Characterization and Induction of Polymorphism In ONgtrlchll sp.
An Honors Thes1 s (10 499)
by
Robert L Meredi th
Robert L Hammersmi th
Ball State Uniyersity
Munci e, Indiana
May, 1966
Graduation Date: Spring 1966
Sec",l/
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Introduction
Oxytdcho is 0 smoll oquot1c, single cell, c1110ted protozoon found in
freshwoter streoms ond lokes which feeds on boctedo ond olgoe. Severol
spectes of Oxytdcho hove been utilized In three moJor reseorch oreos over
the post twenty yeors. These oreos ore: 1) The orgonizotlon, potterning, ond
morphogenesis of surfoce structures (Hommersmith, '760; Hommersmlth ond
Grimes, '81). This includes the role of cytotoctic inhedtonce (Sonneborn,
'63; '75) (0 non-nucleor ond probobly 0 non-nucleic ocld inherltonce system)
In the orgonlzotlon ond molntenance of surfoce structures ond their spot101
ond temporol patterns of development (Grimes, '82; Grimes ond
Hommersmith, '80). 2) The role of cell-cell contoct In the Initlotion of
developmentol progroms (Bonchett., '80; Bonchetti et 01, '82). 3) The
orgonlzotion, ompl1ficotion, and reorrongements of DNA during nucleor
development (Klobutcher, '87; Dowson ond Herrick, '84; Ammermonn, '82).
Oxytrlcho Is on excellent orgonlsm for the Investigotion of cellulor
development. ond cellulor potternlng becouse it possesses not only onterlorposterior polOrlty, but olso dorsol-ventrol ond left-right osymmetry os
defined by the precise plocement of cilioted surfoce structures (Grimes et
01, '80). (Very few cellulor systems possess 011 three definoble oxes.)
Indiylduol slJbcellulor structures within these globol oxes olso show
definoble polorlty ond osymmetrles, thus ollowlng experlmentol
modiflcotion of those structures ond exominotion of how those
modificotions olter or respond to the generol globol potternlng (Grimes,
-
'62).
In oddUton, Oxytrlcho undergoes five unique developmentol progroms
In which the surfoce structures ore essentiolly totolly reploced ond polorlty
-
2
and asymmet.nes reestabl1shed both globally and for IndIvidual structures.
These developmental programs may also Involve simultaneous
development~l
programs associated with the nuclei and DNA content. These
development~l
programs Include: 1) Vegetative d1v1s10n, 1n which one cell
1nduces new cHlary pnmord1a which migrate on the cell
surf~ce
and
ulUmately form two sets of surface structures. The cell then d1v1des
transversely to form two cells 1denUcal to one another with respect to
global pattern1ng. 2) ConJugaUon (8 sexual process), 1n which two cells
undergo cell-ce1l1nteracUons and temporally fuse 1n order to exchange
meloUc products and undergo rec1procal fert111zatlon. Th1s developmental
program also 1nvolves a d1fferenUal degradat10n and redevelopment of
surface structures (H~mmersm1th, '16b). 3) Encystment, 1n wh1ch there 1s a
tot~l
ded1fferent1atlon of all surface structures 1nclud1ng m1crotubules to
form a metabolically InacUve resting cyst (Gnmes, '13; Ricci et al, '85).
Cysts can Withstand drying and other severe envlronment~1 conditions.
When cysts 8re placed In conditions that can support growth they undergo
excystment, redeveloping all surface structures and global
p~ttemlng
1n a
few hours. 4) MorphostaUc regulation, In which cells can replace existing
cHlature In response to Injury or regulate cell size, again reestabl1shlng
typical globel patterning. 5) Polymorphism, In which geneUcally IdenUcal
cells undergo d1fferenUal morphostaUc and dlv1s10nal regulatIon, produc1ng
a minimum of two extreme populations of cells; one very small (dwarfs) and
the other very large (giants). Giants have an altered global pattern such
that the feed1ng structures and cytostome (mouth) are expanded In size so
that they can cannlbal1ze dwarf morphotypes. Dwarfs maintain the typical
surface pattern although reduced In size. Giants and probably dwarfs can
-
undergo d1v1s10n and ma1nta1n extreme dlfferences 1n populat10n s1ze (R1cc1
and Rigg10 '64).
Four of the f1ye developmental pathways (conJugat10n. encystment.
morphostat1c regulaUon and polymorph1sm) occur 1n response to depleUon
of the food supply and starvat10n. S1nce some cell11nes can undergo all
four of these developmental pathways. this 1mpHes that there are
alternat1Ye developmental sw1tches that can be acUyated dunng starvaUon.
The purpose of these studies 1s to lnyesUgate the condlUons wh1ch are
associated with the lnduct10n of polymorphism and the relationship between
cell slze and cannibalism.
Polymorphism In Oxytncha was f1rst descnbed by Dawson ('19) and
later charactenzed in more detan by Giese and Alden ('36). Their
observations suggested that polymorphism was due to a severe and
prolonged starvaUon of the main populaUon of the cell Hne. resulting 1n a
drastic reduction In s1ze followed by a smaller population of s11ghtly larger
cells· cap1tal1z1ng • on a smaller population by cannibalism. AlternaUyely.
R1cc1 and R1gg10 ('64) , suggested that the 1nductlon of polymorph1sm and the
formaUon of g1ants Is solely a funcUon of cell crowding (cell density) and
may be due to poss1ble cell-ce1l1nteracUon or 1nteract10n w1th a secreted
compound, and that cell s1ze 1s not an Important factor.
This study w111 demonstrate that both lnterpretaUons are partially
correct, and that the translt10n between yegetaUye cells and the 1nductlon
of pOlymorphism 1n this species of Oxytncha 1nyolyes at least two discrete
phenomena. Polymorph1sm 1n this species lnyolyes a rap1d Increase In
dlyls10n rate and an Increase In cell dens1ty (possibly lnyolylng a soluble
factor or cell-cell1nteract10n), coupled w1th a radical reduct10n in mean
size wah a smaller population of non-respond1ng (normal s1ze) cens that
--
4
1eter ·cep1teHze· on end thus cenn1bel1ze the smeller populet10n. These
ult1mete1y become glents.
