t
/ . Embryol. exp. Morph. Vol. 30, l,pp. 1-19, 1973
Printed in Great Britain
h
Size determination in Hydra: The roles
of growth and budding
K
By JOHN W. BISBEE 1
^
From the Department of Biology, University of Pittsburgh
r
P
SUMMARY
\~
^
Hydra pseudoligactis cultured at 9 °C for 3-4 weeks are one-and-a-half times larger t h a n
those cultured at 18 ° C . T h e size of Hydra is correlated with t h e n u m b e r s of epitheliomuscular a n d digestive cells in t h e distal portion of t h e animal a n d with the diameters of t h e
epithelio-muscular cells in t h e peduncle.
C o u n t s of mitotic figures and tritiated-thymidine-labeled nuclei and determinations of
increase in mass of Hydra populations suggest that the difference caused by these temperatures does not affect mitosis. A t 9 °C b u d s a r e initiated at a lower rate a n d take longer t o
develop t h a n at 18 °C. T h e surface-areas of buds raised at t h e t w o temperatures a r e similar.
Because Hydra raised at the two temperatures have similar growth dynamics, the differences
in sizes of t h e animals cannot be d u e t o growth rate. T h e observed effect of temperature o n
b u d initiation a n d development is probably relevant t o the increased size of animals raised a t
9 "C, since these larger animals m a y be accumulating m o r e cells while losing fewer to b u d s .
k[
T
y.
T
u
INTRODUCTION
The shape and size of Hydra seems to be a consequence of several dynamic
processes. Growth, budding, cell sloughing, cell migration, and mesogleal
metabolism have possible roles in Hydra morphogenesis (Burnett, 1961, 1966;
Burnett & Hausman, 1969; Brien & Reniers-Decoen, 1949; Campbell, 1965,
1967 a, b, c, 1968; Shostak, Patel & Burnett, 1965; Shostak & Globus, 1966;
Shostak, 1968). This paper deal with the roles of growth and budding in determining the dimensions of Hydra pseudoligactis.
Hydra is essentially a cylinder made up of two cell layers, the epidermis and
the gastrodermis, with an acellular mesoglea between them. The cylinder has a
ring of tentacles and a mouth at its distal end, and an adhesive 'foot' at its
proximal end. The animal is a cellular system with 'input' by cell division, and
'output' primarily by budding; Campbell (1965) and Shostak (1968) have
estimated that 60-85 % of cell loss occurs in buds. Additional cell loss occurs via
cell sloughing at both ends of the animal, and possibly along its length.
Both Stiven (1965) and Park & Ortmeyer (1972) observed that lowering the
ambient temperature increased the size of Hydra. This paper confirms Stiven's
observation for Hydra pseudoligactis, and asks the following questions: Is the
1
Author's address: Department of Biology, Mundelein College, Chicago, 111. 60660, U.S.A.
I
E M B 30
J. W. BISBEE
change in size due to differential increase in one body region or is it uniform
throughout the body column ? Is it due to differential cell size or rate of cell
division ? Is'the change in Hydra size due to differential cell loss ? The approach
taken was to measure body column dimensions and cell numbers and dimensions
on serial cross-sections. Since Hydra size was correlated with cell number, an
effort to understand the mechanism through which temperature alters cell
numbers was made, by determinations of increase in mass of Hydra populations
and counts of mitotic figures and tritiated-thymidine-labeled nuclei. Finally,
budding, as the main form of cell loss, was studied.
MATERIALS AND METHODS
A. Culture methods
A clone was identified by species as Hydra pseudoligactis, according to
Forrest's (1963) key. The tentacles on buds arose successively in a fixed pattern
as pictured infig.10 of Forrest (1963); adults had tentacles approximately three
times column length. The holotrichous isorhizas were narrowly oval with transverse coils. As in Hyman's (1931) original description of Hydra pseudoligactis,
an individual's body column was differentiated into stalk and body (Fig. 1).
