Estimation of Amoeba Cell Volume From Nuclear Diameter and Its Application to Studies in Protozoan Ecology

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Hydrobiologia 284:

229-234,

1994.

( 1994

Kluwer Academic Pu blishers.

Printed in

Belgium.

229

Estimation of amoeba cell volume

from

nuclear diameter and its

application

to

studies

in

protozoan

ecology

Andrew

Rogerson,

Helen G. Butler &

Jeremy

C.

Thomason

University Marine

Biological

Station

Millport, Isle of Cumbrae, KA28 OEG,

Scotland,

U.K.

Received

20

April

1993; in

revised

form 10 August 1993; accepted 14 September

1993

Key

words.

amoebae, ecology, nucleus,

protozoa,

volume

Abstract

To facilitate the estimation of

cell volume

in

uninucleate, naked amoebae (gymnamoebae) the relation-

ship,

log cell volume (him

3

) =

0.882

+ 3.117log nuclear diameter (pm

3

),

is

presented. This links mean

cell

volume

to

mean nuclear diameter and

provides

a

useful tool

for protozoan

ecologists

interested in

es-

timating the biovolume of

amoebae in

laboratory or field samples.

While

it

is virtually impossible to

measure

rigid axes from which volume can be

calculated in

these

amorphous

cells, it

is relatively easy

to

measure

the

diameter of

the nucleus

in

living or fixed

material.

This relationship

has shown that

most

uninucleate amoebae

surveyed

have

volumes ranging between only 188

/lm

3

and

2860

Pm

3

; this range

reflects the

volumes

of

the majority

of

amoebae

in

the

field.

These

small

volumes

are

unexpected

since

many amoebae

have locomotive forms

greater

than

20 m

in

length

giving

the

impression

that

their

cell

volumes should be

correspondingly

large.

This

is not the case, however,

because most amoebae

are

extremely

flat when viewed in profile. The small

cell

volume

of most amoeba

species

has

ecological

implications when

numerical

data

is

transformed to biovolume

and biomass units.

Introduction

With

the

realization

that

flagellates

and

ciliates

are

important

consumers

of

bacteria

in

aquatic

systems (e.g.

Bloem

etal.,

1989;

Bennett

etal.,

1990),

many

recent studies

have

attempted to

quantify their involvement

in

the

flow of carbon

and nutrients

through a variety of aquatic eco-

systems. Despite this

concentrated research ef-

fort,

one

major

group

of

protists, the amoeboid

protozoa, has

been

almost

entirely

overlooked.

An exception

is some

recent

work

indicating that

naked lobose

amoebae

(gymnamoebae)

consti-

tute

a numerically important

component of the

microbial

community

in marine systems

(Roger-

son, 1991; Rogerson

& Laybourn-Parry,

1992;

Butler,

unpubl. data).

Before this new

data on

amoebae can be used to answer questions

about

the

flow

of

material

through populations

in

the

field, abundances must

first

be transformed to

equivalent

biovolume

units (m

3

)

and biological

conversion constants

employed. Accurate cell

volume determinations are also required when

workers wish to use the

available published

rela-

tionships

for protozoa linking, for

example,

cell

volume to

reproductive rate (Fenchel,

1968; Fin-

lay,

1977;

Baldock et al.,

1980)

and

cell

volume to

respiration rate (Fenchel & Finlay,

1983;

Baldock

et al., 1982).

To date,

no satisfactory method

exists for de-



230

termining the cell volume of naked amoebae. The

usual approach for

determining

cell volume in

other groups of protozoa is to measure definable

cell dimensions, such as maximum length and

width, and

to

compute

volume from the closest

geometric

shape. To take a popular

example,

published volume estimates for the ciliate Tet-

rahymena

have used the assumption that cell

shape approximates

an ellipsoid

or

prolate spher-

oid (Curds

&

Cockburn,

1971; Rogerson,

1981;

Baldock et al., 1982). Unlike the

fixed

cell shape

of

most flagellates

and

ciliates, amoebae have

plastic

cell

morphologies, frequently with a

raised

cell

mass surrounded by flattened

hyaline

zones

and

radiating pseudopodia. This makes

the mea-

surement

of

rigid cell

dimensions

virtually

impos-

sible.

One exception

is

Rogerson

(1991)

who

es-

timated cell volume

from

cell dimensions in four

species

of

amoebae

with

relatively

constant

loco-

motive

shape.

