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This is to certify that the

thesis entitled
GENETIC HETEROGENEITY IN A NATURAL POPULATION OF

ACANTHAMOEBA POLYPHAGA FROM SOIL, AN ISOENZYME

 

ANALYSIS
presented by
Lisa Merilee Jacobson
has been accepted towards fulfillment
of the requirements for
MASTER OF SCIENCE BIOLOGICAL SCIENCE

degree in

 

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GENETIC HETEROGENEITY IN A NATURAL POPULATION 0F ACANTHAQQEEA
POLYPHAGA FROM SOIL, AN ISOENZYHE ANALYSIS
BY

Lisa Herilee Jacobson

A THESIS

Submitted to
Hichigan State University
in partial fulfillment of the requirements
for the degree of
EASTER OF SCIENCE
Departnent of Biological Science

1986

ABSTRACT
GENETIC HETEROGENEITY IN A NATURAL POPULATION OF ACANIEAQQEBA

POLYPHAGA FROM SOIL, AN ISOENZYHE ANALYSIS

BY

Lisa Herilee Jacobson

Acanthgggeba pglyphggg, a free-living, bacterial feeder found in
freshwater and soil, reproduces asexually and is norphologicaly
distinguishable from other acanthanoebae. Isoenzyme analyses were
done on 15 random, clonal isolates from soil. Electrophoretic
patterns indicated that enzyme bands occurred in clusters consistent
with that of a diploid organism. The data indicates that natural
populations of A; pglyphggg have a greater genetic diversity than
laboratory isolates of other anoebae, resenbling the heterogeneity

observed for natural populations of bacteria.

Dedicated to my non and dad,

Darryl and Jerrold

ii

ACKNOWLEDGEMENTS

I would like to express my gratitude to all the people who have
assisted me in the completion of this research.

I am especially grateful to Dr. R. Neal Band, my major professor,
for the generous amount of time he spent sharing his wealth of
knowledge with me. His advice, encouragement and direction helped to
make this work possible.

Special thanks are extended to Dr. Herle Heidemann for her
encouragement and friendship over the years as well as her helpful
suggestions as a committee member.

I would also like to thank the other member of my committee, Dr.
Surindar Aggarwal for his helpful suggestions and continued support.

I wish to acknowledge Dr. Donald Straney for his help in
determining how to calculate Nei's Genetic Distances.

Finally, appreciation is extended to Dr. Henretta Band for her

kindness and constant friendship.

iii

TABLE OF CONTENTS

Page
LIST OF TABLES V
INTRODUCTION 1
MATERIALS AND HETHODS 2
Sampling location and collection procedure 2
microorganism identification and clonal isolation 2
Culture methods 3
Gel electrophoresis 3
Data analysis procedures 4
RESULTS 5
Loci identification 5
Allele assignment 5
Null alleles 5
Genotypes 6
Genetic variation 6
Statistics 7
DISCUSSION 8
LITERATURE CITED 11
TABLES 15

iv

Table

11

iii

iv

vi

vii

LIST OF TABLES

GENOTYPES (Site A)

Site A. NEI'S GENETIC DISTANCES (Above the
diagonal)/GENETIC IDENTITY (Below the diagonal)

GENOTYPES (Site B)

Site B. NEI'S GENETIC DISTANCES (Above the
diagonal)/GENETIC IDENTITY(Below the diagonal)

GENOTYPES (Site C)

Site C. NEI'S GENETIC DISTANCES (Above the
diagonal)/GENETIC IDENTITY(Below the diagonal)

STATISTICS FOR GENETIC DISTANCE

Page
15

16

17
18

19

20

21

INTRODUCTION

Free-living, asexual amoebae (e.g. Acanthamoeba, Naggleria) are
widespread in soil and freshwater. Some species are also pathogenic
to man (13, 26). Isoenzyme analyses have been used to clarify the
taxonomy of axenic, laboratory isolates obtained from a variety of
sources and indicate a low genetic heterogeneity for those studied
(23). This is in contrast with isoenzyme analyses of natural
populations of bacteria in which considerable genetic heterogeneity
has been reported (12, 15, 16, 24).

In the present study, isoenzyme electrophoresis was performed on
clonal isolates to determine genetic heterogeneity in natural
populations of A; pglyphggg from soil. The genetic heterogeneity
observed was greater than results obtained for axenic, laboratory
cultures, yet consistent with the heterogeneity reported for bacterial
populations (12, 15, 16, 24).