Also some animals raised at 9 °C in the fall were observed to be sexual, having
rather stout testes with nipples.
Stocks of animals were maintained in Pyrex-brand baking dishes kept in
incubators at 9 ± 1 and 18 ± 1°C. They were fed to repletion three times a
week on freshly hatched Artemia sp. nauplii at room temperature. The culture
solution (Shostak et al. 1965) was poured off daily (after feeding if they were
fed) and replaced with fresh solution already at the appropriate temperature.
Animals were transferred to clean dishes every 4-10 days, with the density kept
below one Hydra per 0-5 ml of solution.
The Hydra used in experiments, drawn from stocks, were raised in finger
bowls. Animals raised for dry-weight determinations and those to be injected
with tritiated thymidine were maintained at a density of one Hydra per 10 ml
of culture solution; all other experimental animals were raised at a density of
one per 20 ml of solution. All groups were incubated for 3 or 4 weeks at the
appropriate temperature and starved 48 h before use. With one exception (a
Hydra raised at 9 °C and used for cell and size determinations), all the Hydra
used were asexual budding animals.
B. Histology
Hydra were placed in 50 x 15 mm Petri dishes with 2-3 ml of approximately
25 °C culture solution at 16.00 h, 52 h after feeding. Within 15 min, having
extended to the approximate proportions of Fig. 1, they were quickly flooded
with hot Bouin's fluid (Pearse, 1960), which prevented them from contracting.
After fixation of all animals for 16-18 h the picric acid was removed by placing
j
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^
A
^
A
i
A
Size determination in Hydra
•*
||
r
3
the animals in LiCO3 in 70 % alcohol. They were dehydrated in an alcohol
series, cleared in xylol and embedded in 56 °C paraplast. Serial sections 10 /im.
thick were cut perpendicular to the long axis of the Hydra. Slides were deparaffinized, hydrated, stained in toluidine blue, dehydrated, and mounted in
permount.
The number of epidermal epithelio-muscular cells and gastrodermal digestive
cells in each section were estimated from counts of their nuclei. These cells were
identified as having cytoplasm extending from the mesogleal surface of the
respective epithelium to the surface of the layer, and as having nuclei with
prominent nucleoli. A cell was considered to be undergoing mitosis if part of
the mitotic figure was visible on the section. Mitotic counts of all cells (as listed
in Campbell, 1967a) were made.
C. Autoradiography
Tritiated thymidine, 0-5 /i\ (Schwartz Bioresearch, Inc., 6-0 Ci/mmole, 1-0
mCi/ml), was injected through the mouth into the Hydra enteron (Campbell,
1965). The animals were returned to culture solution at the appropriate incubation temperature and fixed 6 h later. Serial cross-sections on slides were
dipped in Kodak NTB-3 nuclear track emulsion, stored in the freezer for 5 days,
and developed in D-19. After being stained with toluidine blue, the slides were
mounted and examined at x 200. A nucleus was considered labeled if one half
or more of it was uniformly blackened by silver grains.
D. Mass determinations
The dry weight of Hydra was determined on groups of animals that had been
starved for 48 h. The animals were lyophilized and weighed on an Oertling R20
analytical balance, which can be read to 0-1 mg.
E. Observations of buds
Data on budding were collected by observation often adults at room temperature with a dissecting microscope at x 12, noting the number of buds attached
and detached daily. Newly detached buds were discarded.
RESULTS
A. Size of Hydra
Observations of Hydra cultured at 18 and 9 °C (Figs. 1, 2) show that animals
raised at the lower temperature were larger than those at the higher temperature.
The lengths and diameters of the animals were measured and the circumferences
calculated. The average dimensions at each temperature, their standard deviations, and the locations of the measurements on the body column (axial position)
are shown in Table 1 and Fig. 4.
J. W. BISBEE
Fig. 1. Representative Hydra pseudoligactis raised at 18 °C for 3 weeks, x 10.
Fig. 2. Representative Hydra pseudoligactis raised at 9 °C for 3 weeks, x 10.