Flnelly~
dete will be presented thet suggest thet
the red1ce1 reduction In sIze of the meln populet1on Is Independent of
generetlon of glent or lerge cells.
netertels end nethod.
The cell11ne (TLA 16) used In these exper1ments wes Isoleted
11/20/65 from Twin Lekes et Bloomington, Indlene. Cysts were collected
end cryogenlcelly preserved In l1quld nitrogen In Apr11 1966. Semples of
these frozen cysts were excysted to provide vegetetlve cultures for these
studies. This streln wes chosen for these studies beceuse of 1ts ebl11ty to
regulerly undergo polymorphism.
Texonomlcelly this ce1111ne, besed upon Its c111eture (streight
undulet1ng membrenes end Involvement of postorel clmln morphogenesis),
would be clesslfted es e species of Stylonychle eccordtng to the methods of
Wlrnsberger, Folssner, end Adem ('65). However, hypotr1chs end cHletes In
generel represent e very diverse complex of slbl1ng species. (Sibling
species hev9 tdent1cel morphology, but genetlcelly ere distinct species.)
Previous reseerch ut11lzlng DNA cross-hybr1dlzetlon hes Indlceted thet cells
of the genere Oxytr1che ere exceedingly diverse. Cloned mecronucleer
probes pMA 81 end pMA H1 der1ved from Oxytr1 che fellex mecronucl eer DNA
do not cross-hybr1dlze with Stylonychle or Oxytr1che ~ DNA, but does
weekI y cross-hybr1 dl ze wi th TLA 16 DNA (Hemmersml th & Hem ck
unpubl1 shed). Thus, celll1ne TLA 16 should be considered e species of
Oxytr1che which Is releted to (L fell ex.
Culture procedure:
The cells to be cherectenzed In this study were fed on e diet of
Chlemydomones IQ.. The Chlemydomones wes grown In e complete elgee
medIum (modIfied from Seger end Grenlck, 1953). Flnel concentret1ons of
ell medle components were es follows:
Cheml cel component
FeC13
CeC12
MgS04
KH2P04
K2 HP04
H3B03
ZnS04
MnS04
CoCl2
Ne2Mo04
CuS04
NH~03
NeCltrete
Flnel ConcentreUon
3.7)(10- 5 M
3.6)(10- 4 M
1.2)(10- 3 M
1.5)(10- 3 M
1.1)(10- 3 M
1.6)(10- 5 M
3.5)(10- 6 M
2.4)(10- 6 M
8.4)(10- 7 M
8.3)(10- 7 M
2.5)(10- 7 M
3.7)(10- 3 M
1.7)(10- 3 M
In eddltlon to the ebove Ingredients 4 ml of Vltemln B-12 et e
concentretlon of .1 mg/ml, end .4 ml of Thlemlne et e concentretlon of .5
mg/ml were edded to eech liter of complete elgee medle.
After prepertlon, the medle wes eutocleved for one hour. After
remove) from the eutocleve the medle wes pleced In e previously stenHzed
five gellon bottle. Upon filling of the bottle en epperetus consisting of two
pieces of gless tubing Inserted through e stopper wes etteched to the mouth
of the bottle. One of the tubes wes connected to en ordlnery eQuenum pump
-
6
via a stertle fl1ter (Gelman Acro 50). The other tube was connected to a
piece of plasUc tubing to serYe as a pour spout. All transfer of media was
conducted under a laminator flow hood. The final arrangement used the
positive air pressure generated bU the aQuanum pump to maintain sterl1e
condit1ons.
The OJ<ytncha species under Investlgat10n was grown In a mod1fled
version of the above media. This minimal media lacked the nitrate, citrate,
and phosphate components of the complete media as a discouragement to
bactenal growth. Any Chlamydomonas fed to the Oxytdcha was first spun
down in a cl1nical centnfuge in order to remove It from the complete media.
For all expenments the cells were grown at a temperature of 230C +/- 10C.
DA11.V Growth Curve procedyre:
5h< replications were used for the plotting of dany growth of this
cell line. Ee'ch sealable culture flask was Inoculated w1th five ml of
ChlamydomQMi which had been spun down and resuspended In minimal
media to a volume approximately one-tenth the volume removed from the
complete media. The Chlamydomonas removed from the complete media for
f eedl ng was at maxi mum growth.
Each culture flask was Inoculated with 100 Oxytdcha (isolated by
handt and the f1nal volume was brought to 100 ml by the addition of
minimal media to yield an In1tial cell density of 1 cell per ml.
Each day two one m11111lter samples were taken from each flask. The
Ume of day for sampling was conSistent to within one hour. Upon removal
of the sample the cells were ktlled and fixed with PerenyI's flxaUve. The
cultures were then brought back to a total volume of 100 ml by the addition
of two ml of minimal media.
-
7
After fh(etlon of the semple, the cells were counted vie one of two
methods. The f1rst counting method used thet of direct counting of ell cells.
The two ml semple wes divided 1nto four one-helf ml ellQuots. Eech onehe1f ml of semple wes pIeced 1n e cell culture well wh1ch hed been etched
with e grid on its unders1de. The cells were observed under e 20)( d1ssect1ng
scope, end counted us1ng e hend held counter. The everege of the four velues
obte1ned for eech culture were used to determ1ne the meen number of
Oxytdche per ml for thet dey.
The density to wh1ch these cells grew mede 1t necessery to elter the
count1ng procedure for cell numbers ebove epprox1metely 2000 per m1. For
semples 1n wh1ch cells were of e dens1ty greeter then 2000 cells per ml, the
semple wes dl1uted to e totel volume of four mI. S1x 100 m1croliter
eliQuots were removed from the dl1uted semple us1ng e F1nn p1pette. These
six semples were counted by dlrect microscoplc observet10n using e totel
megniflcet10n of 100)(. The everege of the s1x velues obte1ned for eech
semple wes used to determine the me en cell number per ml1l111ter for thet
dey. Velidity of th1s method with the prey10usly described method wes
performed by counting cells of the seme semple us1ng both methods. The
d1fference between the two wes cons1dered to be neglig1ble s1nce the
verience wes less then three percent.
lerge forms were counted efter completion of the totel count.
