support in which the light should be at the top (24 inches high). The first culture dish should be at exactly 22 inches from the light, the second at 12 inches and the third at 2 inches.  The planarias pl anarias should be let grown for 8 days da ys in the “ideal “i deal conditions”1 and the results should be recorded every 2 days in the form of a table and after 8 days in the shape of  a graph. There should be made 5 trials of this experiment so that the results are more plausible.

QUANTITATIVE ANALYSIS OF LIGHT INTENSITY EXPERIMENT IN BLACK AND BROWN PLANARIA Note: there is a significant grow in the planarias located 2 inches from the light, a medium in the ones located 12 inches from it, and almost no growth in the ones located 22 inches from the light. There is, however, in the dish 22 inches apart from the light a growth of the population of individuals from 6 to 8 and the one in the dish 12 inches from the light, there is a population increase from 6 to 7. 22 inches Day 2

50%

50%

0%

0%

50%

Day 4

43%

43%

33%

0%

0%

Day 6

38%

38%

100%

100%

0%

Day 8

13%

13%

0%

50%

50%

12 inches Day 2

33%

33%

0%

0%

0%

Day 4

33%

33%

0%

50%

50%

Day 6

43%

43%

0%

50%

50%

Day 8

29%

29%

100%

50%

50%

2 inches Day 2

50%

50%

0%

50%

0%

Day 4

0%

0%

50%

100%

50%

Day 6

100%

100%

0%

0%

-- (died)

Day 8

0%

0%

0%

0%

--

 Table 1, 2, 3. Percentage of planarias in the light acc ording to the day at a distance of 22 inches (1), 12 inches (2) and 2 inches (3)

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Despite this data, it is hard to evaluate the preference, due to the fact that the trials are not very organized, and there is a lack of a trend. Since this might be problematic, a new procedure was created in order to fully explore the will of the planarias in the matter of light intensity, using kinesis.

7. LIGHT INTENSITY IN BLACK AND BROWN PLANARIA (USING KINESIS) Again, due to the problem stated before, a new protocol was created to test the preference of the planarias for light or darkness. This method uses kinesis (orthokinesis and klinokinesis), which is a non-directional study of response to stimulus, such as light.

MATERIALS  The materials used during this phase of the methods were: •

1 x Culture dishes (about 5-inches);

•

1 x Brown Planaria in a full-grown state;

•

1 x Black Planaria in a full-grown state;

•

Spring water (no chemicals added);

•

24 inches high light;

•

Scotch tape;

•

Sharpie (2 different colors);

•

Ruler;

•

Aluminum paper;

•

•

 Transparent paper (Graph); Stopwatch or Timer;

PROCEDURE 1. 2. 3. 4. 5. 6.

On the back of each of the dishes tape a circle of transparent paper previously cut with the right measure; Using a ruler and a sharpie, trace one of the radius of the circle, so that this line separates the dish in half; Using the aluminum paper, cover half of the light, so that half of it’s reach is now dark; Position one of the culture dishes to a height of 4, 10.5 and 21 inches of the light; Insert one of the planarias (either black or brown first) in the dish and using a sharpie, trace the path that the planaria makes in the time of 4 minutes; Repeat steps 4 and 5 to the other planaria.

Repeat this whole procedure 3 times (there should be 3 trials of each of the planarias for each of the heights - 18 trials total).

22

QUANTITATIVE ANALYSIS OF LIGHT INTENSITY EXPERIMENT IN BLACK AND BROWN PLANARIA (USING KINESIS) Note: the results for the black planaria were very similar to the ones obtained for the brown ones. For bigger and better pictures, please check the annex section.