Fig. 3. Photomicrograph of a portion of a Hydra pseudoligactis cross-section in the
gastric region. Epidermis is the cell layer on the right; gastrodermis, on the left.
E, Epithelio-muscular cell; /, interstitial cell; D, digestive cell; g, gland cell, x 825.
K
Size determination
%
in Hydra
5
Table 1. Linear dimensions of the Hydra body column^
Region and its [position]:}: on the body column
h
y
L
T
Y
|
Y
f
I
•"
Temperature
Peduncle [1-3]
Budding [4-5]
Gastric [6-10]
Total
9 C
18 X
89(17)
58 (5)
30(14)
21(19)
135(46)
84(16)
253(47)
162(17)
t Averages and (standard deviations) of the number of cross-sections, based on four
animals at each temperatuie.
% Numbers refer to positions on Hydra body column of Fig. 4.
Table 2. Analysis of variance for the body column circumferences at the mesoglea
accumulated for corresponding axial positions at both temperatures
Source of
variation
Treatments
Temperature
Axial position
Temperature v. axial position
Error
Total
Degrees of
freedom
Sum of
squares
Mean
squares
F
19
1
9
9
80
99
38-26
010
37-92
0-24
4-28
42-54
—
010
4-21
003
005
—
—
186
84-20*
0-50
—
—
* Probability less than 1 % that the difference is due to random error.
1. Length
The total lengths and the lengths of regions of Hydra raised at the two
temperatures were determined by counting the serial cross-sections 10 /im thick.
Three regions could be distinguished histologically by the following criteria: the
budding region is the length of the body column that has buds attached; the
gastric region is the body column between the budding region and the tentacles;
and the peduncle is the remainder of the body column. The average lengths of
four Hydra cultured at each temperature are presented in Table 1. Each region
and the total length of the 9 °C Hydra is approximately one-and-a-half times
longer than the corresponding portion in the Hydra raised at 18 °C (peduncle,
89/58 = 1-5; budding region, 30/21 = 1-4; gastric region, 135/84 = 1-6; total,
253/162 = 1-6).
2. Circumference
The circumferences of the Hydra raised at the two temperatures were similar
at representative positions along their length. These circumferences of the body
column at the mesoglea were calculated from measurements made on ten
histological sections at the ten axial positions. Compression during sectioning
caused the sections to be elliptical; the lengths of the major and minor axes were
determined with an ocular micrometer at x 126. These circumferences at each
J. W. BISBEE
9°C
0-76
(007)
0-78
(008)
~»-
< -
-,,
2
1
3
0-68
(008)
0-70
(Oil)
0-80
(0-17)
(0-50)
2-11
2-12
(0-23) (0-29)
.
—-
201
(0-29)
•
•
1-85
(0-25)
• — -
«-
.
4
6
5
1-25
(0-22)
9
8
7
10
.
*—
^
18-
2-12
1-86
(0-38)
116
(019)
0-76
(006)
"
•
1-88
(016)
2-17
(0-25)
2-33
(018)
2-34
(0-25)
215
(0-29)
2-10
(0-40)
Fig. 4. Calculated circumferences of the Hydra pseudoligactis body column at the
mesoglea in mm. Averages and (standard deviations) at the ten axial positions
shown on the diagram are based on five animals at each temperature.
Table 3. Dry weight of adult Hydra
Temperature
...
Sample ...
Number of adults
Average /tg/adult with buds
Average late budsf
attached/adult
Estimated fig of attached late budsj
Corrected /tg/adult
Average fig of
adult/temperature
18 °C
9°C
1
r
II
ir
98
265
99
270
97
131
96
163
2-8
63
202
2-9
65
205
1-4
23
108
1-2
20
143
i
204
j
126
f Late buds = stages 4-6 of Shostak, Bisbee, Ashkin & Tammariello (1968).