This

is,

however, inappropriate for

the majority of amoebae and

only

yields an

ap-

proximation

of

cell volume. Others have fixed

amoebae in

Lugol's

iodine or Bouin's solution

(Baldock

et al.,

1982), a procedure

which

makes

a small percentage of the population form

spheri-

cal

cells

with

only

minor

shrinkage. However, this

method cannot

be

used for field samples

where

most

living

or

fixed

amoebae

are

overlooked, in

cases

where

amoebae

do

not

round-up in

the fixa-

tive, or

for

species

which cannot

be

cultivated in

the laboratory.

One

relatively

constant parameter that can

be

measured

in living amoebae

by light

microscopy

is the

size

of the interphase nucleus. This

can be

viewed by

phase

contrast

microscopy

or, when

nuclei are small or obscured by cytoplasmic

in-

clusions, after staining with the DNA-specific

fluorochrome, DAPI (Rogerson,

1988).

Since

most

amoebae have a single

spherical nucleus

with

a central

nucleolus,

nuclear diameter can

be

measured with relative

ease.

We have employed

both published and

empirically

derived estimates

of mean nuclear size and mean cell

volume

to

derive

a relationship

which

we

believe

has

appli-

cation for

estimating

cell volume

in uninucleate

amoebae from marine,

freshwater and soil

envi-

ronments.

Methods

Many freshwater and soil amoebae, and a few

marine

species,

form

resistant

cysts which are

spherical

and

therefore amenable

to

volume

de-

terminations.

The published literature

on amoe-

bae was

reviewed

and

mean cyst diameter re-

corded

for species

of

naked,

uninucleate amoebae

from soil,

freshwater or

marine

systems (Baldock

etal., 1980; Page, 1968,1983,1988; Page

&

Si-

emensma, 1991). The complete

list

of

species of

cysts

surveyed,

with

strain

designations

for amoe-

bae

measured

in this study,

are as

follows: Acan-

thamoeba

griffini (CCAP 1501/4),

A.

polyphaga

CCAP 1501/3C),

Astramoeba

torrei,

Cochliopo-

dium

actinophorum,

C.

minus,

Comandonia

opercu-

lata, Dermamoeba

minor, Deuteramoeba myco-

phaga,

Echninamoeba

exudans, E. silvestris,

Filamoeba nolandi,

Hartmannella

cantabrigiensis,

H. vermiforms, Heteramoeba clara, H yalodiscus

actinophorus, Gephyramoeba delicatula, Glaeseria

mira,

Mayorella

cultura, Paraflabellula

reniformis

(Strain G, UMBSM), Paratetramitus ugosus,

Platyamoeba placida,

P. stenopodia,

Protacan-

thamoeba

caledonica, Rhizamoeba australiensis,

Rugipes

placidus,

Rosculus ithacus,

Saccamoeba

stagnicola, Sappinia diploidea, Stachyamoeba lipo-

phora,

Vahlkampfia aberdonica,

V.

avara,

V.

en-

terica,

V.

inornata CCAP 1588/2),

V.

lobospinosa,

V.

ovis,

V. ustiana, Willaertiamagna.

In

all cases

cyst

volume was considered to

be

equivalent

to

the volume of a sphere 7r

r

3

),

al-

though

in some

cases

the mean linear dimension

was first

corrected

for cyst wall

thickness

where

a

cyst had

an

obviously thickened, or secondary

wall.

Such

corrections were

applied

in 23% of

cyst

measurements and

were

based

on measure-

ments taken from published micrographs, or from

living

material when available.

For determinations

of

amoeba troph

volume,

the available literature was surveyed (references

as above). In

addition a

range of clonal cultures

of

amoebae were

isolated

from

field

material, or

were obtained

from the

Culture Collection

of

Algae

and

Protozoa [CCAP,

Windermere, En-

gland]. The list of amoebae used for

troph

volume

determinations,

with

strain designations

for

those



231

measured in this

study, are

as

follows:

Acan-

thamoeba

griffini (CCAP

1501/4), A.polyphaga

(CCAP

1501

/3C),

Amoeba

borokensis

(CCAP

1503/7),

A.

lenigradensis (CCAP 1503/6), A. pro-

teus,

Flabellula

calkinski

(CCAP

1529/5),

F. demetica (Strain C, UMB

SM),

Mayorella cant-

abrigiensis, Paraflabellula

reniformis (Strain

G,

UMBSM), Parvamoeba

rugata

(Strain SA,

UMBSM), Platyamoeba sp. (Strain A,

UMBSM), Polychaos fasciculatum,

Rhizamoeba

saxonica

(CCAP 1570/6), Rhizamoeba

sp.