I question the use of isoenzyme analyses for strain and species
classification of laboratory cultures of small amoebae. By using
random isolates from various sources, the genetic heterogeneity of the
parent, natural population for each laboratory isolate is being
ignored. Further, considerable genetic selection takes place in
establishing axenic cultures of amoebae (20, 27).

Results from this study were presented at the American Society

for Hicrbiology Convention, Washington, D.C., March 23-28, 1986.

MATERIALS AND flETHODS

Sampling location and collection rocedure. Random soil samples
were collected from 3 sites (A, B, C) in Dickinson County, Hichigan.
Sites A and C were located in Felch township at lat. 46° 07' N,
long. 87° 54' w and lat. 46° 03' N, long. 87° 55' w
respectively, while site B was located in Norway township at lat.
45° 56' N, long. 87° 56' w. Each site consisted of a 10 by 20
meter area in a northern hardwood forest.

With the aid of a tube sampler (diameter:2 cm) both organic and
mineral layers were collected and subsequently 2 inches from each
horizon was brought back to the laboratory. Amoebae were enriched
from the soil samples using a 96 multi-well plate, soil dilution

technique and Escherichia coli (strain K-12) as the food organism (8).

 

Microorganism identification and clonal isolatign.

 

Hicroorganisms were identified as Acanthamoeba pglyphggg on the basis
of cyst morphology (22). To each well identified as containing 5;
pglyphggg, one drop of LS saline (50 mu NaCl, 4.6 mfl H9504, 0.36 mu
CaClz) (3) was added; then the amoebae were withdrawn with a pipet
and placed in a Petri dish containing LS saline and agar (1.5% w/v).
To obtain single amoebae for clonal isolates, a piece of agar which
contained the smallest number of A; pglyphggg was cut out and placed
on another Petri dish containing LS saline and agar (1.5t w/v)
(LS-agar). At this point, had a single A; pglypgggg_been isolated,

it was cultured with E; coli (strain K-12) at 23°C on L5 agar. If

 

however, an isolate had not been achieved, amoebae were subcultured to

the bottom of a fresh Petri dish containing LS-agar, with g; 59;;
spread over the dish, which was slanted during incubation. The slant
allowed the amoebae to migrate towards the top and distribute widely
over the plate which eased the isolation of individual organisms.
Culture methods. E; ggli was cultured in 200 ml Tryptone
broth(0.5 9 NaCl, 1.0 g Tryptone, Difco Laboratories, Detroit,

Hichigan u.s.n. and a o to 100 ml) for 18 n at 37°C. The

2
organisms were harvested and washed 2 times in LS saline, suspended in
30 ml of LS saline and distributed in 10 ml volumes to 250 ml plastic
tissue culture flasks (Corning Science Products). Subsequently,
clonal isolates of A; pglyphggg were inoculated into the tissue
culture flasks containing E; coli and incubated at 23°C. Once the
peak of population growth had been reached (approx. 48-72 h), the
amoeba cultures were harvested by centrifugation, washed once in LS
saline and pelleted. Culture pellets were then frozen and stored over
liquid nitrogen.

9g; Electrophoresis. A vertical discontinuous polyacrylamida gel
was used (14): (resolving gel (7.5%), resolving gel buffer (pH 8.9)
and spacer gel (5%), spacer gel buffer (pH 6.7). Samples were removed
from liquid nitrogen, thawed and suspended in 1 ml sample solution (10

ml, 5.98 g Trizma base/100 ml, pH 6.7, 80 ml H 0, 10 ml glycerol).

2
After centrifugation in a microcentrifuge (Fisher Nodal 235B) for l
min, 50 ul of sample supernatant (approx. 2-4 x 104 cells) was
loaded into each gel electrophoresis well. Electrophoresis was run
for approximately 4 h at 300 V with pH 8.3 electrode buffer (14).