% Stiven (1965) estimated that day-old buds at his cooler temperature (15 °C) weigh 22-5 fig,
and at his warmer temperature (25 °C), 16-7 fig.
axial position of Hydra cultured at the two temperatures did not differ significantly when tested with the F statistic (Table 2). The averages and standard
deviations for each temperature are presented in Fig. 4.
3. Mass of Hydra
The dry weights of adult Hydra were determined by lyophilizing duplicate
cultures of approximately 100 adults previously raised for 18 days at either 18
or 9 °C. The measurements were corrected for the weight of attached buds as
follows: the numbers of late-stage buds attached (stages 4-6 of Shostak, Bisbee,
Ashkin & Tammariello, 1968) were estimated from observations of ten Hydra
in each culture the day the adults were lyophilized; these buds were considered
a day or more old; Stiven's (1965) estimates of the dry weights of day-old buds,
multiplied by the number present were subtracted from the adult dry weights.
These data are presented in Table 3. Adults incubated at 9 °C averaged 204fig
and those at 18 °C, 126/*g; this is a 62 % increase in mass, which is significant at
the 5% level.
Size determination
in Hydra
Table 4. Cells per unit distance and cell diameters
in longitudinal and cross-sections of Hydraf
t
Y
K
Longitudinal
sections
Epithelio-muscular cells
Digestive cells
Cells/mm
Cell diameter (/tm)
Cells/mm
Cell diameter Qim)
28 (5)
36 (6)
51 (7)
20 (3)
Crosssections
35 (9)
31 (10)
60 (10)
17 (3)
f Averages and (standard deviations) for thiee of each kind of section in the gastric region.
L
B. Hydra cell number and size
Cell numbers and cell sizes were determined to learn whether the larger Hydra
cultured at 9°C have more cells, larger cells, or both.
First, the dimensions of the epithelio-muscular cells and the digestive cells
were compared along each of their longitudinal and circumferential axes. If
these cells were cylindrically shaped, cell diameters could be calculated from
either longitudinal or cross-sections. The numbers of epithelial cells per unit of
length and per unit of circumference in each of three cross and longitudinal
sections in the gastric region were calculated. Their reciprocals (distance at the
mesoglea divided by the number of nuclei counted over that distance) are cell
diameters. These data are presented in Table 4 as means and standard deviations.
The numbers of epithelio-muscular cells per mm (and, of course, their reciprocals, epithelio-muscular cell diameters) in the two types of sections were not
significantly different, nor were the number of digestive cells per mm (and their
reciprocals, digestive cell diameters).
The epithelio-muscular cells are considered to be contiguous, as are the digestive cells, as illustrated in Fig. 3 (except that in the epidermis, interstitial cells
may be inserted between the epithelio-muscular cells at their basal ends, while
in the gastrodermis, gland and mucous cells intervene occasionally). Thus the
epithelio-muscular cells and the digestive cells are essentially equi-dimensional
along the longitudinal and circumferential axes and can be considered as
roughtly compact cylinders contiguous with each other.
In what follows, the numbers and diameters of epithelial cells in the two layers
are calculated from data from cross-sections.
1. Cell number
The numbers of epidermal epithelio-muscular cells and gastrodermal digestive
cells in each of ten cross-sections at the axial positions were counted. These
numbers of cells per axial positions multiplied by the numbers of sections in
each tenth of the Hydra body column gave the numbers of cells per axial tenth.
The averages and ranges of the estimated cell numbers per axial tenth at each
J. W. BISBEE
• 9
o 18
i
c
c
I
41
S 3
<1
*
* *
'r
1—OH
I-O—1
i
*
10
Fig. 5. Epithelio-muscular cell number in Hydra pseudoligactis. Means and ranges
of estimated cell numbers per axial tenth in five animals at each temperature are
presented. • Probability less than 5% that the difference is due to random error;
• * probability less than 1 %.
temperature are presented graphically in Figs. 5 and 6, where the cell numbers
are plotted as a function of the axial tenths. These estimates of cell numbers
were compared statistically, considering the estimates for each tenth from all
the Hydra as a block. The first question asked was, 'Is there an interaction
between the numbers of cells per axial tenth and temperature ?' The 'interaction *
term calculated here represents the differences between the means for temperatures with different axial tenths and differences between means for axial tenths
with different temperatures. Since this term is significant for both cell types, all
further comparisons had to be performed either for different axial tenths with
only one temperature or different temperatures at the same axial tenth.