Oeterm1net1on of whet wes consldered to be e member of the lerge
morphotype wes mede by observet10n of s1ze 1n reletlon to the rest of the
cells 1n the semple.
Morphometd c meesurements:
6
After the counting of deny semples, wet mounts of the semples were
prepered, end meesurements of cell length, width, end where possible
thickness, were teken for e rendomly selected fifty cells using en oculer
micrometer et 450)( megniflcetlon on en Amerlcen OpUcel model flftU
m1croscope. Meen, stenderd error, stenderd dev1eUon, frequency
distribution" end histogrems, were obte1ned using e Mec1ntosh Stetvlew
512+ stet1st.icel progrem. During morphometriC meesurements cells were
elso observed for generel corticel end nucleer eppeerence, end eny etypicel
cells were noted.
fml[ .bOW: growth
curve procedure:
The procedure for the four hour growth curve wes essent1elly the
seme es thet used for the delly growth curve. Four repllcetes were used
with prepertlon ldentlcel to thet described ebove. Sempl1ng followed the
seme two-one ml procedure w1th the except10n of reeddlt10n of mlnlmel
medle. The reeddlUon of mlnlmel medle wes excluded In order to evold
dl1utlon of eny possible culture condlUons which might contribute to the
generetlon of polymorphism. Semples were teken every four hours for e
period of four deys. The flnel volume of eech culture efter compleUon of
sempl1ng W8S fifty ml1Hl1ters.
The count1ng procedure used for the four hour semples wes the six
repl1cete direct microscopic count described ebove. After completion of
counUng the cells were pIeced In seeled vlels for future morphometriC
meesurlng.
--
JRB 72 growth curve procedure:
-
9
The procedure used for the compenson growth curve wes Identlcel to
thet descnbed for the deny growth curve. Semples were teken et the seme
time deny to wahln one hour. The counting of the semples employed both
methods described In the deny growth curve procedure. Counts were teken
for both ect1Ye cells~ end resting cysts.
Anelysls g{~:
The dete obtelned for the eforementloned experiments wes enelyzed
on en Apple Meclntosh Plus personel computer using e StetVlew 512+
stet1sticellgreph1cs progrem (Bre1nPower Inc. Celebeses, CAl.
Rasults
ImiUl GrowttJ. Curve:
Results for the twenty four hour growth curve rayeel en Inl11ellog
phese fission rete of epproxlmetely one fission per dey. The eyerege cell
density eUelned for the six rep11cete cultures wes 2643 cells per ml11111ter.
Culture number two obtelned e meen cell dens1ty of 4050 cells per
mlll1Hter.
-
10
-
RA 16 retal ~1att. 8nva CWw
~2S00
~
1
3
•
2000
1SOO
..
==
1000
~
I
SOO
Of~~-----------------------------------~p-
o
____________~~~~~~~~~~~~~
S
10
1S
20
2S
~
m
3S
40
4S
SO
Fig. 1 Mean of the total population for all six replicate cultures.
Refer to appendix for graph of all six replicates.
Upon reech1ng peek cell dens1ty, ell cultures show e greeter then log
phese decreese in cell number reech1ng e meen cell density of 726 cells per
m1ll11lter 1n two or three deys.
Follow1ng the collepse of the culture, the populet1on begen to go 1nto
cycl1c fluctueUons of cell density. These fluctueUons show e crest to
trough Ume period of eppro)(1metely one week. However, t1mlng of
fluctuetlons 1s not 1n d1rect correlet1on with eny set Ume freme.
-
11
·-
TlA 16 letal ..., D. l ..... ..., tt.n 25
of In CulVts
.L-., MNn of CulVts )( 2S
O~, MNn
~
•
2000
lSOD
o~~----.---------"---------------------~
~~~~~~~~~_ _-T~~~~_ _~_ _~~~
o
S
10
1S
20
2S
Dav
!O
JS
40
4S
so
Fig. 2 Mean total for all cultures vs. mean large cells for all cultures. large values
are 25X for clarity of comparison.
The generaUon of a dlchotomy of cen slzes ls flrst apparent at the
polnt of maximum cell density. The peak density of the larger cells occurs
at approximately the same Ume as the bottoming out point for the total cell
population. The population of the larger cens show a cycl1c fluctuation Just
as the total cen population, wlth fluctuations of the larger morphotypes
lagging out of phase with the population as a whole.
WhtJe the graph of average population growth is valuable, results of
cycl1c fluctuations in individual cultures frequently act to cancel the effect
of the six repl1cates. Thus the fluctuations for individual cultures tend to
be of a larger nature than is revealed in the graph of average populaUon
growth. (See appendix A)
In some cultures, a sl1ght lag in fission rate was observed during the
period of log phase growth. This lag Is most noticeable in cultures one and
-
four. In culture one this lag occurs between 1371.5 cens per ml and 1724
ce11s per m1. Culture four shows a slml1ar lag in growth between 1537 cens
per ml and 1644.5 cens per m1.
12
RA 16 c...... 81 Tet.l Pef YS L.... Pef x 2S
.L.-gt, Culbn ·1 )( 2S
OCulbn 1 MNn ./ml
2SOO
22SO
2000
17S0
_ 1!500
.@
12S0
•~ 1000
7SO
!500
2SO
O~~
______________________________
M _ _ _ _ _ _ _ _~
-2S0~~~~~~~~~~~~~~~,-~~~~~~
o
10
1S
20
2S
Dau
ao
3S
40
4S
SO
Flg.:5 Comparison of the large cell morphology with total cell population for Culture - 1
Note the lag In cell growth between days seven and eight.