Gels were stained for acetyl esterase (7), propionyl esterase (7) and

tetrazolium oxidase (2). All reagents used in the enzyme staining
procedures were obtained from Sigma Chemical Company. For comparison,
5; pplyppggg, American Type Culture Collection strain #30487, was used
in this study.

pats analysis procedure . Loci were coded by number from the
most anodic banding cluster, designated number 1 and so on to the most
cathodic cluster (23). Allele number was assigned based on isoenzyme
patterns expected for heterozygotes in the case of enzymes which are
monomers, dimers, trimers or tetramers (10). The calculation of
genetic distances was based on Nei's formula (17) and was done with
the aid of a computer program (9) and a correction to that program

(11).

RESULTS

Laal identification. Isoenzyme analyses indicated a clustering
of the electrophoretic bands for acetyl esterase (AE), propionyl
esterase (PE) and tetrazolium oxidase (T0). Based on these clustering
patterns, loci were assigned by number from the most anodic to the
most cathodic for each of the enzymes. The number of loci observed
for each enzyme varied by soil site (Tables 1, 3 and 5). Three loci
were observed for PE from sites A and C, while four were observed from
site 8. Four loci were observed for AE from sites A and B; three from
site C. Three loci were observed for T0 from site A and 8; four from
site C.

Allala aasigpaen . All loci were assigned allele numbers in
accordance with the isoenzyme patterns expected in heterozygotes.
Alleles were assigned with numbers increasing from the most anodic
electrophoretic band to the most cathodic. Heterozygous monomers
consisted of two electrophoretic bands, dimers three bands, trimers
four bands and so on, with the homozygote represented by the two outer
bands of the respective heterozygote.

Data given below showed that all of the clonal isolates differed
from one another, although isolate B5 was identical to the banding
pattern of A. polyphaga strain #30487 from the American Type Culture
Collection. Replicate electrophoretic gel runs showed identical
banding patterns.

gall allele . Absence of enzyme activity was noted for each of

the enzymes studied from the three sites. The alleles responsible for

this loss of activity ("null alleles") were assigned a number in
relation to the corresponding allele assignments for each locus.

Genotypes. Genotypes were described based on the allelic
information for each clonal isolate (Tables 1, 3 and 5). The
homozygotes had been designated by one allele and the heterozygotes by
both alleles. A range of variation existed in the genotypes between
the isolates at a given site. Between isolates A1 and A2 (Site A),
six of ten loci showed the same alleles in identical frequencies, two
showed one allele the same yet in different frequencies and the
remaining two loci had unlike alleles. Between isolates C1 and C3
(Site C), only two of ten loci had genotypes with the same alleles in
identical frequencies. The remaining eight loci had different
alleles. Differences in the genotypes between the other isolates
varied between these two extremes.

Genetic variation. Genetic similarities ("identities") were
determined and subsequently used to calculate genetic distances
between clonal isolates from each site (Tables 2, 4 and 6). Values
ranged from 0.122 to 0.751 over the three sites. These two values
corresponded to the allelic comparisons made between isolates C1 and
C3 and isolates A1 and A2 respectively.

Genetic distances ranged from 0.286 to 2.106 corresponding to
comparisons between A1 and A2 and C1 and C3 respectively. The greater
the proportion of loci with the same alleles, with identical
frequencies between the two isolates, the greater the genetic

similarity and subsequently, the smaller the genetic distance.

Statistics. The data indicated large differences between clonal
isolates at each of the soil sites. Yet there is no statistically
significant difference in mean genetic distances between the three

sites (Table 7).

DISCUSSION

Isoenzyme electrophoresis is a technique for the study of genetic
variation within a species (25). Electrophoretic banding patterns
provide data in the form of allelic variants for specific genes. This
information can be readily converted to allelic frequencies which are
subsequently applied to the chosen formula for genetic variation.

A measure of heterozygosity (18) would be determined if one
wished to calculate genetic variation at each enzyme locus. A measure
of genetic distance (17) would be calculated to determine genetic
variation between isolates over all loci.

In previous studies, a measure of heterozygosity was used to
express genetic variation when each individual electrophoretic band
was the product of a separate genetic locus as seen in haploid systems
(12). On the other hand, genetic distance was calculated when a
clustering of the electrophoretic bands suggested a diploid structure
for the genome (23). Calculation of genetic distances was similarly
used in this study to interpret the data. However, genetic distance
calculations are not dependent on ploidy or mating scheme (17).