Comparisons of the estimated cell numbers for the two temperatures at each
axial tenth show that in the upper budding region and gastric region, the
estimated cell numbers per axial tenth differ significantly, with more in the
animals raised at 9 °C. That is, as shown in the abscissa of Fig. 5, the Hydra
raised at 9 °C have significantly more epithelio-muscular cells in the upper
budding region and lower gastric region (axial tenths 5, 6, and 7) than the Hydra
cultured at 18 °C; there are significantly more digestive cells (Fig. 6) in the upper
Size determination in Hydra
9 C
18 C
i
•*
5
6
7
*
8
* *
9
*
10
Axial tenths
Fig. 6. Digestive cell number in Hydra pseudoligactis. Means and ranges of estimated
cell numbers per axial tenth in five animals at each temperature are presented.
• Probability less than 5% that the difference is due to random error ; • • probability
less than 1 %.
budding region (axial tenth 5) and upper gastric region (axial tenths 8,9, and 10)
of Hydra cultured at 9 °C than those raised at 18 °C. The gastric region is approximately the distal one-half of the Hydra, between the tentacles and the stalk of
the peduncle. The budding region is the proximal one-fourth of the gastric
region, distinguished functionally by the presence of buds. Thus the Hydra
raised at 9°C have more cells in the distal portion of the animal.
2. Cell size
Cell size can be expressed as cell diameter. The epithelial cell diameters at the
mesoglea for each cross-section were calculated by dividing the circumferences
in mm at the mesoglea by the number of nuclei counted in that section. The
averages and ranges of the cell diameters at each axial position of Hydra raised
at the two temperatures are presented in Fig. 7. Comparisons of the cell diameters accumulated for corresponding axial positions at both temperatures
showed that the interaction term was significant. Therefore comparisons of the
cell diameters for the two temperatures are required at each axial position. There
were significant differences only in the epithelio-muscular cell diameters of the
10
J. W. BISBEE
40
20
0
T
If
B
• 9 C
o 18 C
3 .120
<u
I 100
I
80
°
60
40
20
0
4
5
6
Axial position
7
10
Fig. 7. Epithelio-muscular cell diameter (A) and digestive cell diameter (B) in Hydra
pseudoligactis. Means and ranges of cell diameters per axial position in five animals
at each temperature are presented.
peduncle (positions 1-3, Fig. 7 A). These on Hydra cultured at 9°C were significantly larger than the cells in the corresponding axial positions of the 18 °C
Hydra. The diameters of the digestive cells at the axial positions (Fig. 7B) were
similar throughout the body columns of the Hydra raised at the two temperatures.
Also, the diameters of the epithelio-muscular cells (Fig. 7 A) in the budding and
gastric regions (positions 4-10) of the Hydra cultured at the two temperatures
were similar.
Thus, the increased size of the Hydra raised at 9 °C is correlated with increased
number of epithelio-muscular and digestive cells in the distal portion of the body
column, and increased size of the epithelio-muscular cells in the proximal
portion.
C. Cell division
Three ways of estimating growth in Hydra were used: (1) the number of
mitotic figures was counted in histological sections: (2) the number of tritiumlabeled nuclei was counted in autoradiographs of sections of Hydra injected
with tritiated thymidine; and (3) the increase in mass of the Hydra populations
was determined.