Pictures 1,2 and 3. 1 st trial à 4, 10.5 and 21 inches (from left to right); the side illuminated is the one to the left of the blue line, the other is dark,

Pictures 4, 5 and 6. 2 nd trial à 4, 10.5 and 21 inches (from left to right); the side illuminated is the one to the left of the blue line, the other is dark,

Pictures 7, 8 and 9. 3 rd trial à 4, 10.5 and 21 inches (from left to right); the side illuminated is the one to the left of the blue line, the other is dark,

23

Klinokinesis according to trial and distance 0.25

    )    c    e    s     /    s    n    r    u     t     (

0.2

   e0.15    u     l    a    v    s     i 0.1    s    e    n     i     k    o0.05    n     i     l     K

4 inches 10.5 inches 21 inches

0 1st Trial

2nd Trial

3rd Trial

Graph 2. Data obtained from the results in the section above (Klinokinesis)

26

CONCLUSION

Flatworms aid scientists study cancer  A U.S. study shows a key genetic pathway for cell growth and division is similar in both humans and flatworms, or   planarias. Planarias, normally living in freshwater, are known for their  ability to regenerate. (…) Now research suggests the planarian can help scientists understand the basis of human development and disease. Scientists at the University of Utah and the Forsyth Institute at Harvard University report planaria contain a gene highly  similar to the human gene PTEN (…) . They found PTEN was  present in many planaria cell types, including stem cells.  Additionally, disruption of PTEN pathways in the cell  resulted in abnormal growths, drastic changes in body  shape and, eventually, death. The scientists said their findings demonstrate planaria are a new animal model to use in researching the biology of  human stem cells and cancer cells In Facts on File – Science Online 7 

 The problem proposed relied on the possibility of the use of planarias as a source of  stem cells. This evaluation requires a lot of different methods, and the approach taken in the methods and base of this report is a very basic approach based on the concept that if something verifies a set of essential conditions, it is fit to be used. In general, all experiments made with the planarias went very well, and the general results were, mostly expected, even if the values obtained weren’t exactly the expected.  There were, however and as always, some problems. Right in the beginning of the experiment, there was the problem with the feeding of the brown planarias (they were being overfed). This problem was easily overcome using a simple design experiment, and the quantities adjusted, but this tells us a limitation of the planarias: they cannot be overfed, otherwise they might suffer from it (they might even died). This is probably due to the presence of a cavity with only one opening, being harder to control the amount of food that comes in and excrements that goes out; this leads to a continuous consume of food and, therefore, to a continuous absorption that cannot be supported by the cells.

27

 The “Reproduct ive Study in Black and Brown Planarias” gav e us a lot of information on the ability for a planaria to regenerate, and which (black or brown) regenerates faster, and therefore is better for the purpose of the problem. As taken from the observations and qualitative analysis of the results, the brown planarias have a greater ability to regenerate (in average they regenerate 8 days quicker than the black ones, in full development state3). Despite this difference in regeneration, both rates are very good results, given that it is very hard (if not impossible) to recover from an injury in half the human body. In the study about the resistance to a different concentration of salts (1), the results were a little bit different than expected. The concentration of salts in this experiment in which the planarias were inserted in equals the concentration of salts normally inside of the cells (0.9% in volume). All the planarias died (no matter what species).  This is probably due to the fact that the concentration of salts inside of the planaria cells is smaller than 0.9%1, which made some of the water inside the planarias go out (osmosis) and therefore the planarias die from dehydration. In the next trial (resistance to different concentration of salts (2)), the planarias reacted normally, surviving and growing at the same rate for both the black as the brown, despite the fact that the concentration is only 0.09% in volume. This step could be a problem, although genetic modification can be a solution to it4.  The “Resistance to a different concentration of alcohol (1)” showed, in a very similar way to the concentration of salts, not so good results for the hypothesis. All the planarias subjected to a concentration of alcohol equal to 0.1% died. This, however, is a very high concentration of alcohol though. In order for one cell to have a concentration of alcohol of 0.1% (in volume), a male individual of about 160 pounds would have to consume approximately 1.5 gallons of pure alcohol (ethanol in this case was used). The second trial of this series (“Resistance to a different concentration of  alcohol (2)”), the results were better for the hypothesis. All the organisms survived to the concentration, which means that 0.01% in volume of alcohol is tolerable to the planaria cells, something that, if required depending on the place where the cell is going to be positioned, can be modified using the genetic coding process 4. Light intensity is a very important factor for the evaluation of the possibility of use of  stem cells, since some of them can be used to cure or substitute cells in a skin cancer as well as other skin diseases. The results in the first trial by themselves were, in a certain way, inconclusive, although in the light of the second trial, they gain a new meaning. We can see an increase in the values of Orthokinesis and Klinokinesis, as the dishes get closer to the source of light. There is, however, a preference for the dark  side of the plate in each of the individual dishes. So what do the planarias prefer really, since light doesn’t appear to be such a possibility? There was another variable that was unintentionally inserted into it: temperature. As planarias get closer to the source of  light, the temperature of the water increases (as it absorbs light and energy), and the planarias appear to have a more vivid movement (give the values above), but there is still a preference for darkness over light! This result is confirmed by all of the trials in the second, and most of the trials in the first experiment in this area. There is a preference for warm and dark places, rather than room temperature and illuminated,