RA 16 c.""'e
8
4 Tet.l P., ........ Pef x 2S
OCulV.4 Mtan e/m1
.....gt, CulV. e 4 )( 2S
2000
:@ 1SOO
e
~ 1000
SOO
o~~----------------------------------------soo~~~~~~~~~~~~~~~,-~~~~~~
o
10
1S
20
2S
Dau
30
3S
40
4S
SO
Fig. 4 Comparison of the large cell morphology with total cell population for Culture -4
Note the lag In cell growth between days seven and eight.
Celculet10ns indicete thet if this leg were to occur in ell cultures, it
would not be detected unless the sempl1ng period coincided to e high degree
--
13
with the onset tmd termln8t1on of the phenomemt This poss1b111ty wes
InvesUgeted by the use of e four hour sempl1ng of the celll1ne dunng log
ph8se growth.
£.ow: Jmw: growth curve:
Results of sempl1ng four repl1cete cultures every four hours do Indeed
reveal a lag In culture growth. This lag begins at 1569 cells per m11111tter,
and continues to 2210 cells per m111111ter dunng a 30 hour penod. Dunng
this Ume the mean fission rate drops from 8 norm81 rate of approximately
1.06 fissions per day, to approxlmetely .16 fissions per day for the duraUon
of the lag. This lag penod is not constant, but Is coupled w1th a senes of
population fluctuaUons. At 1569 cells per m1, the growth rate for the
culture begins to level off, between 34 hours end 44 hours the total density
of the culture increases by onlyl1.91 as comp8red to an increase of 43.11
for the previous ten hour penod (24 hours to 34 hours). After this initial
plateau the cultures actually go Into a stage of decreasing cell number.
Between 46 and 52 hours the cell number drops by 16.91. This sudden
decreese Is followed by e temporery Increase In the next four hour penod,
end then a second decre8se between 56 8nd 64 hours. This final dip et 64
hours is fonowed by a repld increese of 2641 in the thirty two hour period
between 64 hours end 96 hours. Dunng this repld Incre8se, the cultures
show a mean fission rete of epproxlmetely 1.5 fissions per d8y. They reech
e mean velue of 6492 cells per milliliter at peak cell dens1ty. This peek
population then gives wey to e co1l8pse In cell density without spending eny
Ume 8t e plateau. This beh8vlor Is suggestive of a repld onset of cell deeth
--
following the peek populetlon. (Refer to figure five)
-~
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-
8
<>x
.
i
.f
l
1
.!
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Q)
[:
~
::J
I- ~
U
..,
.&:,
~
e
(!)
L.
::J
I.!
~
.1 j
No.
0
:x:
~
i
L.
::J
0
LL.
~
~
0
.!
!
In
~
•
i
~
-.
O<l
------------------------------~~o
-
14
Dunng, and Immediately following the lag in culture growth, the cells
exhibit a behavior slmllar to an avoidance reacH on. The cells appear to have
an aversion to contact with other cens, and exhibit a rapid retreating
movement upon making contact with each other. Microscopic observation of
cells dunng this penod reveal a vanety of atypical cell fragments. Many
fragments show atYPical c111ature. and a vanety lack the adoral zone of
membranelles (AZM). In addition, some cells show cleavage furrows for
cytokinesis on only one side.
This data thus lends strong support to the observed lag In culture
growth dunng the dany sampl1ng curve. Calculations of four hour sampling
Indicate thtlt It would be possible to generate tI dal1y sampl1ng curve which
would Indeed fal1 to show the observed lag between 1600 cells per ml, and
2400 cells per mI.
JIm 12 .dG1.lV growth curve:
The results of da11y sampl1ng of this cell11ne were done for use as a
control, and companson for the TLA 16 cell l1ne. This growth curve shows a
mean maximum vegetive cell density of 4235.5 cells per m111t11ter, with the
peak density obtained being 4466.6 cells per m1. This cell line reaches Its
maximum density seven days after beginning of the culture, and thus grows
at a similar rate as the TLA 16 line. However, un11ke the TLA 16 I1ne, JRB
72 begins encystment at a cell density of 1461.2 cells per ml111Hter.
Encystment occurs over a three day penod with the maximum period of
encystment occurring between day seven and day eight dunng which time
roughly 661 of the cells undergo encystment. By day nine of culture growth
virtually all vegetative cells have encysted.
1:5
5000
4SOO
... 12 Greva . . EMt....t CWw
OHNn • t.ns
eHNn • ~sts
~4000
!,1SlO
-1
3000
8ZDl
.....
&12000
~ lB
•Ii 1000
fB
O~__-o~~~~~~__~__~~2=~"_""'_""'_""'_M""
~~~~------------~------~--------~
-2
o
2
.. 6 8 10 12
Fig. 6 Mean cell density of vegetative cells for JRB 72 vs. mean cyst density.
Comper1son of the totel cell number efter dey eight with the totel
cyst number efter dey eight reveels thet HUle or no cell deeth occurs dur1ng
the process of encystment.
It 1s 1mportent to note thet the cell density for encystment of JRB 72,
wh1ch begins et 1461 cells per mils within 6.61 of the 1569 cell per ml
density of TLA 16. Thus the first eppeerence of cysts for the comper1son
cell11ne closely corresponds to the beginning of the leg In culture growth
for the cell11ne under comper1son. This Is perheps representettve of e
cruclel developmentel swltch1ng point. It Is know thet TLA 16 Is cepeble of
encystment, but the exect conditions wh1ch ellow for encystment ere es yet
unknown.
The following dete for ell JRB 72 cultures wes used to celculete the
curve of mean velues presented ebove.
-
16
OA a/ml
6000
... 72 In"" . . bet....,
eA ovsts a/ml
DB a/ml
.B ovsts a/ml
ew..
,aC a/ml
&C ovsts a/ml
~O
a/ml
.0 ovsts a/ml
~
~
~4000
a
-3000
I
82000
"
~ 1000
0~--~~-D~~~~~~~s8~~--1ooo~
··2
__~--~-r~~~~--r-__~--~-r~~
12
10
6
e
2
4
o
0-..
