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/ the.::'I:'

~ D c. J.-Jf~. , • ztf I q '2;:)' .M~J7

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 onterlor­

posterior 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

- and asymmet.nes reestabl1shed both globally and for IndIvidual structures.

These developmental programs may also Involve simultaneous

2

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

-- 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.

4

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 H 1 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 K2HP04 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

-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.

6

- 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 one­

he1f 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.

7

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:

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:

6

-

-

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.

9

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.

-

-

~2S00 ~ • 2000 -11SOO

3 == 1000 .. ~

RA 16 retal ~1att. 8nva CWw

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.

10

·-

-

TlA 16 letal ..., D. l ..... ..., tt.n 25 O~, MNn of In CulVts .L-., MNn of CulVts )( 2S

2000

~ • lSOD

o~~----.---------"---------------------~ ~~~~~~~~~ __ -T~~~~ __ ~ __ ~~~

o S 10 1S 20 2S !O JS 40 4S so Dav

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.

11

-

2SOO

22SO 2000 17S0

_ 1!500

.@ 12S0 • ~ 1000

7SO

!500 2SO

RA 16 c. ..... 81 Tet.l Pef YS L .... Pef x 2S OCulbn 1 MNn ./ml .L.-gt, Culbn ·1 )( 2S

O~~ ______________________________ M ________ ~

-2S0~~~~~~~~~~~~~~~,-~~~~~~ o 10 1 S 20 2S ao 3S 40 4S SO

Dau

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.

2000

:@ 1SOO e

~ 1000

SOO

RA 16 c.""'e 8 4 Tet.l P., ........ Pef x 2S OCulV.4 Mtan e/m1 ..... gt, CulV. e 4 )( 2S

o~~-----------------------------------------soo~~~~~~~~~~~~~~~,-~~~~~~

o 10 1 S 20 2S 30 3S 40 4S SO Dau

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

12

--

--

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)

13

-

Q)

[: ::J U .&:, .., ~ e (!)

L. ::J 0 :x: L. ::J 0

LL.

i ..,f <>x

I .!

N-o.

-. O<l

~ -

8 -

i

~

i

~

------------------------------~~o

. .f l 1 .! I ~

I ~ - ~ .1 j ~ ~

~ 0

.! ! In • i ~

- 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.

14

-

-

5000

4SOO

~4000 !,1SlO

-1 3000

8ZDl ..... &12000 ~ lB • Ii 1000

fB

... 12 Greva .. EMt .... t CWw

OHNn • t.ns eHNn • ~sts

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.

1:5

-... 72 In"" .. bet .... , ew..

OA a/ml DB a/ml ,aC a/ml ~O a/ml eA ovsts a/ml .B ovsts a/ml &C ovsts a/ml .0 ovsts a/ml

6000

~

~ ~4000 a -3000 I 82000 " ~ 1000

0~--~~-D~~~~~~~s8~~--

-1ooo~ __ ~--~-r~~~~--r-__ ~--~-r~~ ··2 o 2 4 6 e 10 12 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.

16

--

-~

2500

2250

2000

i 1750

I 1500 j 1250

~ 1000

• 750 I: ~ 500

250

Culture ·1 Tota. population vs Length (Microns) x25 .Culture - 1 total Olenglh Mean x 25

O~~------------------------------------250+--r----r-----r-,r---....----,----.---r--.--,.--""T"'""""--r-----r-,r---..---.,

o 5 10 15 20 Day

25 30 35

Fig.8 Mean total population for culture -1 vs. mean population size (length). Population size Is 25X to aid clarity in comparison.

40

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

17

-

-

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 .

100

90

M 80

) 70

I 60

50

40 ..... • 30 == • 20 u

10

0

-10 0

.......... _y •. Lar .. ~ eCultur •• 11.-91 OLtntth MNn

10 15 20 0..,

Fig. 9 Mean population length vs. mean population of the large morphotype.

40

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.

16

.-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).

19

-

-

l ..... YS. VNa fer c. ..... -1 Cernw. ... CMm. .. t-.189; R q-.622

100 .Width MNn OL~ MNn

90

80

I 70

60

S)

40

30

20

10 , 10 1:5 20 ~ 30 3S Dav

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.