(Strain

M, UMBSM), Saccamoeba limax, Stereomyxa

sp. (Strain

I,

UMBSM), Triaenamoeba

bulla,

Vahlkampfia

inornata

(CCAP 1588/2), unidenti-

fied valkampfiid (Strain B, UMBSM), Vannella

sp.

(Strain

V,

UMBSM),

Vexillifera

minutissima

(CCAP

1590/3), Vexillifera sp. (Strain

Ve,

UMBSM).

Amoebae were maintained in

the

laboratory

using standard

culture

procedures

(Page, 1983,

1988). The

volumes of these

non

cyst-forming

trophic

amoebae were

estimated by shaking

an

amoeba culture

5 ml)

to

round cells,

before

add-

ing 3 drops of

the

fixative

Lugol's iodine. Linear

measurements

of

cell

diameter

(at least 30

rounded

cells

per

species) were

made

using an

eyepiece graticule at a magnification

of

x

400

or

x 1000.

Only

recently fixed cells that had

com-

pletely rounded in the fixative were measured and

cell shape was assumed to be spherical with 4

exceptions: Lugol's iodine

fixed Amoeba proteus

were compressed to

a known

depth

in a counting

chamber (Rogerson, 1981), A. borokensis

approxi-

mated a prolate

spheroid

(volume

=

7rab

2

,

where

major axes are

a and

b respectively), A.

leni-

gradensis

and Rhizamoeba

saxonica

approximated

a

cylinder (volume

= 7rr

2

, where

r is

the

radius

of the base

and

is the length).

Nuclear sizes

of

amoebae

were

recorded

from

published

works (Page,

1983, 1988; Page &

Si-

emensma,

1991). When

published

nuclear dimen-

sions

were not

available,

these were measured in

amoebae fixed in

Lugol's

iodine or

after

staining

with

the

DNA-specific fluorochrome

DAPI

(4',6-

diamidino-2-phenylindole, Sigma Chem.

Co.,

England)

using the

methods given in

Rogerson

(1988).

The nucleus

of

most

amoebae

is

spherical

(Fig. 2a), but

in

the few species

with an irregularly

shaped nucleus,

for example those slightly

oval

in

form, the mean

length of

the major and

minor

axes was calculated and this

value

was

used as an

approximation

of

nuclear 'diameter'.

In

the

case

of Vahlkampfia

inornata

(CCAP

1588/2), Acanthamoeba

griffini

(CCAP

1510/4),

A. polyphaga

(CCAP

1501

/3C)

and Parafiabellula

reniformis (Strain G) comparisons were made be-

tween cell

diameters

of

cysts

and trophs fixed in

Lugol's iodine.

One

species of

amoeba,

V. inor-

nata,

was rounded in

Lugol's

and prepared for

Scanning Electron Microscopy

SEM)

by post-

fixing in

gluteraldehyde

(2%

v/v)

and

osmium

tetroxide

(0.5 o w/v) for 30

min.

at 4

C. Cells

were

dehydrated

through

an alcohol series

(30-

100% / v/v), critical point

dried,

gold/palladium

sputter-coated and examined in

a

JEOL JSM-

5200 SEM.

Results

and

discussion

The ability of many amoebae

to form spherical

cysts

(Fig. 2b)

allows

the

easy

determination of

cell

volume.

This

is

not

possible

in

the case of

trophic amoebae

which

are highly

irregular in

shape. The

mean

diameters

of

amoeba cysts,

de-

termined empirically

or

recorded from

published

works,

were used to estimate

cell

volumes. These

were

related

to mean

nuclear diameter

after log

transformation to

give

the relationship shown

in

Fig.

1

(line

A:

y=

1.022

+ 2.767x; R

2

= 0.829,

p

<0.05).

Supporting data for this relationship were

ob-

tained by

measuring

the

volumes of rounded

amoebae

fixed in Lugol's

iodine. The

species

of

amoebae

used

are given in Table

1 and

the

data

points

are included

in Fig. 1

(line

B:

y= 0.895 +

3.2

82

x;

R

2

= 0.919, p<0.05). Lugol's

fixation generally yielded some spherical cells (e.g.

Vannella sp., Fig. 2c), but for

any

given

species

it

should be noted that only a small percentage of

cells in a population (around 10%)

formed

spheres suitable

for

measurement. This is a major

reason why the method is impractical

for

deter-

mining the

volumes

of

amoebae

in field

samples.