Fig. 7 Growth and encystment of all four replicate cultures for JRB 72
MorohometrLc. dna.:
Morphometric meesurements were teken using the cell semples
obte1ned for counttng during the deny growth sempllng. For eech dey used
for morphometriC dete, fifty cells from the culture were meesured et
rendom. These meesurements were then enelyzed by computer.
Anelysls of size meesurements for culture one reyeel e repid decline
In oyerell cell size during the period of post leg growth (both beginning on
dey eight). This size reductton occurs in both length end width. During
normellog phese culture growth the cells heye 0 meen size of 89.28 (r =
1.501) micrometers In length, end 52.36 (r = 1.069) micrometers in width.
17
--
Culture ·1 Tota. population vs Length (Microns) x25
.Culture - 1 total
2500
Olenglh Mean x 25
2250
i
I
j
2000
1750
1500
1250
~ 1000
•I:
~
750
500
250
O~~-----------------------------------250+--r----r-----r-,r---....----,----.---r--.--,.--""T"'""""--r-----r-,r---..---.,
10
15
5
20
25
30
35
40
o
Day
Fig.8 Mean total population for culture -1 vs. mean population size (length).
Population size Is 25X to aid clarity in comparison.
Beginning just after the lag in log phase growth, the cell population
begins to reduce Us mean size. The size decl1nes at a rapid rate, reaching
51.66 (r =.976) microns in length, and 22.77 (r =.537) microns In width in
the first 24 hours after the conclusion of the lag. The populat10n reaches a
minimum mean size of 42.05 (r =.909) microns in length, and 16.95 (r =
.616) microns In width 46 hours after beginning its initial size decrease.
This represents a 531 reduction in overall population length, and a 63.61
reduction In overall population width. This size reduction corresponds very
closely to the peak cell density obtained by the culture. Following this
reduction to minimum size, the population rapidly beg1ns to 1ncrease its
mean size reaching 70.92 (r = 2.136) m1crons In length and 16.47 (r =.633)
microns In width 1n only 24 hours.
The size reduction of the population occurring on day nine and ten
corresponds directly w1th the appearance of the larger morphotypes. The
-~
f1rst appeanmce of the large morphology Is on day n1ne when the mean
-
16
populetlon size Is 51.66 (r =.976) microns In length, end 22.77 (r =.537)
mlcrons In width. The peek density for the lerge morphology occurs one dey
efter the totel populeUon reeches Its minimum me en size .
.........._ y•. Lar. . ~
100
eCultur•• 11.-91
OLtntth MNn
90
M
80
)
70
I•
60
.....
==
u•
50
40
30
20
10
0
-10
0
10
15
20
0..,
40
Fig. 9 Mean population length vs. mean population of the large morphotype.
Around dey twenty five there is e repld decreese In meen populeUon
size. The meen size drops from 74.35 (r = 1.054) in length end 36.49 (r =
.613) In width to 43.62 (r = .694) microns In length end 21.09 (r = .537)
microns In width. This Is the smell est size reeched efter the InlUel
minimum meen size which occurs et peek populetion density. This reduction
in size Is perheps representetlve of e second Induction of the phenomene
which triggered the InlUel repld fission rete end reducUon of cell size. The
renge In size for the populetlon prior to this second size reduction Is 77.74
microns to 66.6 microns for cell length, end 36.34 microns to 32.56 microns
for cell width. It should be noted thet et no Ume does the populeUon
-
reeUeln the me en size of pre-leg log phese growth.
.-
19
Using the dete obtelned by the f1fty rendom semple meesurements, 1\
1s possible to determIne the vel1dlty of the ·observers dlscreUon· method
used 1n cetegorizlng e cell es e member of the lerge morphotype or e normel
morphotype. UsIng two stenderd devletlons for determlneUon of lerge cells,
the following greph Is genereted:
&,MtH ....... fa r . . . . . . . .~ ., 50 .s ........ 1ar.. (2 ID) ,-.253
6
elln9th 19 2 so
0 191Xp1Ct.d !Iv ~
5
46
•
~
3
2
1
0
-1
0
5
10
15
20
25
30
35
40
Dav
Fig. 10 Observed number or cells or the large morphology In the random sample (n ·50) vs. the
predicted number or large cells in the sample based upon values observed during counting.
For the ebove dete, p =.253, 1nd1cet1ng thet the cherecterizet10n mede
by observet10n 1s 1n egreement w1th the dete obte1ned by the morphometriC
meesurement of e rendom semple of f1fty cells.
The dete shown for the chenges 1n populet1on s1ze, ere shown only for
the verieble of length for the purpose of brev1ty. As shown by the following
greph, the correlet1on of length end w1dth 1s qu1te h1gh, end enelys1s
1nd1cetes thet ell behev10rs cherecterized by length ere dupl1ceted 1n the
behev10r of cell w1dth. MorphometriC meesurements for cultures three end
four reveel results very simi1er to those obtelned for culture one (See
eppendl)( B).
20
l ..... YS. VNa fer c...... -1
100
.Width MNn
Cernw.... CMm.. .t-.189; R q-.622
OL~
MNn
90
80
70
I
60
S)
40
30
20
,
10
10
1:5
20
Dav
~
30
3S
Fig. 11 Length vs. width for culture -1. Correlation Coefficient· .789
AnelYS1S of the lerge cell type (cells greeter then two stenderd
devletlons from the meen), reveel thet upon first eppeerence of the lerger
cells, they ere not even es lerge es the meen populetton size pnor to the
collepse of the culture. Dunng the first eppeerence of lerger cells on dey
nine, the lerge cells heve e meen cell length of only 65.5 microns, this Is In
contrest to the meen cell length for the populetlon of 51.7 microns. On dey
ten, when the populetlon me en hes dropped to Us lowest point of 42.1
microns, the lerge cell morphotype 1s et Its ell time low of 54.9. On dey
eleven, the lerge cell populetlon hes essumed e more typlcelly glent size,
reechlng e meen cell size of 101.1 microns In length. For ell cultures
enelyzed, the lergest glent observed wes eppro)(lmetely 225 microns in
length, the smell est dwerf wes eppro)(lmetely 16 microns In length. This
represents e venetton in cell volume of eppro)(lmetely 12501.
