Inhibition of Planarian Regeneration

Brigham Young University BYU ScholarsArchive

Theses and Dissertations

1968-08-01

Douglas Pierre Bingham Brigham Young University - Provo

Follow this and additional works at: https://scholarsarchive.byu.edu/etd

Part of the Life Sciences Commons

BYU ScholarsArchive Citation Bingham, Douglas Pierre, "Inhibition of planarian regeneration" (1968). Theses and Dissertations. 7627. https://scholarsarchive.byu.edu/etd/7627

This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. INHIBITION OF PIANARIAN REGENERATION 'I/ l,/

A Thesis

Presented to the

Department of Zoology and Entomology

Brigham Young University

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

Douglas P. Bingham

August 1968 This thesis by Douglas P. Bingham is accepted in its present form by the Department of Zoology and Entomology of Brigham Young University as satisfying the thesis requirement for the degree of Master of Science.

8 Au1,;u_{z /t?ef Date

Typed by Lillian Zang iii

ACKNOWLEDGMENTS

Grateful acknowledgment is made for the valuable suggestions and help given by the chairman of my advisory committee, Dr. A. Lester Allen, and other members of my committee, Dr. Clark Gubler, and Dr. James North.

Financial support for my study was provided by a National Defense Education Act Fellowship. The Depart- ment of zoology and Entomology, Brigham Young University, supplied laboratory space, equipment, supplies, and the experimental animals. TABLE OF CONTENTS

ACKNOWLEDGMENTS ...... iii

LIST OF TABLES •• • • • • • • • • • • • • • • • • • V LIST OF FIGURES ...... vi INTRODUCTION 1 • • • • • • • • • • • • • • • • • • • • \ REVIEW OF THE LITERATURE. • • • • • • • • • • • • • 3

MATERIALS AND METHODS • • • • • • • • • • • • • • • 6 1--

Maintainance of Stock Animals. . . • • • 6

DMSO Tolerance • • • • • • • • • • • • • • • 7

Preparation of Homogenized Tissues • • • • • 7

Test Conditions. • • • . . . • • • • • • • • 9

Procedure for Measuring the Brain. • • • • • 11

Statistical Analysis • • • • • • • • • • • • 13

RESULTS • • • • • • • • • • • • • • • • • • • • • • 14 ~

Inhibition of Cephalic Regeneration • • • • • 14

Effect of DMSO .• " •.• • • • • • • • • • • • • 25

Inhibition of Asexual Reproduction • • • • • 25 DISCUSSION • • • • • ...... • • • • • • • • • 26 CONCLUSIONS AND 31 S2) SUMfJ'ARY • • • • • • • • • • • • • • ' LITERATURE CITED • • • • • • • • • • • • • • • • • • 33 V

LIST OF TABLES

Table Page

1. Tolerance of regenerating Dugesia tigrina to various concentrations of DMSO ••• . . • • 8 2. Culture media for Dugesia tiqrina during regeneration •••••••••••••• • • • • 10 3. Brain length in microns in regenerated Dugesia tigrina cultured at 13°c for 16 days • • 16 4. Brain length in microns in regenerated Dugesia tigrina cultured at 20°c for 10 days {experiment #1) ••••••• . . . . . 17 s. Brain length in microns in regenerated Duqesia tiqrina cultured at 20°c for 10 days {experiment #2) ••••••• • • • • • 18 6. Brain length in microns in reg8nerated Dugesia tigrina cultured at 23 C for 9 days • • 19 7. Bartlett's test for homogeneity of variances in control planaria grown at different temperatures for different lengths. of time. . . 21 8. F-test for the equality of true means of four -normal populations of regenerated control planarian brain lengths under the assumption of homogeneous variances ••• . . . 22 9. Combined results of cephalic regeneration in Dugesia tigrina at varying temperatures. • • 23 10. Tests of the equality between the true means of planaria cultured in different media using a 5% chance of rejecting a true hypothesis ••• 24 vi

LIST OF FIGURES

Figure Page

1. Areas of transection for removing the head or tail of a planarian •••••• • • • • 12 2. The part of the brain labeled C was measured to determine the extent of brain regeneration •••••• • • • • • • • • • • • • 12 3. The normal appearance of the brain tissue of a planarian cultured in river water or 1% DMSO in river water •••••••••• • • • 15 4. The appearance of the brain tissue or lack of brain tissue in a planarian cultured in media containing extract from homogenized planarian heads •••••••••••••• • • 15 s. Comparison of the means and the 95% confidence intervals between regeneration cultures at 13°c. for 16 days •••••• • • • 16 6. Comparison of the means and the 95% confidence intervals between regeneration cultures at 20°c for 10 days (experiment #1) • • 17 7. Comparison of the means and the 95% confidence intervals between regeneration cultures at 20°c for 10 days (experiment #2) • • 18 8. Comparison of the means and the 95% confidence intervals between regeneration cultures at 23°c for 9 days ••••••• . . . 19 9. Comparison of the means and the 95% confidence intervals between combined regeneration cultures •••••••• • • • • • 23 1

INTRODUCTION

This thesis describes my study of inhibition of brain regeneration in planaria. The flatworm (Dugesia tigrina) possesses a remarkable ability to regenerate lost parts, including the head. The primary objective of my study was to determine the extent to which an inhibiting substance, located in the head, would prevent regeneration of the brain. A secondary objective was to study whether DMSO (Dimethyl Sulfoxide) would facilitate the action of the inhibitor.