^
Size determination in Hydra
11
30
• 9 C
o 18 C
20
10
H
II
3
4
5
6
Axial position
7
10
Fig. 8. Mitotic figures in the epidermis of Hydra pseudoligactis. Means and ranges
of mitotic figures per axial position in three animals at each temperature are
presented.
r
t
1. Mitotic figures
The numbers of mitotic figures in the epidermis and gastrodermis were
counted in a section at each of the ten axial positions on three Hydra at each
temperature. The means and ranges for the epidermis are presented graphically
in Fig. 8. The numbers of mitotic figures per axial position in the epidermis of
Hydra raised at the two temperatures were compared statistically. The differences were not significant (Table 5).
The numbers of mitotic figures in the gastrodermis averaged 0-21 per axial
position, with a range of 0-3.
2. Tritiated-thymidine-labeled nuclei
Tritiated thymidine was injected through the mouth into the Hydra enteron;
the animals were returned to culture medium at the appropriate incubation
temperature and fixed 6 h later. The tritiated-thymidine-labeled nuclei were
counted in cross-section autoradiographs at the ten axial positions of five Hydra
raised at each of the temperatures. The means of labeled nuclei and their standard deviations per axial position in the epidermal and gastrodermal layers are
presented in Fig. 9.
The numbers of labeled nuclei per axial position in the epidermis of Hydra
incubated at the two temperatures differed significantly only in axial position 5,
12
J. W. BISBEE
Table 5. Analysis of variance for the mitotic figures in the epidermis accumulated
for corresponding axial positions at both temperatures
Source of
variation
s
Degrees of
freedom
Sum of
squares
Mean
squares
19
1
9
9
40
1344
1
1070
273
1015
2359
1
119
30
25
004
4-76*
1-20
—
__
Treatments
Temperature
Axial position
Temperature v. axial position
Error
Total
59
F
J
* Probability less than 1 % that thedifference: is due to random error.
Temperature
9 LC
18 C
i
(1)
°
2
(2)
18
(19)
53
(33)
70
(33)
60
(28)
54
(33)
42
(25)
30
(23)
21
(22)
(0
(2)
21
.(30)
24
(29)
22
(24)
44
(20)
40
(18)
37
(18)
29
(15)
25
(13)
-——
5
4
L
N-*
-
•
6
7
•
J
Number of labeled nuclei in the epidermis per axial position
3
V
8.
1
A
_
9
H
______
-^^.
4
c
IS C
18
c
o
i
(i)
(i)
°
°
(1)
(1)
4
(5)
7
(7)
12
(9)
9
(5)
9
(4)
3
(2)
5
(6)
4
(3)
3
(4)
(3)
5
(8)
9
(6)
8
(9)
7
(4)
7
(3)
5
(1)
1
1
1
j
Number of labeled nuclei in the gastrodermis per axial position
Fig. 9. Numbers of 3H-thymidine-labeled nuclei in the epidermis and gastrodermis
of Hydrapseudoligactis. Averages and (standard deviations) at the ten axial positions
shown on the diagram are based on five animals at each temperature.
where the animals raised at 9 °C had more. In terms of the total of the gastrodermal labeled nuclei for both temperatures at each axial position, the numbers
for the two temperatures did not differ significantly at any axial position.
3. Change in mass of Hydra populations
Three groups often newly detached buds at each of the two temperatures were
used to initiate six populations of Hydra. All parents and detached buds were
retained at a constant density (one individual per 10 ml of culture medium) for
1
Size determination in Hydra
13
Table 6. Population mass and number of Hydra raised at 9 and 18 °C
i
0
V
Populations
Temperature
18°C
9°C
Dry weight (mg)
Number
Dry weight (mg)
Number
i
8-9
65
80
33
8-3
92
6-2
26
9-2
91
7-4
30
8-8
83
7-2
30
Table 7. Analysis of variance of population masses
Source of
variation
Temperature
Error
y
Average
Determination
Total
Degrees of
freedom
Sum of
squares
Mean
square
1
4
3-8
2-1
3-8
0-5
5
5-9
F
7-60f
t For these degrees of freedom, F at the 5 % level = 7-71.