Table

I. Comparisons between cell diameters of Lugol's fixed

amoeba

trophs

and cysts. Four

species of amoebae

were ex-

amined:

Acanthamoeba

polyphaga (CCAP 1501/3C),

Acan-

thamoeba

griffini

(CCAP 1501/4),

Valhkampfia

inornata

(CCAP 1588/2)

and Parafabellula

reniformis

(Strain G).

Means of

30

measurement determinations

with

standard

error

of

the mean in parenthesis.

Species

Feature measured Cell diameter

(m)

A. polyphaga Cyst

19.28 0.37)

A. polyphaga

Troph 18.94 0.47)

A. griffini

Cyst

14.45(0.17)'

A. griffini

Troph

16.07(0.39)'

V. inornata

Cyst 11.98(0.54)2

V. inornata Troph

16.68(0.26)2

P. reniformis

Cyst

6.27(0.16)

P. reniformis Troph

6.37(0.20)

' Significant

difference

t-test,

p<

.05).

2 Significant

difference

t-test, p 0.05).

0

0

1 2

Log diameter

of

nucleus

Fig.

1. Scatter plot

of log,, cell

volume (,um

3

)

versus log,,

0

nuclear

diameter (jim) for

naked uninucleate amoebae.

Open

squares

represent cysts.

Solid

squares

represent Lugol's fixed

trophs. Regression equations

for A (cyst data),

B (troph data)

and C (total

data) are given

in the text.

This

problem is

further accentuated

by

the

fact

that although amoebae

are

common,

they are

fre-

quently

overlooked

because

they attach to biotic

and abiotic surfaces.

Scatter in

the

data

in Fig. 1

is due to variation

in

both

nuclear diameter

measurements and

vol-

ume

determinations.

In

the

case

of the nuclear

measurements,

although

a previous

study has

shown

that

DAPI-stained

nuclei

and

nuclei

mea-

sured

in vivo are

equivalent (Rogerson,

1988), it

was found

that Lugol's fixed

nuclei

in Vahlkampfia

inornata

and Paraflabellula

eniformis

were signifi-

cantly

larger than

DAPI-stained

nuclei

(using the

fixative

gluteraldehyde).

Clearly

the

method

used

to

determine

nuclear size can

affect

the

accuracy

of

the measurement for

some amoebae

and will

contribute

to variation

in the data.

Similarly while

most amoebae

have spherical

nuclei with a

cen-

tral nucleolus

(Fig. 2a), some

species

have

slightly

elongate

nuclei

which

are

less amenable

to

'diameter' estimation. Others,

such as

P

renifor-

mis

and

Flabellula

citata,

show indistinct

nucleoli

when stained with DAPI

(Rogerson, 1988) sug-

gesting

a

more open nuclear

arrangement

with

a

concommitant

increase

in

nuclear

size/cell

vol-

ume ratio. Unusual nuclear

configurations, like

the parietal

nucleoli in

some

amoebae,

or polyp-

loid nuclei,

would also

have

contributed

to scat-

ter in

the relationship.

Variation in the

data can

also

be

attributed

to

the volume determinations.

For

example,

com-

paring the diameters

of

Lugol's

fixed

trophs and

cysts

(Table 1), the mean diameters

of rounded

cells

ofAcanthamoeba

griffini and

V. inornata

were

significantly

greater (t-test,

p<0.05) than mean

cell diameters

of

cysts

of

the same species. This

was because

many of the

apparently

'spherical'

cells

were

in

fact

slightly

flattened

(SEM,

Fig.

2d)

and,

if

inadvertantly

included in

the calculations,

gave

a higher

mean cell

diameter

for

these species.

This

overestimation

of cell diameter

for some

spe-

cies

of

amoebae

probably accounts

for

the

fact

232

1

8

6

-

4

U

o

2

3



233

Fig. 2. (a)

Light

micrograph of spherical nucleus

(arrow)

and central nucleolus

of

a marine

Vannella sp. (b) Spherical cysts

(arrow)

of

Vahlkampfia

inornata.

(c)

Rounded-cell

of

Vannella sp.

after

fixation

in

Lugol's

iodine.

(d)

Scanning

electron micrograph

of

V.

inornata

cells fixed

in Lugol's.

From this side elevation,

some cells

are clearly

spherical (s); others are slightly

flattened (f).

Scale

bars = 10 pm

throughout.

that

slightly more

of the

troph data

points (63

%,

solid squares,

Fig. 1)

than

the

cyst data points lie

above regression

line C.