A better understanding of the mechanism of regeneration can be obtained by studying substances and procedures which influence the process. This research sheds more light on the controlling mechanisms of regeneration through an analysis of the action of an aqueous extract of planarian heads upon planarian brain regeneration. Once the critical materials in this extract are isolated and characterized, it may be possible to label or mark them and find their site of action in the animal. It may be possible in the future to use this information in the study of humans and other higher organisms whose meager capacity for regeneration may be limited by the action of naturally occurring inhibitors. 2 Earlier work using Dugesia lugubris and Polycelis nigra has shown that it was necessary to traumatize the blastema to allow the penetration of extracted inhibitory material present in the planarian environment (Lender, 1956, 1960). To avert mechanical traumatization to the blastema in the current study, DMSOwas added to the aqueous solution of extracted inhibitor to determine whether or not it would allow greater absorption of external material into the blastema. DMSOwas chosen for this purpose because it is known to act as a carrier and to enhance permeability across skin barriers (Kligman, 1965: Leake, 1966). These earlier workers found a con- siderable variation in the extent of inhibition: I wanted to see if DMSOwould reduce this variability. The study reported here is only the beginning of a large and complex problem. It indicates the presence of a naturally occurring material in the head region which inhibits brain regeneration. The characterization of this material is left to future research and investigation. 3

REVIEW OF THE LITERATURE

The power of regeneration in planaria has been observed for more than 150 years by such workers as Pallas, Shaw, Draparnauld, Dalyell, Johnson, Duges, Faraday, Charles Darwin, Harvey, Wyman, and Zacharies, all of whom did research on regeneration during the l800's (see Bronstedt, 1955). However, the mechanism of regeneration was not investigated until the early 1900 1 s by Morgan (see Bronstedt, 1955), "the father of modern investigation on planarian regeneration", and by Child (see Bronstedt, 1955) who was the first to study regeneration on a physiological basis. The mechanism of regeneration has been studied in mor~ recent times by Wolff and Dubois (1947), Dubois (1948), Kolmayer and Dubois (1960), Wolff (1961), Woodruff and Allison (1964), and Chandlbois (1965). Despite all the research that has been done by these investigators there has been no general agreement on some of the basic aspects of regeneration and so the mechanism by which planaria regenerate is still unsettled. In order to try and understand the phenomenon of regeneration, various unnatural inhibiting agents have been used to alter regeneration in the planarian. It was found by Bronstedt (1961) that lowering the temperature 4 will have little effect on the formation of the blastema, but the differentiation processes are slowed down and eventually halted. It was also noted by Morgan (1901) that raising the temperature above 29.7°c caused inhibition of regeneration, and at 32°c and above the regenerating planaria eventually died. The inhibitory effects of anaesthetics, many organic compounds,. irradi- ation, and starvation are reviewed by Child, Rulon, Dubois, and Morgan (see Bronstedt, 1955). Smith and Hamn {1963} have studied the effects of metabolic inhibitors on planarian regeneration. Gabriel (1965) reported the inhibition of planarian regeneration caused by b-mercaptoethanol and actinomycin B. Henderson and Eakins {1959) have made extensive studies on the effects of pH, norrral metabolites, antimetabolites, enzyme inhibitors, antimitotic agents, nerve-active agents, and lipoic acid on planarian regeneration. Natural means have also been employed to inhibit regeneration. Rand and Brown (1926) and schewtschenko (Raven, 1959) have shovm that head grafts in the anterior region of a planarian will inhibit regeneration of the host head in varying degrees depending on how far anterior the graft is placed. They have also shown that the more anterior the grafts are placed the more unacceptable the head grafts seem to be to the intact planarian host. Rand and Ellis {1926) have shown that in double-headed forms there is 5 inhibition of regeneration when one head is removed. Using extracts made from homogenized planarian heads, Lender (1955, 1956, 1960, 1962, 1965) has retarded the regeneration of the brain in decapitated Dugesia lugubris and Polycelis nigra. Sengel (1959, 1965, 1967a) used this same technique to retard pharyngeal regeneration with extracts from the pharyngeal zone of planaria. Planaria are not the only animals that seem to contain natural inhibitors of regeneration. Embryonic development of tissues and organs has been retarded with extracts from adult tissues and organs, including frog brain and heart (Clark and Mccallion, 1959a), frog ectoderm (Shaw, 1966), and chick brain (Clark and Mccallion, 1959b). Retardation of regeneration has also been demon- strated with organ extracts in other animals: nemertian worms (Tucker, 1959), bamboo worm (Smith, 1963), and salamander limb (Batchler, Hofheins, and Trunnell, 1964). 6

MATERIALS AND METHODS

The animals used in this experiment are of the species Dugesia tigrina and were obtained from the Dahl Biological Supply co. (Berkeley, Calif.). The specimens are easily maintained in spring or river water, or defined

salt solution (Henderson and Eakins, 1959). The regeneration

time is short (Morgan, 1901: Needham, 1952: Bronstedt, 1961), they have the ability to completely regenerate lost parts

(Hamburger, 1967), the amount of brain regeneration can be measured histologically (Flexner, 18971 Lende~ 1956, 1960), and they are economical to maintain. Planaria have an exceedingly high capacity to regenerate and are used more than any other animal for regeneration experiments. Hay (1966) and Hamburger (1967) give a brief review of the regeneration capacity of animals of other phyla.