*"
Y
f
f
\
3 weeks, after which the total population mass (dry weight) was determined for
each of the six groups. These data are presented in Table 6. The numbers of
individual Hydra per population were highly significantly different, averaging
83 animals at 18 °C and 30 animals at 9 °C. The total population masses, which
averaged 18 % greater at 18 °C than at 9 °C, were not significantly different
(Table 7). Thus the increase in mass at the temperatures was similar but distributed differently.
D. Bud initiation, development and size
Three aspects of Hydra budding were studied: (1) rate of bud initiation, (2)
duration of bud development, and (3) size of newly detached buds. Two groups
of Hydra were raised at each temperature; one group was fed once a day, and
the other every other day.
The rate of bud initiation is the average number of new buds appearing on a
parent per day; the number of buds initiated on day n is determined by subtracting the number of buds attached on day n— 1 from the sum of the attached
and freshly detached buds on day n. The duration of bud development is the
length of time between bud initiation and establishment of the bud as an
independent individual.
Table 8 presents the data on bud initiation. Hydra cultured at 9 °C initiate
significantly fewer buds than animals raised at 18 °C. Feeding schedule, as
well as temperature, altered the rate of bud initiation significantly (Table 9).
The Hydra at 9 °C averaged one-third as many bud initiations per day as the
18 °C animals when they were fed once a day (twice per 48 h). When the parents
were fed once per 48 h, about one-sixth as many buds were initiated at 9 °C as
14
J. W. BISBEE
Table 8. Budding in Hydra pseudoligactisf
Temperature
No. of
feedings/48 h
9°C
18°C
2
1
2
1
No. of buds
initiated/dayj
Duration of
bud development
(days)
Surface area
of newly
detached
buds (mm2)§
0-60
0-20
1-78
1-28
15||
16«I
4|l
5';
2-8
—
2-6
t Averages for variables.
% Observations made for the first 6 days after Hydra had been fed three times on appropriate schedule.
§ Averages for four buds at each temperature; average for 14 additional buds at 18°C =
2-7 mm 2 .
|| Parents observed for 7 days.
if Parents observed for 14 days.
——
Table 9. Analysis of variance for initiated buds accumulated
for corresponding feedings at both temperatures
Source of variation
Treatment
Temperature
Feeding
Temperature v. feeding
Error
Total
Degrees of
freedom
Sum of
squares
Mean
square
F
3
1
1
1
20
23
893
770
121
2
264
1157
770
121
2
13
.
59-23*
9-31*
015
.
Probability less than 1 % that the difference is due to random error.
at 18 °C. However, this difference (one-third v. one-sixth as many buds initiated
- the interaction between temperature and feeding) was not significant (Table 9).
The durations of bud development and the surface areas of newly detached
buds are also presented in Table 8. Buds at 9°C took approximately three times
as long to develop and detach as buds at 18 °C. The feeding schedule did not
seem to alter this variable.
The surface areas of newly detached buds were calculated from measurements
of length and width made on 2 x 2 transparencies. The surface areas of these
buds at the two temperatures (Table 8) were not significantly different. Thus
Hydra raised at 9 °C initiated fewer buds than Hydra cultured at 18 °C; these
buds took longer to develop, but were similar in size to buds on animals raised
atl8°C.
*
*
Size determination in Hydra
*
DISCUSSION
9
*
*
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k
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^
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i
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15
A. Size and temperature
Hydra are well known for their spontaneous movements of the body column
and tentacles, and the contraction of these body parts when the surrounding
water is agitated. These behavioral characteristics make precise determinations of
Hydra body column dimensions difficult. Certainly dry-weight determinations
are an acceptable means of expressing Hydra size. Measurements from photographs have been used to ascertain the dimensions of Hydra (e.g. Shostak,
1968). My determinations of Hydra body column dimensions are based on
measurements of fixed animals (Webster & Hamilton (1972), for example, use
the same methods as mine for Hydra length). The Hydra at the two temperatures
were handled identically to minimize histological artifacts. Thus the data
collected are meant to be relative, not absolute.