28

as though and almost acquired data1. This is very good for the hypothesis, because the use of these cells inside of the body depends on their ability to resist to this kind of  temperature variation.  The conclusion of this study cannot be given in terms of yes, the hypothesis is verified, or no, it is not, because this study is based in a very small sample of conditions. However, one could say probably the hypothesis is confirmed due to the results. Almost all the results obtained verify the hypothesis proposed. There is an independent study (quoted in the beginning of the Conclusion section) that defends the hypothesis that planarias could indeed be a possible source for stem cells, for having the same gene (PTEN), which is very important in regeneration 7. In a very near future, more studies will be made in this area, for sure, due to its importance in our daily life, but for now this experiments made in this context can only conclude that those studies are not going to be in vain, because there is a good chance that this can be the solution to this problem.

29

REFERENCES

1. 2. 3.

4.

5.

6. 7.

30

Carolina TM Protozoa and Invertebrates Manual , Carolina Biology Supplies © 2006, printed in the USA, pages 18 to 20. Care of Lubriculus, of Lubriculus, Carolina Biology Supplies © 2006, printed in the USA, page 1. Planaria Regeneration Activity, at http://www.hhmi.org/biointeractive/activities/planaria/planaria_regen_activity.pdf  (consulted on December 19, 2012) ScienceDirect.com – Opinion of the biologist at  http://www.sciencedirect.com/science/article/pii/S0958166902002987 (consulted on December  19, 2012) PLOS Computational Biology at  http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.1002481&image URI=info%3Adoi%2F10.1371%2Fjournal.pcbi.1002481.g002;jsessionid=855126C8811F0728615 BC62504466606 (consulted on January 7, 2013) Britannica Academic Version Online at  http://www.britannica.com/EBchecked/topic/495880/regeneration (consulted on January 7, 2013) Facts on File, Science Online – Flatworms aid scientists study cancer at  http://www.fofweb.com/Science/LowerFrame.asp?SID=5&iPin=UPI-2008092214580800&rID=1&RecType=News+Article&InputText=planaria&SearchStyle=Keyword&RecTyp e=News+Article&CurTab=News&RecCountVal=1&TopTermPrincCount=17&BioCount=3&ExpA ctCount=0&ImgCount=1&NwsCount=2 (consulted on April 2, 2013)

ANNEX

Picture 10. 1st trial à 4 inches from the light; the side illuminated is the one to the left of the blue line, the other is dark,

31

Picture 11. 1st trial à 10.5 inches from the light; the side illuminated is the one to the left of the blue line, the other is dark,

32

Picture 12. 1st trial à 21 inches from the light; the side illuminated is the one to the left of the blue line, the other is dark,

33

Picture 13. 2nd trial à 4 inches from the light; the side illuminated is the one to the left of the blue line, the other is dark,

34

Picture 14. 2nd trial à 10.5 inches from the light; the side illuminated is the one to the left of the blue line, the other is dark,

35

Picture 15. 2nd trial à 21 inches from the light; the side illuminated is the one to the left of the blue line, the other is dark,

36

Picture 16. 3rd trial à 4 inches from the light; the side illuminated is the one to the left of the blue line, the other is dark,

37

Picture 17. 3rd trial à 10.5 inches from the light; the side illuminated is the one to the left of the blue line, the other is dark,

38

Picture 18. 3rd trial à 21 inches from the light; the side illuminated is the one to the left of the blue line, the other is dark,

39