Maintainance of stock Animals

A healthy stock of planaria (Dugesia tigrina} was maintained by growing them in glass beakers containing Provo River water in a refrigerator set at 13°c. The water was changed at regular weekly intervals. The planaria would not live in tap water, distilled water, or demineralized water. 7

At weekly intervals small pieces of calf's liver were dropped into each container, and the planaria were allowed to feed on the liver for 2~3 hours at room temperature. When the planaria finished feeding, I removed the liver, cleaned the containers, added fresh water, and put the planaria bac}:: into the refrigerator.

DMSOTolerance

Before starting the experiment~ the planaria were tested as to their tolerance to various concentrations of DMSO (Tab. 1). I decided to use a 1% solution of DMSO in river water because it did not inhibit regeneration nor kill the planaria.

Preparation of Homogenized Tissues

A small piece of clean stainless steel razor blade held in a hemostat was used to remove either the head or the tail of a planarian while it was moving along the bottom of a plastic petri dish containing a few ml of river water (Fig. 1). The heads from twenty medium- sized planaria (1.5 cm long) were placed in 5 ml of either river water.or 1% DMSO in river water depending on whether or not DMSOwas to be used later in the experiment. The tails from twenty medium-sized planaria were placed in 5 ml of either river to.Tater or 1% D:'-i1S0 in river 8

Table 1. Tolerance of regenerating Dugesia ~igrina to various concentrations of DMSO in river water.

Effect on the planaria Concentration of DMSO Starting No .. showing No. dead no. of inhibition of by 72 hrs. planaria regeneration*

!.:f/4 6 0 0 1% 6 0 0

2% 6 3 0

3% 6 6 O** ' 4% 6 6 O** 8% 6 6

*Examined after 10 days of regenerating at 20°c. **All died between the.second and fourth week. 9 water again depending on whether DMSOwas to be used later in the experiment. The heads and tails were chilled to 4°c and homogenized in a Thomas Tissue Homo- genizer. The homogenized tissues were then centrifuged at 10,000 times gravity for 30 minutes at 4°c in an International Centrifuge Model B-20. The supernatant fluid was removed, labeled as to whether it was head or tail supernatant, and stored at 4°c. The pellet was resuspended in distilled water and recentrifuged. The supernatant fluid of the second centri- fugation was discarded and the pellet was suspended in either 5 ml of river water or 1% DMSOand river water, depending on what fluid had been used to homogenize the tissue the pellet was derived from. The pellet suspension was labeled 11debris" and saved for future use.

Test Conditions·

Decapitated planaria were allowed to regenerate in groups of five under each of the conditions listed in Tab. 2. Cultures 1 through 6 were ·tested at the following temperatures and days: 13°c for 16 days1 20°c for 10 daysi and 23°c for 9 days respectively. Cultures 7 and 8 were tested at 20°c for 10 days. 10

Table 2. Culture media for planaria during regeneration.

Planarian environment Culture no. Ml of river Other materials that were water used added to the water.

l* 15 None

2** 15 1% DMSO

3 10 5ml of supernatant f-luid from centrifuged homogenized planarian heads

4 10 1% DMSO and 5 ml of supernatant fluid from centrifuged homogenized (in 1% DMSO) planarian heads.

5 10 5 ml of resuspended pellet from centrifuged homogenized planarian heads

6 10 1% DMSO and 5 ml of resuspended pellet from centrifuged homogenized (in 1% DMSO) planarian heads

7 10 5 ml of supernatant fluid from centrifuged homogenized planarian tails

8 10 5 ml of resuspended pellet from centrifuged homogenized planarian tails

*This was the control for the cultures not using DMSO in the water.

**This was the control for the cultures using a 1% DMSO in river water solution for the regeneration medium. 11

Procedure for Measuring the Brain

Each regenerated planarian was killed in an extended condition by placing several worms with a little water around each (planarian) on a glass plate and then applying a drop or two of 2% nitric acid upon them

(Braithwaite, 1962). The planaria were then transferred to a container of fresh Zenker's Fixative (Guyer, 1953r

Lender, 1956, 1960), removed from the fixative solution after 30 minutes, and then washed in running tap water for 3-6 hours. The washed planaria were sectioned on an

American Optical Cryocut at 30 microns (Fig. 2). Egg albumin was used to adhere the planarian sections to glass slides. The sections were stained with

Mallory's Triple Connective Tissue Stain (Guyer, 1953r

Lender, 1956, 1960), dehydrated in a series of alcohols, and mounted in permount. An Olympus binocular compound microscope with an occular grid and lOOX magnification was used to measure the brain size (Fig. 2). The brain was identified by its location, feathery appearance, and yellow. to orange staining properties. Measurements were recorded to the nearest 20 microns and subsequently analyzed statistically. 12

Figure 1. The planaria were decapitated by removing the head with a transection at A. The tails were removed with a transection at B.