-
21
-
L. . . c.n 8tn D ............1att. 8tn
• Populltion MNn ~
0 L9 «:.ns (> 2 61»
130
120
110
;
I
100
90
80
70
60
S)
40
~
1~
20
Dav
Fig. 12 large cell morpho type length compared to mean population length for culture #1. Values
for the large morphology are accurate to +/- 1 micron.
The second dip in meen cell size which occurs eround dey 24 for the
totel populet1on corresponds with e slml1er dip in meen cell size for the
lerger cell type. The 1erge cell type comes extremely close to Us minimum
size, Just es the populet10n es e whole comes close to return1ng to 1ts
minimum me en size. Th1s would seem to suggest thet e second inducUon of
whetever triggered the polymorphic behevior eround dey ntne, hes en effect
upon the lerge morphotype which ts present In the culture et dey twenty
four.
Regression enelysis upon the dete obteined for the lerge populetlon,
end the populeUon es e whole, show thet through out culture growth, the
1erge cell populetlon conUnues to grow reedtly In meen cell length, while
the populeUon es e whole recovers towerd orlgtnel size very slowly.
-
22
0
120
110
."
I
000
100
0
90
00
0
0
0
0
0
0
0
0
eo
o
0
0
0
• •• •• • • • • •
•
•
•
70
60
50
•
0
0
••
40
S
10
1S
20
D..,
2S
30
3S
Fig. 13 linear regression for mean cell size of the large morphotype. as compared to the mean
of the total population for culture -1. For the larger cells m •.672. for total population
m • .291. Note: Days 1 through 3 have been deleted In this analysis to avoid gMng the
total population a negative slope value.
Observat1on of population distnbution In the histograms of appendix C
show that the population undergoes cycl1c widening and t1ghtenlng about the
mean. As show on days such as 19,26, and 27 for culture one, a widening of
the populat1on Is due to not only an increase In cells of the large
morphotype" but also an increase In the frequency of extremely small cells.
Careful observation of the histograms would seem to suggest that the
polymorphism In this species of Oxytdcha generates not two, but three
dlst1nct populat1ons. However, due to the necessity of controlling for
vanables such as cell division, and fragmentat10n during canntbal1zatlon,
the suggestlon of the histograms are elusive of empirical analysis.
Discussion
The Induct10n of polymorphism In this strain of Oxytdcha represents
an alternative developmental pathway In genet1cally Identical cells. In this
2:5
stUdy we cherecter1zed the trensltlon of vegetetlvely growing cells Into the
polymorphic phese end heve escertelnlng whet steps mey be Involved In the
induction end trens1tlon to polymorphlsm# end suggest possibly why the
other developmentel progrems (conjuget1on end encystment) usuelly Induced
under slml1er nutr1ent conditions do not occur.
Polymorphism end formetlon of glents hes been descr1bed for severe1
other c111etes Including other hypotr1chs# these Include: Stylonychl0 SD.#
Oxytr1chell1fer1e, Oxytdcha hymenostoma, Blephadsma, and Tetrehymena.
From these studies several hypotheses have been advanced In order to
explain the formation of giants. Both Dawson ('19) and G1ese ('36) suggested
that polymorph1sm and formation of giants occurred In response to severe
and prolonged starvation. Dawson, utl1lzlng Oxytdcha hymenostoma
suggested that severe starvat10n 1nduced the release of soluble metabol1tes
wh1ch Induce a small proportion of cells to enlarge their oral structures
through reorganization, this then leads to connibal1sm of smaller cells.
Giese howe\ler, found no evidence for soluble factors with Stylonychla , and
suggested a rare cannlbal1stic event allowed some cells to undergo
reorgan1zaUon thus enlarging the oral structures which then leads to
further cannlbal1sm and the establ1shment of a giant population by dlv1slon.
Ricci et 01. ('64) argue that Induction of polymorphism is not due to severe
storvotlon but rether Is due to oyercrowdlng of cells and perhaps the
release of a soluble factor or cell-cell contact. They also suggest thot the
number of giants formed Is directly proportional to cell density and that a
minimum cell density of 360 cells per m11111iter are necessary for any giant
formation. This conclusion was based on exper1ments where different cell
densities were achieved via centr1fugatlon. However, none of the above
exper1ments were based on carefully determined cell density and
-
24
morphometric meesurements dunng vegetetlve log phese growth end the
Induction of polymorphism. Consequently, meny of the dynemlc population
and size fluctuetlons were not observed In these e)(per1ments. Our date,
over e forty-five dey per1od~ suggest thet et least three distinct phenomene
ere essoc1at.ed w1th the trens1t10n of vegetative cells to polymorphic forms,
end thet certain fluctuetions In popu1et1on end size ere not correlated w1th
the generetlon of glents.
Cells of this streln (TLA 16) exhlb1t epproxlmately e one fission per
dey log phase growth untl1 e population dens1ty of epproxlmetely 1200 to
1500 cells per mIls reeched. Upon reaching this cell density the fission
rate dropped to en averege of 0.15 fissions per day for the ne)(t 32 hours.
This Is the (:ell density that most strelns of Oxutdche being Inducing
conjugation end encystment es developmentel pathweys. (See figures 5 end
6). Direct observation of cells dur1ng this leg penod showed thet cells of
the TLA 16 streln were undergoing cell-to-cell contect which Induced
severe evoldence reect10ns# spinning behev10r# end heighten ect1vlty. This
ectivlty suggest thet some type of cell-cell 1nterect1 on Is occurring dunng
this penod. Both direct cell-cell contect end Interectlon with releesed
soluble components ere known to be necessery for the Induction of
conjugetion In Oxutnche end other hypotnchs (Ricci, '81; Hlwetesh, '81).