By calculating the values for genetic distance it was evident
that genetic heterogeneity did exist for the 15 clonal isolates at
each of the 3 8011 sites. Since random isolates taken from 3
different soil sites did not differ in terms of mean genetic distance,
the values obtained were not unique to a particular site. The data
for these 15 clonal isolates represented a single species, A;

pplyppaga, rather than a mixture of Acanthamoeba species. This amoeba

9

species can be readily identified by a unique cyst morphology (22).
The cyst protoplast tends to be angular so that a mixture of square
and triangular protoplasts, as well as other shapes, are seen in a
population of cysts. The variation that existed among the isoenzyme
patterns of the 15 clonal isolates also provided evidence that a
single species was studied. If the clonal isolates had represented
more than 1 species, a few repetitive banding patterns between
isolates of the same species would have been observed. The only
similarity that was found in the isoenzyme patterns was that isolate
BS was identical to the banding pattern of A; pplyppaga strain #30487
from the American Type Culture Collection.

The genetic heterogeneity observed for the 15 A; palyppaga
isolates was high. Genetic distances were calculated to be between
0.286 and 2.106 over the 3 soil sites. These values were higher than
those calculated by Pernin (23) for axenic laboratory stocks of
Naagleria. The species Na lovaniensis, considered to be the most
genetically variable of the species studied, had genetic distances
ranging from 0.11 to 0.146. These low values for genetic distance may
be due in part to axenic selection which has been observed in the
cellular slime mold Dictyostelium (20).

The values of genetic distance far Na lovaniensis (23) which
ranged from 0.11 to 0.146 were determined by comparison between strain
6, an isolate-from a thermally polluted water source in the U.S.A.,
and strain 8, an isolate from a hydrotherapeutic swimming pool in
Belgium; and between strain 1, an isolate from a thermally polluted

water source in France, and strain 7, an isolate from a

1'0
hydrotherapeutic swimming pool in Belgium, respectively. It would be
useful to determine the amount of genetic heterogeneity within a
population of a Naagleria species from a single source and with clones
that had not been selected for axenic growth. An isoenzyme study of
axenic, laboratory isolates of Acanthamoeba (6) also would have
benefited for similar reasons.

The results of this study indicate that there is greater genetic
diversity in this asexually reproducing species than in sexually
reproducing diploid animals (1). In the absence of normal genetic
recombination, the dispersal of new genotypic elements within the
population may be inhibited which would prevent new species from
forming and would consequently lead to greater heterogeneity within
the original species.

The basis for this heterogeneity may be some type of
recombination such as defective DNA repair proposed in fungi (21).
Perhaps genetic variation could be explained by the presence of
plasmids, found in wild-type strains of the cellular slime mold
Dictyostelium discoideum (19) or by the presence of transposons, also

reported in Ql discoideum (4,5).

LITERATURE CITED

Avise, J. C., and P. J. Ayala. 1975. Genetic differentiation in
speciose versus depauperate phylads: evidence from the
California minnows. Evolution. 30:46-58

Ayala, F. J., J. R. Powell, N. L. Tracey, C. A. Hourao, and S.
Perez-Salas. 1971. Enzyme variability in the Drospppila
williatopi group 1V. Genie variation in natural populations

of Droapppila gilliston . Genetics 70:113-139.

Band, R.N. & S. Hohrlock. 1969. The respiratory metabolism

of Acantpapaeba gaysodes during encystation. J. Gen.
microbiol. 59:351-358.

Cappello, J., S. H. Cohen, and H. F. Lodish. 1984.
Qictyastelium transposable element DIRS-l preferentially
inserts into DIRS-l sequences. H01. Cell. Biol. 432207-2213.
Cohen, S. 8., J. Cappello, and H. F. Lodish. 1984. Transcription
of Dictypsteliaa discaideum transposable element DIRS-l. Hol.
Cell. Biol. 4:2332-2340.

Daggett, P-H., D. Lipscomb, T. K. Sawyer, and T. A. Nerad. 1985.
A molecular approach to the phylogeny of Acanthamoeba.
Biosystams 18:399-405

Daggett, P—H and T.A. Nerad. 1983. Procedures for isoenzyme
electrophoretic analysis. American Type Culture Collection.
Rockville, Haryland, U.S.A.

Darbyshire, J. F., R. E. Wheatley, N. P. Greaves, and R. H. E.

I1

10.

11.

12.

13.

14.

15.

16.