Figure 2. The part of the brain labeled C was measured to determine the extent of brain regeneration. 13

Statistical Analysis

For each experimental condition the median, mode, range, midrange, standard deviation, variance, and coef- ficient of variance were determined. A 95% confidence interval was plotted for each condition. The cumulative t-distribution test was used to test the hypothesis made by comparing the experimental conditions to the controls (Ostle, 1963). All of the results from the various temperatures and days of regeneration were pooled and the same statistical procedures were applied to the pooled results. 14

RESULTS

Inhibition of Cephalic Regeneration

Comparisons between culture groups

At all temperatures there was a marked difference in the amount of cephalic regeneration observed between the control planaria and the planaria cultured in media containing extract from planarian heads (Figs. 3 & 4). The planaria cultured in media containing extract from planarian heads regenerated about 50-60"/4 as much brain length as the control planaria (Tabs. 3, 4, 5, and 6). The planaria cultured in media containing resuspended debris from centrifuged homogenized planarian heads or tails showed about 80-90% as much regeneration of brain length as the control planaria. The planaria cultured in extract from planarian tails regenerated about 80"/4 as much brain length as control planaria (Tab. 5). The 95% confidence intervals for the mean length of regenerated brain tissue showed the interval for the true mean brain length of planaria which regenerated in extract from planarian heads to be completely separate from the interval of the true mean brain length for the control planaria (Figs. 3, 4, 5, and 6). 15

Figure 3. The normal appearance of the brain in a planarian cultur ed in river water or 1% DMSO in riv er water. (B=brain lengt h measured in microns~ N.C.= nerve cord)

Figure 4. The appearance of th e brain or lack of the brain in a planarian c ultur ed in media containing extract from homo genized planarian heads. (N.D.B.=no d etectab l e brainr N.C.=nerve cord) 16 Table 3. Brain length in regenerated Dugesia tigrina cultured at 13°c for 16 days.

Culture no. statistical analysis l* 2 3 4 5 6

No./sample 7 7 10 9 8 6

Mean 220 197 132 100 195 163 Median 230 190 120 120 190 170

Mode 250 170 130 130 150 170

variance 1233 1291 4573 3725 1771 427

st. dev. 35.1 35.9 67.6 61.0 42.1 20.7

Coef. var. 16.0 18.2 44.6 61.0 21.6 12.7

*See table 2 on page 11 for clarification of nos.

Figures. Comparison of the means and the 95% confidence intervals between regeneration cultures at 13°c for 16 days.

Culture Brain length measured in microns no. Mean= -t- 95% Conf. Int.= ---

1 • - .

5 • •

6 • I • 0 50 100 150 200 250 17

Table 4. Brain length in microns in regenerated Dugesia tigrina cultured at 20°c for 10 days (experiment #1).

Culture no. statistical analysis 1 2 3 4 5 6

No./sample 5 5 5 5 4 4

Mean 198 192 122 140 175 180

Median 200 220 130 140 170 170

Mode 170 220 130 130 200

variance 870 970 920 100 433 600

st. dev. 29.5 31.1 30.3 10 20.8 24.5 Coef. var. 14.9 16.2 24.9 7.1 11.9 13.6

Figure 6. Comparison of the means and the 95% confidence intervals between regeneration cultures at 20°c for 10 days (experiment #1).

Culture Brain length measured in microns no. Mean= I 95% Conf. Int.=--

4 I ,

6 0 50 100 150 200 250 18

Table 5. Brain length in microns in regenerated Dugesia tigrina cultured at 20°c for 10 days (experiment #2).

Culture no. statistical analysis 1 3 7* 8*

No./sample 6 8 7 7 Mean 213 91 173 177 Median 210 80 170 170 Mode 210 170 170 variance 1066 641 657 291 St. dev. 32.7 25.3 25.6 17 .o

·Coef. var. 15.3 27.8 14.8 9.6

*Total results under this condition.

Figure 7. Comparison of the means and the 95% confidence intervals between regeneration cultµres at 20°c for 10 days (experiment #2).

Culture Brain length measured in microns no. Mean= I 95% Conf. Int.= --

3 -1

8------0 50 100 150 200 250 19

Table 6. Brain length in microns in regenerated Dugesia tigrina cultured at 23°c for 9 days.

Culture no. Statistical analysis 1 2 3 4 5 6

No./sample 7 6 7 12 Lost 3

Mean 227 231 126 123 197

Median 250 230 120 130 170 Mode 270 230 130 130 170 variance 2324 1277 762 2240 2133

st. dev. 48.2 37.1 27.6 47.3 46.2

Coef. var. 21.2 16.0 22.0 38.5 23.4

Figure 8. Comparison of the means and the 95% confidence intervals between regeneration cultures at 23°c for 9 days.