However# encystment probebly occurs In response to stervetlon end does not
require cell-celllnterection or Interection with releesed fectors. The
evidence for this Is two fold; first, If meting Immeture (Incompetent) cells
ere mixed with meting competent cells, the competent cells cen Interect
end be Induced to conjugete wherees the Incompetent cells encyst es food 1s
depleted; secondly, 11 log phese cells (either meting Incompetent or
competent) ere Indlvlduelly Iso1eted Into medle lecklng food, they repldly
-
encyst. Thw;, conjugation can be viewed as an Induced developmental
pathway caused by cell1nter8ctions, and encystment may be viewed as a
default developmental pathway ceused in response to starvation if
conjugation is not induced. Both conjugation and encystment can occur in
the seme culture. Two observations on TLA 16 during the lag phase and
subsequent growth phase are of interest; first, if starvation is e cond1tion
for Inducing encystment, why do cells of TLA 16 not normelly encyst even
under extreme starvation (cells of TLA 16 ere capable of encysUng under
some non-defined cond1tions); and secondly, cells of TLA 16 are possibly
capable of inhib1tlng both conjugation and encystment in other related
Oxytricha species when mixed together (Hemmersmlth personal
communlcaUon). These results suggest that the events that occur during
the lag period may represent an add1tional developmental pathway
controlled by cell-cell contect, a released soluble factor or both. Recent
research on releted hypotrichs, Euglotes and Onychodromus, indicate that
these cells can Induce sp1ne formation 1n response to both Interspecific and
lntraspeclf1c release of a soluble morphogens by predators (Kuhlmann &
Heckmann '65), (Folssner et al" '67), and (W1cklow, '66), Associated with
the development of these spines is radical alteration of mlcrotubules and
aSSOCiated cytoskeletel structures (Jerka-Dz1adosz et a1., '67), Thus, these
morphogens have the ab111ty to restructure total cellular orgen1zetion end
petteming, Future experiments ere being des1gned to test the ebl1lty of
ls01eted cell fluid on 1nhibltlon of conJugetion end encystment.
Follow1ng the leg period 1s e greeter then log phese growth period (1,5
11ss10ns per dey) lest1ng 34 hours. During th1s t1me the populetion
lncreeses epprox1metely 264 percent reechlng e density of epproxlmetely
6500 cells per mlll11lter, Four 1mportent events occur during this time
-
26
period. 1) Cells undergo 8 2.1 fold reduction In size (69.26 microns verses
42.05 microns) wah hlstogr8m distribution showing a tight clustering 8bout
the me8n. 2) The sIze of the m8cronucle1 ch8nges r8dlc8lly. This Is coupled
wah 8 r8dlC8l decre8se 1n DNA content. 3) During this time period 8nd In
the subseQuent POPu18t10n collapse# there are 8bnorm8l d1v1s10ns resulting
In the genef8tlon of cell fr8gments wah 8bnorm81 c1118ture and In some
C8ses 8bsence of c1118ture. M8ny of these 8bnorm81 divisions Involved
8berr8nt 8nd Incomplete cytokinesis, uncoupled from typlC81 surf8ce
reorg8nlz8Uon. A s1m118r phenomenon, though less common, occurred
during P8rt of the 18g period. 4) Wah the r8dic81 decre8se In size of the
m81n popu18t1on, there Is the 8ppe8r8nce of 8 second population of larger
cells. However, these cells do not constitute a population of giant cells,
their size be1ng 561 the size of veget8t1ve cells (51.7 microns verses 69.26
microns for veget8tlve cells 8nd 42.05 mIcrons for the me8n population).
These results suggest th8t some type of Inter8ctlon may be occurring during
the 18g period th8t condalons or determInes the majonty of the cells to
undergo the r8pld cell division presum8bly without DNA synthesis. Thus,
wh8tever events 8re occurring during the lag period, a apparently can
disassociate normally coordinated cell cycle processes. The few cells
which do not radIcally alter sIze presumably are not respondIng to the lag
penod stImulus or at least do not respond as strongly. Other studies on
Induction of polymorphism have not reported thIs phenomenon. Dark fIeld
microscopy of these larger cells also showed the same general proportions
for the oral c111ature as see In vegetatively grown cells. This Is contrary to
the suggestion of RIccI and RiggIo ('64) that cell crowding Induces first a
change In the size of the oral structures which then leads to cannlba1tsm.
27
The smell cell size genereted by the repld cell division Is correleted
with the meximum cell density. In the four hour sempling expenment Jcell
denslt1es es high es 6500 cells per mlll111ter were observed on e single
feeding of Chlemudomones J end densities es high es 24JOOO cells per
milll1iter heve been observed. No other hypotnch hes ever been reported to
normelly echieve such high denslt1es. The meximum biomess JhoweverJIs
probebI y reeched dun ng the leg pen od.
Following the meximum cell density there Is e repld decreese
(collepse) In the cell populetlon without e stetlonery phese of growth. This
decreese Is probebly due to cell deeth end not solely cennibel1sm. There ere
two 11nes of evidence for this: firstlYJ observet1on of collepsing cultures
indlcete the presence of e number of cell fregments with verylng degrees of
ci1ieture end possibly nucleer metenel; end secondlYJ cultures heve been
observed to reech high cell densities then totelly die out within 24 hours
without the formetlon of lerge cells (Hemmersmith &. Meredith unpubl1shed).
Bectenel cultures heve long been known to show e slmller phenomene
follOWing stetlonery phese growthJ end this deeth Is ettnbuted to the
eccumuletlon of toxic metebol1c products (StenlerJ'63). HoweverJfor this
Oxutnche streln, this cell deeth is probebly due to ebnormel division end
cytokinesis end the generetlon of lnvleble fregments. These fregments ere
probebly cep1tel1zed on, end cennlbel1zed in cultures with non-responding
slightly 1erger cells.