20

-

-

L ... c.n 8tn D ............ 1att. 8tn

130 • Populltion MNn ~ 0 L9 «:.ns (> 2 61»

120

110

; 100

I 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.

21

120 0

110 000 0

100 0 0 o 0

" 00 0 .. 90 0 0 0

I 0 0 0

0 eo • •• • • • •• • • • 70 • • 60

0 0 50 • • 40 •

S 10 1S 20 2S 30 3S D..,

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

22

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 severe 1

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

2:5

- 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 1 nterect1 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

24

- 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

-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.

26

-.

The smell cell size genereted by the repld cell division Is correleted

with the meximum cell density. In the four hour sempling expenmentJ cell

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 biomessJ howeverJ Is

probeb I 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). HoweverJ for 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 mlcronsJ even though populetlon denSities fluctuetes

27

-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.

28

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

-

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 V eclenoYledQes the _stance of OUvia Keller I Jemie WooldridQe I 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.

29

-Literature Cited

Ammermann, D. 1962. DNA and Protein content of Different Hypotrich 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 content. J.. Em. l..Qal. 221: 251-254.

Bannchetti, R., and RicCl, N. 1966. The doblet of OxgtriCh6 bil6ri6(Cl1iata, Hypotrichid~). I. Morphology and development. Protistologica. 22: 161-166.

Dawson, J.A. 1919 An experimental study of an amicronucleate OXgtriCh6. I. Study of the normal animal, with an account of cann1balism. J.. f}m.. IQ21., 29: 473-514.

Dawson, D., and Herrick, G.A. 1964. Telemeric Properties of C4A4 Homologous Sequences in M1cronuclear DNA of Oxgtrich616//6X. Cill30: 171-177.

Giese, A.C. 1936. Cannibalism and gigantism in Bleph6rism6. Trans. Am. M1crosc. Sm~ 57: 245-255.

Giese, A.C., ~nd Alden, R.H. 1936. Cannibalism and giant formation in StglOllgchi6. J.. f1UL.l..Q.Ql., 78: 117-134.

Grimes, G.W. 1973. DifferenUat10n During Encystment and Excystment in Oxgtrich6111//6X. J. Protozool., 20: 92-104.

Grimes, G.W. 1962. Pattern Determination 1n Hypotrich Ciliates. Amer. zmn., 22: 35-46.

Grimes, G.W", and Hammersmith, R.L. 1960. Analysis of the effects of encystment and excystment on incomplete doblets of OXlltrich616//6x J. Embruol. 59: 19-26.

Hammersmith, R.L. 1976. The Redevelopment of Heteropolar Doublets and Monster Cens of OJJ'IItriCh616//6X after Cystment. J.. Cill5..tl. 22: 563-573.

Hammersmith, R.L. 1976. Differential Cortical Degradation in the Two Members of Early Conjugant Pa1rs of Oxgtrich616//6X. J.. fU.l..Qal. 196: 45-70.

-Hemmersm1th, R.L., end Gr1mes, G.W. 1961. The Effects of Cystment on Cel1s 01 OXlltrlch6111//lIx possessing Supernumerery Dorsel Bristle Rows. J.. EmbryoJ. 63: 17-27.

Hiwatash1, K. 1981. SeKual Interactions of the Cell Surface in P,v8RJ8Cium. in Sexualloterections in Eukal1l0tic Microbes. ed. by D. H. O'Dey, end P. A. Horgen. Acedemic Press.

Klobutcher, L.A. 1967. Micronucleer Orgenizetion of Mecronucleer genes in the Hypotr1c:hous CUiete OXII/rlchll novII. J... ProtozooJ. 4: 424-429.

Kulmenn, H.W. & Heckmenn, K. 1985. Interspecific morphogens reguleting prey-predator relationships in protozoa. Science. 227: 1347-1349.

Ricci, N. 1981. Preconjugant cell interactions in llAytrich6 billllill (Ciliate, Hypotrichidi~): 0 two-step recognition process leading to cell fusion and the induction of' meiosis. In Sexual Interactions in Eukadotic Microbes. edited by P.A. Horgen and D. H. O-Day. Academic Press, New Vork. pp. 219-350.