Culture Brain length measured in microns no. Mean= I 95% Conf. Int.=--

3 ,

4 • 5

6 0 · 50 100 150 200 250 20

Comparison of culture conditions when the results at various temoerature were combined.

Because-the mean lengths of regenerated brain cultured at various temperatures seemed quite close, I applied the uBartlett•s test for homogeneity of variance" and "the F.:.test for the equality of means of k normal populations" (Ostle, 1963:134-7) to the results of the control planaria from each temperature to see if I could combine all the samples from the various temperatures. Statistical analysis indicates that there are no significant differences in the variances or the means of the samples cultured at the various temperatures (Tabs. 7 & 8). The statistical analyses of the combined measurements are found in Tab. 9 and Fig. 7. Data in Tab. 9 and the statistical analyses in Tab. 10 indicate that planarian brain regeneration in the presence of head extract is inhibited to a significant degree. The hypotheses of equality of the means between cultures were tested using a "student T test" to determine the boundries necessary to insure a 5% or less chance of rejecting a true hypothesis. The hypotheses of equality were rejected (Tab. 10) and it is evident that head extract significantly inhibits brain regeneration. I found from the statistical inference tests that the mean brain length of planaria cultured in extract from planarian heads did not equal the mean brain length of 21

Table 7. Bartlett's test for homogeneity of variance in control planaria grown at different temperatures for different lengths of time.

Sample y? 1/D.F. s? D • F • ) J. D.F.* J. ( log sf ) (

19°c 7400 6 o.167 1233 18.546

23°c 13943 6 o.167 2325 20.196

20°c 2560 4 0.250 870 11.758

20°c 5334 5 0.200 1066 15.140

Sum 29237 21 0.784 65.640

Chi Square Calculation

s 2 =29237/2.=1392

B=(3.1336)(21)=65.8056

Chi Square =2.3026(65.8056-65.640) 3 :::0.3813

Chi Square(o.gs)( 3 )=7.81***

Interpretation

Chi square is less than the Chi Square(o.gs)( ) so 3 I accept the hypothesis that there is homogeneity of the true variances of the control planaria cultured at different temperatures for different lengths of time.

*D.F. is the degrees of freedom **Controls from the second experiment of planaria that regenerated at 20°c for 10 days. ***Chi Square limit above which I would have rejected homgeneity of variances. It was determined from ostle (1963:525). 22

Tables. F-test for the equality of true means of four normal populations of regenerated control planarian brain lengths under the assumption of homogeneous variances.

source of Degrees of Sum of Mean F- variation freedom Squares Square ratio

Mean 1 1166400 1166400

Among 0.92 groups 3 2644 1322 Within groups 21 30156 1436

Total 25 1199200

Interpretation

F( 0 • 95 )( 3 )(Zl)=3.08 is much greater than the F=0.92 value obtained from the foregoing calculations.

I accepted the hypothesis that the true means of · the four conditions the control planaria were grown under are equal. •

*This value was obtained from Ostle (1963:538). 23

Table 9. Combined results of cephalic regeneration in Dugesia tigrina at varying temperatures.

Culture no. Sta tis:tical analysis 1 2 3 4 5 6*

No./sample 25 18 20 27 12 13

Mean 216 213 118 119 188 176

Median 210 210 120 130 180 170

Mode 170 170 130 130 200 170

variance 1267 1292 2157 1455 1136 876

st. dev. 37.0 35.9 46.4 38.0 33.7 29.6 Coef. var. 17.1 16.9 39.3 32.0 17.8 14.8

*Combined results of culture nos. 7 & 8 are on table s.

Figure 2. Comparison of the means and the 95% confidence intervals between combined regeneration cultures.

Culture Brain length measured in microns no. Mean= I 95% Conf. Int.=

1 , •

2 • 3 • • 4 • 5 • • 6 0 50 100 150 200 250 24

Table IO. Tests of the equality between the true means* of Dugesia tigrina cultured in different media using a 5% chance of rejecting a true hypothesis.

Results of the testing

Hypothesis Limits** t•-value*** Acceptance

U1=U2 +2.117 0.21 yes

u3=u4 +2.050 -0.09 yes u5=u6 +2.184 0.94 yes

U1=U3 ±2.053 8.70 no

u1=u7 ±2.306 3.52 no

u1=us +2.150 2.30 no

ul =uf:3 +2~229 3.98 no

u2=u4 +2.087 8.30 no u2=u6 +2.130 3.14 no u6=u4 +2.113 5.20. no U7::U3 +2.273 4.27 no

U5::U3 +2.130 5.43 no

U9::U3 ±2.192 5.54 no

U9=U7 +2.447 0.34 yes

u5=u8 ±2.276 0.94 yes

*In this table the letter u represents the true mean for the condition indicated by the subscript.