I2

Inkson. 1974. A rapid micromethod for estimating bacterial
and protozoan populations in soil. Rev. Ecol. Biol.
11:465-475.

Green, D. H. 1979. A BASIC computer program for calculating
indices of genetic distance and similarity. J. Hered.
70:429-430.

Harris, H. 1976. Handbook of enzyme electrophoresis in human
genetics. North-Holland Publishing Co., Amsterdam.

Hillis, DuH., and D. C. Cannatella. 1983. Calculation of
indicas of genetic distance, genetic similarity, and average
homozygosity: correction of Green's computer program. J.
Hered. 74:115.

Howard, D. J., G. Bush, and J. A. Breznak. 1985. The
evolutionary significance of bacteria associated with
gpagalatla. Evolution 39:405-417.

JOhn, D. T. 1982. Primary amebic meningoencephalitis and the
biology of Naagleria fowler . Ann. Rev. Hicrobiol.
36:101-123.

Haizel, J. V. 1971. Polyacrylamide gel electrophoresis of
viral proteins, p.179-246. la Hethods in Virology-1971.
Academic Press, New York.

Hilkman, R. 1973. Electrophoretic variation in Escherichia
gall from natural sources. Science 182:1024-1026.

Husser, J. H., E. L. Hewlett, H. S. Peppler, and R. K. Selander.
1986. Genetic diversity and relationships in populations of

Bordetella spp. J. Bacteriol. 166:230-237.

I3

17. Nei, H. 1972. Genetic distance between populations. Am.
Nat. 106:283-292.

18. Nei, H. 1978. Estimation of average heterozygosity and genetic
distance from a small sample of individuals. Genetics. 89:583
-S90.

19. Noegel, A., D. L. Walker, B. A. Hetz, and K. L. Williams. 1985.
Presence of nuclear associated plasmids in the lower eukaryote
Qictygstelium discoidagp. J. Hol. Biol. 185:447-450.

20. North, H. J., and K. L. Williams. 1978. Relationships between
the axenic phenotype and sensitivity tocv-aminocarboxylic acids
in Dictyostellum discoideum. J. Gen. Hicrobiol.

107:223-230.

21. Orr-Weaver, T. L., and J. W. Szostak. 1985. Fugal recombination.
Hicro. Rev. 49:33-58.

22. Page, F. C. 1967. Re-definition of the genus Agapgpaaggpa with
descriptions of three species. J. Protozool. 14:703-724.

23. Pernin, P., H—L. Cariou, and A. Jacquier. 1985. Biochemical
identification and phylogenetic relationships in free-living
amoebas of the genus Naggleria. J. Protozool. 32:592-603.

24. Selander, R. K., and B. R. Levin. 1980. Genetic diversity and
structure in Eacherichia gall populations. Science
210:545-547.

25. Selander, R. K., D. A. Caugant, H. Ochman, J. H. Husser, H. N.
Gilmour, and T. S. Whittam. 1986. Hethods of multilocus enzyme
electrophoresis fer bacterial population genetics and

systematics. Appl. Environ. Hicrobiol. 51:873-884.

26.

27.

I4

Visvesvara, G. S., D. B. JOnes, and N. H. Robinson. 1975.
Isolation, identification, and biological characterization of
Agapgpaaggpa,pglyppaga from a human eye. Am. J. Trop. Hed.
Hyg. 24:784-790.

Williams, J. L., R. H. Kessin, and P. C. Newell. 1974.
Parasexual genetics in Dictyosteliaa discoidaap: mitotic
analysis of acriflavin resistance and growth in axenic medium.

J. Gen. Hicrobiol. 84:59-69.

TABLE 1. GENOTYPES (Site A)**

 

CLONAL ISOLATES

 

 

LOCI A1 A2 A3 A4 A5
PE 1 1 1 2 1/3 2
PE 2 1/3 2 2 1/3 1/2
PE 3 1 2/3 4* 1/2 2
AB 1 2* 2* 2* 1 2*
AE 2 1/2 2 3* 1 3*
AB 3 2* 2* 2* 1 2*
AE 4 1 1 1 1/2 2
TO 1 1/2 1/2 2 2/3 3
TO 2 1/2 2 2 1/2 3*
TO 3 2* 2* 2* 1 1

*Null alleles.