On

the other

hand,

be-

cause cysts

have highly

compacted

cytoplasm,

in

which

cell components

are

reduced through the

breakdown

of

organelles

and

storage granules,

cell volumes

based on

cyst diameters alone

could

underestimate

the biomass

of some amoebae;

in

Fig.

1,

65%

of

these data points

are

below

re-

gression

line C.

In

this

study

only

recently en-

cysted

amoebae were measured

to

try

to limit this

potential

reduction

in volume

with time.

Although

the

troph regression

line B

appears

to

have a

steeper

slope

than the cyst line

A, these

slopes

are

not significantly

different

(ANCOVA, p<0.05)

and the entire data

set

gives

the

best relationship

between mean

cell

volume

and mean nuclear

size.

This regression

line (Fig. 1,

ine C)

allows

amoeba

log mean cell volume (Pm

3

)

to be

predicted

from

y=0.882+3.117x;

R

2

=0.879,

p<0.05, where

x =

log

mean nuclear

size.

It should

also be noted

that the

generalised

isometric

relationship (y =

x

3

found

in this study implies

that

the

size of the

amoeba nucleus determines

the size

of the cell.

The isometric

equation

presented here

y =

0.882 +

3.117x) is

a

useful

tool for

microbial

ecologists

and the

ability to

estimate

cell

volumes

from

the diameters

of

nuclei

will

be of particular

interest to

protozoologists working

with

gym-

namoebae. The range

of nuclear sizes

surveyed

ranged from

1.2 pm for

Parvamoeba

rugata,

the

smallest marine

amoeba

described with

a

mean

locomotive

length

of

only

3.9

m (Rogerson,

1993),

to a

nuclear dimension

of

40 pm for

A.

pro-

teus. From the

relationship

presented, this

nuclear

size

range

implies

a

range

of volumes

extending

from

13.4 Pm

3

to

754099 pm

3

. However

it

is clear

from

Fig.

1 that the majority

of naked

amoebae

(70%

of the

data set) have

mean

nuclear diam-

eters

within

a much tighter

range,

i.e. between

2.8

and

6.7

pm (overall

mean

4.5 +

1.3

pm; n

=

41),

implying

volumes between

188 m

3

and 2860

pm

3

(mean

828

pm

3

).

In

our

experience

the vast ma-

jority

of

amoebae isolated

from

field

samples

have

nuclei within

this

narrower

range, implying

that

cell volumes

around

this mean

better

represent

the majority

of

common gymnamoebae; large

amoebae within

the

size range of A.

proteus are

seldom

encountered

in

field

material.

The narrow

volume range

for most common amoebae

is

sur-

prising

given that

many cultured forms

appear

large,

particularly

those

cells with

a

broad ante-

rior

hyaline

zone. For

example

two species of

amoebae

studied

here,

Rhizamoeba

saxonica

and



234

Paraflabellula

reniformis,

had mean

locomotive

form lengths of 26.5 m and

20.5 /im respectively

yet their computed volumes

were only

668

m

and

189um

3

.

Ciliate or flagellate protozoa of

comparable

lengths

would have

volumes

of

sev-

eral

thousand

t

3

. It

is

this extreme flattening of

cells, producing

small

biovolumes, that

enables

amoebae

to inhabit small interstitial spaces

in

sediments. Not

surprisingly, the

most

conspicu-

ous

protozoa in benthic sediments

are naked

amoeba and heterotrophic

flagellates (Butler, un-

publ.data).

The

general applicability of the relationship

presented

here to other free-living protozoa (cili-

ates

and

flagellates) has not

been fully examined,

since

their cell volumes

can

be

determined

from

cell dimension determinations.

However

it is in-

teresting to note

that the

mean nuclear diameter

of

the ciliate

Tetrahymena pyriformis

is 13.0

A.m

(this study), implying

a

cell volume

of 20 037

m

from regression

equation C

(Fig. 1). This

is simi-

lar

to

the

published

value of 21500 /m

based on

cell parameter measurements (Baldock

et al.,

1982). If application

of the

relationship

to

other

protozoa is justified, then the

common practice of

counting

protozoa

by

fluorescence

microscopy

in

conjunction with DNA-fluorochromes

could

yield

additional information on the

total

biovolume of

heterotrophic protozoa

in a sample.

Acknowledgements

This

work

was partly funded

by an award to

A.R.

from the Central Research Fund (University

of

London) and a

NERC studentship

to

H.G.B.

We

thank

Mr

P. Wilson for technical assistance.

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Documents

Estimation of Amoeba Cell Volume From Nuclear Diameter and Its Application to Studies in Protozoan Ecology