**These limits were computed from the table on page 528 of Ostle (1963).

***A statistitian, Bob Burge, suggested using the test in which I assumed the true variances of each culture to be different, sot' was computed (Ostle 1 1963:120). 25 planaria cultured in suspended debris of planarian heads and tails or extract of planarian tails (Tab. 10).

The added effect of DYSO to inhibit cephalic regeneration

The presence of DMSO in the culture conditions had no positive or negative effect on the amount of cephalic regeneration (Tabs. 3, 4, 6, 9, & 10), and only a behavioral difference was caused by the presence of sublethal amounts of DMSO. The planaria would attach themselves more securely to the sides and bottom of the containers than would those which did not have DMSO in the culture media.

Inhibition of Asexual Reproduction

It was noted that the decapitated planaria undergo 100% asexual reproduction when allowed to regenerate in river water. However, planaria that were allowed to regenerate in river water containing planarian head extract showed about 50'/4 asexual reproduction. This observation was not noted until it was too late to collect any significant results. 26

DISCUSSION

The 45% decrease found in the average brain length of: D. tigrina that had been allowed to regenerate in water containing extract from other planarian heads did not agree with the findings of Buchanan {1938), who claimed instead a stimulation of head regeneration in decapitated planaria treated with extract of planarian head homogenate. However, he did not investigate the amount of brain regeneration which took place but only the macroscopic regeneration of the head. I found that the heads regenerated macroscopic- ally equally well under all conditions employed.

My results agreed with those of Lender (1956 & 1960) who found about 66% inhibition of brain regeneration in Dugesia lugubris which were allowed . to regenerate in water containing extract from other planarian heads. Lender may have obtained more inhibition because he traumatized the blastema of the regenerating planaria every two days to allow better penetration of any inhibitory materials present in the environment. In Lender's work with D. lugubris and Polycelis nigra and in my work with~- tigrina the active inhibitor has been a·water soluble material from the anterior region of the animal. The idea of a water soluble inhibitory material agrees with the fin~ings of other 27 workers on organ inhibitors. Sengel (1967b) found the inhibitor of the pharynx in planaria to be in the water soluble fraction of the homogenized planarian pharyngeal zone. Clark and Mccallion (1959a, 1959b) used cell-free extracts of homogenized organs to inhibit chick brain, frog brain, and frog heart development. Tucker (1959) centrifuged out cellular debris from homogenized nemertian worms and found the active inhibitors of anterior and posterior regeneration to be in the water soluble extracts of similar body parts of bamboo worm by using cell free extracts of the respective body parts in the culture media in which the worms were allowed to regenerate. Contrary to my results and the results cf other investigators men- tioned above, Bachelor (1964) reported inhibition of salamander limb regeneration to be in the acetone-soluble fraction of the homogenized salamander limb tissue and not in the water soluble fraction. Lender (1956, 1960) found only about 5% inhibition

> of regeneration when he substituted cellular debris in the culture media for extract of head homogenate. In my study there was 13% inhibition of regeneration when the planaria were allowed to regenerate in cultures containing debris from centrifuged homogenized planarian heads. I may have had more inhibition of brain regeneration inn. tigrina, since I washed the debris only once before putting 28 it in the culture media. Lender (1956, 1960) reported a 4% stimulation of brain regeneration when he added supernatant fluid from homogenized, centrifuged planarian tails to his culture media with decapitated planaria. My results do not agree with his findings since I noted 19% inhibition of brain regeneration in those planaria that regenerated new heads in culture media containing extract from planarian tails. I cannot explain this difference unless there is a species difference. The 1% DMS0 solution did not change the amount of inhibitory action of the extract from planarian heads. This did not surprise me since the best results using DMSO to enhance penetration of other molecules across skin barriers employs a concentration of between 60-100% DMS0 (Leake, 1966). The 1% DMSO solution which I used in my research was probably not sufficiently concentrated to cause significant changes in the permeability of the blastemal membrane of the regenerating planarian. Inhibition of regeneration by organ.extracts has been attributed by some to bacterial contamination (Fulton, 1959). I did not add anything to the cultures of regenerating planaria to stop bacterial growth. How- ever, the high degree of inhibition of regeneration noted in cultures containing extract from planarian heads could not be attributed to bacterial growth since less bacterial 29 growth was noted in those cultures than the cultures con-

taining cellular debris. The culture containing extract from planarian tails showed equal amounts of bacterial growth, but far less cephalic inhibition than those cultures containing the extract from planarian heads. I cannot conclude that bacterial inhibition of regeneration of the brain was not involved, but if it was, it would only account

for that degree of inhibition found in cultures containing

cellular debris or tail extract.

The experimental data of this study support the hypothesis that organs act as controllers, when present,

to prevent the development of homologous organs and suppress

the continued growth of organs after maturity. The brain

inhibits the regeneration of another brain and induces the

development of eyes (Lender, 1952, 1956). The pharyngeal zone of the planarian suppresses th~ development of a

second pharynx (Sengel, 1967b). When an organ loses its ability to suppress the development of a similar organ

there arises a double structured organism (Sengel, 1967).

I noted a second type of inhibition from extracts of planarian heads other than the inhibitor of brain regeneration.