Ricci, N., end Benchettl, R. 1982. Cell differentietion in OXlllrichll bllllrill (Protozoa, Cil jeta) . .6.i.Ql. CilL 45: 158.

Ricci, N., end Benchetti, R. 1982 Cell differentietion in OXlllrichll bllllrill. J... Protozool., 29: 455 (Abstract).

Ricci, N., end Riggio, D.C. 1984 Cannibals of OXlltrichllbllllrill (Ci11ate, Hypotrichido): A Crowding-Dependent Cell Differentietion. J. ~ ZQ.Q.L 229: 339-347.

Ricci, N., Verni, F., end Roseti, G. 1985. The cyst 01 OXII/rlchll bllllrill (C111ete, HYJ,otr1chide). I. Morphology end significance. Trans . .Am.. Microsc. So&.. 104: 70-78.

Sonneborn, T.M. 1963. Does preformed cen structure pley en essentiel role 1n cell heredity? in J. M. Allen (ed.), The nautre of biOlogical diversity. pp. 165-221. McGraw-Hill, New Vork.

Sonneborn, T.M. 1975. Positionel informetion end neerest neighbor interections: 1n reletion to spetial petterns 1n c1lietes. Ann . .6.Utl.. 14: 565-584.

-

--

W1cklow, B .. J. 1966 Developmentel Polymorph1sm Induced by Intrespec1f1c Predetion in the C111eted Protozoen Onychodromus qUlldrlcomulus. J... Protozool.,35: 137-141.

W1rnsberger, E., Foissner, W. & Adem, H. 1965. Morphologicel, Biometric, end Morphogenetic Comperison of Two Closely Releted Species, Slylonychill vorll)( end S. puslulllill (Clliophore: Oxytrichidee). ~ Protozool. 32: 261-266.

- - If) IE IE ..... ..... • • • fi i ff ..,~ ., ., i~ aa <3.

(j)

~ ;:, .., -;:, u --., L. 0 '- - -CD IE IE .......... ~ • • ;:, i i u if s::. ~E .., Nin

~ ., .,

0 ~ ~ L. ;t::;t:: (!) aa :::D eo -., 0

<[

X

'C C CD

If) C. C. -<[

- -IE IE .......... • • i i e..:::::::::: 0

ff -- .. ., ., }j - -~ ~ uu o.

o

-

-

;3000 i zsoo j 2000 ~ 8 1500

~ UXX)

500

Appendix B Morphometric date for culture -3 end culture -4

c..,.. 4t3 letal p.,.1attM Ys ....... Otter--) xZ5 .eulV. ~ Total Pop OMt. Ltngth ~

O+-~~~--~---r--~--~--~--r-~~~--~--~

M

27!50

zsoo 22!50

!5

) 2000

In!50 ~ 1!500 8 1250

S; 1000 (J

7!50

500

10 1!5 20 DIIJ

c..,.. -4 letal,.,.1attea YS L ..... (Hierea) xZ5 .eulV. 84 Total pop OMtan I.tngth x 25

~+-~~~--~---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.

-

II!!

~ ... I s.. .2 1: ..... ---• u

II!! s.. • ... I h -~ ---• u

140

120

100

80

60

40

20

120

100

80

60

40

20

AppendIx B Morphometric data for culture -3 end culture -4

....... He •• s ...... p.,.1d_ f. c.Jt.r. 83 Olfn9th MI.- .Cul ~ Ur9f

....... He •• s ....... p.,. ..... c.Jt.r. -4 .CulVt -4 Ur9f OMlIn Ltngth

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

100 .Width MI_ OLtngth Mlln

90

80

M 70 ) 60 I SO f 40

30

20

10 5 10 15 20

Dav

...... YS. VNtll f. C81tw. -4 c.rr ...... CMfflDt.t-.972; R ~.94:1

100 .Width MI_ OMlIn Ltngth

90

80

M 70 ~

60 .,

I SO i 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; R squared· .823. For culture -4 correlation coefficIent - .972; R squared - .945. These graphs compare with ngure twelve in the text.

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