**Abbreviations represent the loci coding for the various
enzymes, the numbers representing multiple forms of the
enzyme. Alleles are numbered from the most anodic to the
most cathodic. Homozygotes are designated by one allele

and heterozygotes by both alleles.

15

TABLE 2. Site A. NEI'S GENETIC DISTANCES(Above the

diagonal)/GENETIC IDENTITY(Below the diagonal)*

(A Site)

 

CLONAL ISOLATES

 

 

A1 A2 A3 A4 A5
A1 ---- 0.286 0.685 0.834 1.820
A2 0.751 ---- 0.434 1.378 1.417
A3 0.504 0.648 ---- 1.719 0.916
A4 0.434 0.252 0.179 ---- 1.087
A5 0.162 0.243 0.400 0.337 ----

*Null alleles included.

16

TABLE 3.

GENOTYPES (Site B)**

 

CLONAL ISOLATES

 

LOCI BI 82 B3 B4 BS
PE 1 1/3 1/2 2 2 2
PE 2 1/3 4* 2 1/2 1
PE 3 2/3 4/6 7* 3/5 1/5
PE 4 1 3 4* 2 2
AE 1 3* 1/2 3* 1/2 3*
AB 2 1 2* 2* 2* 1
AE 3 2* 2* 2* 2* 1
AB 4 1/4 2 1 3 3
TO 1 1/2 3 5* 3/4 2/3
TO 2 2 2/3 2 2 1/2
TO 3 2* 1 2* 1 2*

*Null alleles.

**Abbreviations represent the loci coding for the various

enzymes, the numbers representing multiple forms of the

enzyme .

Alleles are numbered from the most anodic to the

most cathodic. Homozygotes are designated by one allele and

heterozygotes by both alleles.

I7

TABLE 4. Site B. NEI'S GENETIC DISTANCES(Ab0ve the

diagonal)/GENETIC IDENTITY(Below the diagonal)*

(B Site)

 

CLONAL ISOLATES

 

 

81 82 B3 84 BS
81 -—- 1.609 0.906 1.642 0.899
82 0.200 ---- 1.498 0.636 2.001
B3 0.404 0.224 ---- 0.938 1.305
84 0.194 0.529 0.391 ---- 0.857
85 0.407 0.135 0.271 0.424 ----

*Null alleles included.

18

TABLE 5.

CLONAL ISOLATES

GENOTYPES (Site C)**

 

LOCI C1 C2 C3 C4 C5
PE 1 1 2 3* 1 1
PE 2 1 1/2 1 1 1
PE 3 2 1/2 1 3* 2
AE 1 1 2/3 4* 4* 1
AB 2 2/4 1/3 3 1/3 3
AB 3 1 2 1 2 2
TO 1 2 1 1 3* 2
TO 2 2/3 1/2 4* 4* 2/3
TO 3 1/3 1/2 2 1/2 1/3
T0 4 1 2* 2* 2* 2*

*NUll alleles.

**Abbreviations represent the loci coding for the various

enzymes, the numbers representing multiple forms of the

enzyme a

most cathodic.

heterozygotes by both alleles.

19

Alleles are numbered from the most anodic to the

Homozygotes are designated by one allele and

TABLE 6. Site C. NEI'S GENETIC DISTANCES(Above the

diagonal)/GENETIC IDENTITY(Below the diagonal )*

(C Site)

 

CLONAL ISOLATES

 

 

C1 C2 C3 C4 C5
C1 ---- 1.638 2.106 1.824 0.377
C2 0.194 --- 0.896 _0.819 0.837
C3 0.122 0.408 ---- 0.752 1.445
C4 0.161 0.441 0.471 ---- 0.758
C5 0.686 0.433 0.236 0.469 ----

*Null alleles included.

20

TABLE 7. STATISTICS FOR GENETIC DISTANCE*

 

5111: um GENETIC DISTANCE .t so
A 1.0576 1 .521
8 1.2291 1 .444
c 1.1452 .t .565

 

One-way ANOVA: (For sites A, B & C)

D.F. H.S.

Between 2 .0735

Within 27 .2628
= .2798 (NS)

 

*Abbreviations: SD, standard deviation; ANOVA, analysis of variance;
D.F., degrees of freedom; H.S., mean square; F, F-test; NS, not

significant at the 0.05 level.

21

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