This second inhibitor suppressed asexual regeneration in decapitated planaria. I might explain that removal of the head of a planarian normally stimulates 10~¼ asexual reproduction under these experimental conditions. I-did not note 30 this inhibitor in time to record any data on it during this study, but it may prove to be a key to a better understanding of the control over asexual reproduction in planaria.

I agree with Lender that the inhibitor of brain regeneration is a neural secretion of the brain and that it is distributed in an anterior-posterior gradiant.

This would explain why a graft of a planarian head on the anterior of the planarian host does not take while a graft of planarian head onto the posterior of a planarian host survives very well.

I believe a better understanding of the control of regeneration could be gained by isolating the inhibitor involved in brain regeneration and by using immunological and radiological techniques to locate the site of inhibitory action imposed by the inhibitory substance. Once the control of regeneration in planaria is understood it may reveal the key to manipulating the control of regeneration in higher forms of animals. 31

CONCLUSIONS AND SUMM..2\...~Y

In this study the extent to which an inhibitory substance located primarily in the head of the planarian Dugesia tigrina prevented brain regeneration in decapitated planaria was investigated. Dimethyl Sulfoxide was used in conjunction with the extracted planarian brain inhibitor in hopes of enhancing the action of the inhibitory substance. The decapitated planaria were allowed to regenerate in river water or 1% DMSO in river water containing various combinations of extracts of homogenized planarian heads or tails, and debris of centrifuged, homogenized planarian heads or tails. The extent of brain regeneration in decapitated planaria was recorded after regenerating at 13°c for 16 days, 20°c for 10 days, and 23°c for 9 days. When an extract of homogenized planarian·heads was added to the culture media containing regenerating decapitated planaria, a 45% decrease in the mean length of regenerated brain tissue was observed. The debris of centrifuged, homogenized planarian heads or tails caused a 13% decrease in the mean length of brain tissue when added to the culture media of regenerating decapitated planaria. A 19~~ decrease in the mean length of brain tissue was observed in decapitated planaria after regenerating in river water plus an extract from homogenized 32 planarian tails. I concluded from the results of this study that in the region of the brain in Dugesia tigrina there exists a substance that prevents the development of another brain. The removal of the brain from the intact animal also removed the inhibitor of brain development and the organism was free to regenerate a new one. This inhibitory substance acts as the controller or one of the controllers of brain regeneration in Dugesia tigrina. 33

LITERATURE CITED

Batchler, J. w., c. c. Hofheins, and J.B. Trunnell. 1964. Agent of inhibition of regeneration in salamanders.

Proc. Utah Acad. Sci., Arts, and Letters, 41:143-144.

Braithwaite, L. 1962. The taxonomic problem of Polycelis in the u. s. Master's thesis, Department of zoology, Brigham Young University, Provo, Utah. Bronstedt, H. v. 1955. Planarian regeneration. Biol. J Rev~, 30:65-126.

Bronstedt, H. v. and A. Bronstedt. 1961. Influence of

temperature on the rate of regeneration in the

time-graded field in planarians. J. Embryol. exp. Morph., 9(1):159-166. ~ Buchanan, J. w. 1938. The effect of planarian extracts and exudates upon head regeneration in Euolanaria

dorotocephala. Physiol. Zool., 11:144-154. \ Chandebois, R. 1965. Cell transformation systems in

planarians, pp. 131-142. Inv. Kiortsis and H. A.

L. Trampusch, (ed.) Regeneration in animals and

related problems. North-Holl. Publ. co., Amsterdam.

Clarke, R. B. and D. J. Mccallion. 1959a. specific inhibition of differentiation in frog embryos 34

by cell-free homogenates of adult tissue. Can.

J. zool., 37:129-132.

Clarke, ·R. B. and D. J. Mccallion. 1959b. Specific inhibition of neural differentiation in the

chick embryo. Can. J. Zool., 37:133-136. Dubois, F. 1948. Demonstration de la migration des

cellules de regeneration des planaires par la

methode des greffes et des irradiations combinees. c. R. Acad. Sci., 226:1316-1317. Flexner, s. 1897. The regeneration of the nervous system of Planaria torva and the anatomy of the

nervous system of double headed forms. J. Morph.

14:337-346. Fulton, c. 1959. Re-examination of an inhibitor of regeneration in tubularia. Bio. Bull., 116:232-238.

Gabriel, A. 1965. Effets du B-mercaptoethanol et de

l'actinomycine B sur la regeneration de la planaire

Dugesia gonoceohala. pp. 149-159. Inv. Kiortsis

and H. A. L. Trarnpusch, (ed.) Regeneration in animals and related problems. North-Holl. Publ.

Co., Amsterdam.