Within 24 hours efter the 1n1t1el collepse of the culture, the meen
populet10n s1ze 1ncreeses 68J to e me en of 70.92 microns end lerge cells
84J to e meen of 101.1 microns. The meen populet10n s1ze for the next 12
-.
deys is epproxlmetely 72 microns with venetlons of plus or minus
epproxlmetely 5 mlcronsJeven though populetlon denSities fluctuetes
-
28
between 300 tmd 1200 cells per ml1111 tter. The large cell population also
fluctuates between approximately 10-to-40 cells per m1ll111ter dunng this
time and generelly shows peak populations 24 hours after a peak 1n total
population. On day 24 there 1s a second major decre8se in cell s1ze
(approx1m8t.ely 74.35 microns to 43.62mlcrons) followed by an Increase to
approximately 78 microns within 48 hours and then a stabll1z1ng of the
populat10n s1ze to approx1mately 75 m1crons even though there are further
fluctuations In both total and 18rge cell populations. This second major
decrease In cell size also was accompanied by generetion of 8 number of
cell fregments. These results suggest four things: 1) Change in total
population density Is not directly correlated with alteretlons in cell size.
2) Increases in large cell population 1s not dependent on cell s1ze or
decreases in cell size. Th1s Is contrary to the observation of G1ese ('38),
and Giese and Alden ('38), but slml1ar to the results of Ricci and Riggio
('84). 3) The large decrease 1n size on day 24 probably represents a second
inductive event simllar to the Initial decrease in size and is possibly due to
accumulation of an Inductiye factor or interections. 4) The repld increase In
cell size following the lnductiye event occurs due to a release of the cells
from the Inducing Influence. This Idea Is supported by the observation that
when very small cell are Indlyldually Isolated Into non-nutnent media, they
rapidly Increase In size even though they are In a st8rYed cond1tlon.
Future research will be directed toward charactenzlng whet fectors
are assoclat.ed wah the induct10n process and analYZing the rates of DNA
synthesis dunng log phase growth, the lag penod, and the growth phase
following the lag penod. Seyerel other theoretical Questions also should be
addressed. Very small cells possess a much lower DNA content but when
Isolated can redevelop normal size and form a new population also capable
29
of underg01ng polymorph1sm. Th1s suggests thtlt there 1s some mechtln1sm
for DNA copy control within these cells. AddlUontllly, s1nce g1t1nts
represent
tI
tottllly d1fferent developmenttll form from vegettlt1ve cells,
there mtly be proteins tlnd RNA specIes spec1f1c to the g1t1nt form, tlnd these
components would consUtute developmenttllly s1gn1f1ctlnt molecules.
Experiments tire currently beIng des1gned In order to Invest1gtlte these
questIons.
Acknowl edgments:
This project 'tiI$ supported tn pert bva B.5.U. ecdemic veer Qrant to R. L. Hammersmith
from the office of research/and the Honors Co11. underQreduete fellwship prOQram.
The author Qretefu11 VeclenoYledQes the _stance of OUvia Keller Jemie WooldridQe and
Keith Johnson for their help Yith morphometric meuuremenb. I 'WOuld al$O Ub to thanle Dr.
Robert L. Hammersmith for his invelueble essistance Yith this research and the preperation of
this thesis.
I
-
I
-
Literature Cited
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Cl111ates. EuroD. J.. Cilllnol. 3: 22-24.
Banchetti, f(~., Cetera, R., Nob111, R., R1cci, N., and Seyfert, H.M. 1962. Matingtype dependent cell pairing 1n Oxgtrich6 hgmsllostom6. IV. Macronuclear DNA
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Appendix B
Morphometric date for culture -3 end culture -4
c..,..
4t3
.eulV.
letal p.,.1attM
~ Total Pop
Ys . . . . . . .
Otter--) xZ5
OMt. Ltngth ~
;3000
ij zsoo
~
2000
8
1500
~
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500
O+-~~~--~---r--~--~--~--r-~~~--~--~
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c..,..
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84 Total pop
27!50
YS
L..... (Hierea) xZ5
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zsoo
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In!50
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8
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1250
S; 1000
(J
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500
~+-~~~--~---r--~--~--~--r-~~~--~--~
!5
10
15
Total population 'Is. length In micrometers for culture #3 (upper) and culture #4 (lower). length is 25X
to aid clarity in comparison. These graphs compare with figure eight In text.
-
AppendIx B
Morphometric data for culture -3 end culture -4
140
....... He• •s ...... p.,.1d_ f . c.Jt.r. 83
Olfn9th MI..Cul ~ Ur9f
120
II!!
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1:
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u
100
80
60
40
20
120
....... He• •s ....... p.,...... c.Jt.r. -4
.CulVt -4 Ur9f
OMlIn Ltngth
100
II!!
...•s..
Ih
--u•
~
80
60
40
20
10
15
20
Dav
Mean population length vs. mean population of the large morphotype for culture -3 (upper) and culture -4
(lower). These ~Iraphs compare with figure nine in the text.
-
Appendix B
Morphometric data for culture -3 and culture - 4
u.,u. Y5. VNtll f . C81twe ~ c..relattelt c..fftotRt-.907; R~.82
.Width MI_
100
OLtngth Mlln
90
80
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I
f
70
60
SO
40
30
20
10
5
10
15
20
Dav
. . . . . . YS.
100
VNtll f . C81tw. -4
.Width MI_
c.rr...... CMfflDt.t-.972; R ~.94:1
OMlIn Ltngth
90
80
M
.,~
I
i
70
60
SO
40
30
20
10
5
10
15
20
30
DI\I
length vs. width for culture -3 (upper) and culture -4 (lower). For culture -3 correlation coefficient =
.907; Rsquared· .823. For culture -4 correlation coefficIent - .972; R squared - .945. These graphs
compare with ngure twelve in the text.
---.,.. .......,... _...
Appendix C-l:
:
Histograms for Culture One
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Day 22
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Appendix C-1:
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Appendix C-2:
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Histograms for Culture -...
Four
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Histograms for Culture Four
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Appendix C-2:
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Histograms for Culture Four
.........IIz:-.--.. .
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Appendix C-2:
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Histograms for Culture Four
-... .....
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Day 27
2
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Appendix C-2:
Histograms for Culture Four
-"''''111:-'''.
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_......... "' •• :&,..e ..... _ ...
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