Guyer, G. M. 1953. Animal micrology. Univ. of Chicago

Press, Chicago. 35 Hamburger, v. 1967. Regeneration. In Encyclopedia Britanica, 19:73-81. Hay, E. D. 1966. Regeneration. Holt, Rinehart and Winston, New York. Henderson, R. F. and R. E. Eakins. 1959. Alteration of regeneration in planaria treated with lipoic acid. J. exp. Zool., 141(2):175-187. Kligman, A. M. 1965. Topical pharmacology and toxic- ology of Dimethyl sulfoxide--Part I. JAMA, 193 (10):140-148. Kolmayer, s. and F. Dubois. 1960. Neoblasts er limita- tion du pouvoir de regeneration cephalique la planaire Dendrocoelum lactrum. J. Embryol. exp. Morph., 8:376-384. Leakes, c. D. (ed.), 1966. Biological action of Dimethyl Sulfoxide. Annals N.Y. Aca. Sci., 141:1-671. Lender, T. 1952. Le role inducteur de cerveau dans la regeneration des yeux d'une planaire d 1eau douce. Bull. Bio. Fr. Belg. 86:140-215. Lender, T. 1955. Sur l'inhibition de la regeneration du cerveau de la planaire Polycelis nigra. c. R. Acac. Sci., 241:1863-1865. Lender, T. 1956. L 1 inhibition de la regeneration du cerveau des planaires Polycelis nigra et Dugesia lugubris en presence de broyats de tetes ou de queues. Bull. Soc. Zool. Fr. 81:192-199. 36

Lender, T. 1960. L 1 inhibition specifique de la differ-

erenciation du cerveau des planaires d 1 eau douce

en regeneration. J. Embryol. exp. Morph., 8(3):

291-301.

Lender, T. 1965. La regeneration des planaires, pp. 95- 111. Inv. Kiortsis and H. A. L. Trampusch, (ed.) Regeneration in animals and related problems, North- ✓ Holl. Publ. Co., Amsterdam.

Morgan, T. H. 1901. Regeneration. MacMillan Co.,

London. Needham, A. E. 1952. Regeneration and wound healing.

John Wiley & sons, New York.

Ostle, B. 1963. statistics in research. 2nd Ed.

Iowa State University Press, Ames, Iowa. Rand, H. w. and A. Browne. 1926. Inhibition of regeneration in planarians by grafting: Technique of

Grafting. Proc. nat. Acad. Sci., Wash., 12:575-581. Rand, H. w. and M. Ellj_s. 1926. Inhibition of regeneration in two-headed or two-tailed planarians. Proc.

nat. Acad. Sci., Wash., 12:570-574. Raven, c. P. (ed.). 1959. An outline of developmental physiology. Pergamon Press, New York. Senger, c. z. 1959. La region caudal d'une planaire est- elle capable d'induire la regeneration d 1un pharynx?

J. Embryol. exp. Morph., 7:73-85. 37

Sengel, c. z. 1965. Inhibition de la regeneration du pharynx chez les planaires, pp. 193-201. In v. Kiortsis and H. A. L. Trampusch, (ed.) Regener- ation in animals and related problems, North-Holl.

Publ. Co., Amsterdam. Sengel, c. z. 1967a. Recherches sur 1 1 inhibition de la regeneration du pharynx chez les planaires. Part I.

J. Embryol. exo. Morph., 18(3):91-105. Sengel, c. z. 1967b. Recherches sur 1 1inhibition de la regeneration du pharynx chez les planaires. Part II. J. Embryol. exp. Morph., 18(3):107-119. Shaw, c. P. 1965. Ectodermal or presumptive skin dis- integration in the frog embryo as a response to

specific tissue factor(s). Dev. Bio., 13:1-15.

✓ Smith, s. D. 1963. Specific inhibition of regeneration in Clymenella torguata. Bio. Bull., 125:542-555. vsmith, z. z. and c.s. Hamn~ 1963. Effects of metabolic inhibitors on planarian regeneration. Bio. Bull.,

125:534-546.

Tucker, M. 1959. Inhibitory control of regeneration in

nemertian worms. J. of Morph. 105:569-601. Wolff, E. 1961. Migration et contacts cellulaires dans

la regeneration. Exp. Cell Res. 8:suppl:246-255.

Vwolff, E. 1962. Recent researches on the regeneration of planaria, pp. 53-84. In. D. Rudnick, (ed.) Regener- ation. Ronald Press, New York. 38

Woodruff, L. s. and A,. L. Burnett. 1965. The origin of blastemal cells in Dugesia tiqrina. Exp. Cell Res.,

38:295-305.

• ABSTRACT

This study investigated the extent to which an inhibitory substance, located primarily in the head of the planarian, Dugesia tigrina, prevented brain regeneration in decapitated planaria. Dimethyl Sulfoxide was used in conjunction with the extracted planarian brain inhibitor in hopes of enhancing the action of the inhibitory substance. When decapitated planaria regenerated a new head in culture media containing the extract of homogenized planarian heads, there was a significant decrease in the length of the regenerated brain. Less inhibition of brain regeneration was noted in decapitated planaria which regenerated in media containing an extract of homogenized planarian tails. The removal of the brain from the intact animal also removed the inhibitor of brain development and the organism was free to regenerate a new brain to replace the lost one. This inhibitory substance acts as the controller or one of the controllers of brain regeneration and development in Dugesia tigrina, and by studying it more closely a better understanding of planarian regeneration can be attained.