The dry weights of adult Hydra raised at the two temperatures were corrected
for the variable presence of buds. Hydra incubated at 9 °C had an average dry
weight 1-6 times greater (62 % greater) than those at 18 °C (Table 3). Similarly,
the mean total length of the Hydra body column on animals raised at 9 °C
(determined by counting histological cross-sections) was 1-6 times greater than
Hydra raised at 18 °C ('Total' column, Table 1). On the other hand, Hydra
raised at the lower temperature did not differ in representative circumferences
along their length from Hydra raised at the warmer temperature. Thus, temperature seems to have affected the general size by influencing length rather than the
overall shape or form of Hydra.
Stiven (1965) has considered the relationship of size to temperature in three
species of Hydra: Hydra pseudoligactis, Chlorohydra viridissima and Hydra
littoralis. He expressed size in terms of calories per 1-day-old bud. The mass of
buds rather than of adults was measured, since the presence of different numbers
of buds on adults would have led to considerable variation in mass measurements. He assumed that bud size was proportional to the relative sizes of the
species, and concluded that a lowering of the temperature from 25 to 15 °C
increased the mass of three species. For H. pseudoligactis, Stiven observed a 39,
33 and 35 % increase, respectively, of calories, protein and dry weight, when
lowering the culture temperature from 25 to 15 °C.
Also, Park & Ortmeyer (1972) have reported on the size of Hydra littoralis
adults and newly detached buds raised at several different temperatures. Adults
raised at 10 ° C have a dry weight 66 % greater than those at 21 °C, while buds at
this lower temperature were 113 % larger than buds at the higher temperature.
Thus my observations on the size of Hydra at two temperatures add another
example to the literature implicating temperature among normal size-regulating
mechanisms of organisms. It is well known that some animals (notably homeotherms) are larger at the more polar extremes of their ranges (Ray, 1960). The
applicability of this generality to poikilothermic animals is less clear cut (Ray,
16
J. W. BISBEE
I960; Vernberg, 1962). Ray found that many poikilotherms are larger when
grown at a lower temperature. He surveyed 36 species and found that 27 of
them were larger when they were cultured or lived at lower compared to higher
temperatures. On the other hand, Rensch (1959) suggests that poikilotherm's
body size will decrease towards colder climates. He describes examples that both
support and refute his generalization.
Representatives of homeotherms may be larger in colder climates because
maintenance energy per unit of body weight is usually smaller in a large animal
than in a small one. This difference in metabolic rate can be explained by the fact
that surface-area in relation to body weight decreases with increasing size of the
animal (Davson, 1964). Ray notes the arguments of some studies that an
attempt to generalize about poikilotherm body size and temperature is meaningless. Their suggested explanation for this, that these animals produce no significant metabolic heat, is not entirely true. Certainly Hydra have a negligible
temperature difference from their environment. Because any heat produced is
rapidly conducted to the environment, they stay at ambient temperature. Therefore, ascribing some adaptive significance to observations that Hydra are larger
when raised at a lower temperature, does not seem to be too pertinent.
B. Size of Hydra
Several possibilities exist to explain the changed size of Hydra raised at
different temperatures. The possibility of an overall increase in cell size has been
ruled out. One explanation could involve cell division, which is the only 'input'
that can explain the increase in cell number in the Hydra system. Another might
involve 'output' in the form of cell loss during budding. Also Stiven (1965) has
suggested that the decrease in size of Hydra species between 15 and 25 °C is
'very likely explained by the corresponding decrease in food intake' he observed.
Any of these possibilities or some combination might be the correct explanation.
1. Cell number and growth
The increased size of Hydra raised at 9 °C is correlated with increased number
of epithelio-muscular and digestive cells. Only in the peduncle of 9 °C Hydra do
the epithelio-muscular cells of the epidermis differ in diameter. The increased
size of these cells in the peduncle is correlated with the increased size of this
region, since the numbers of epithelial cells in the peduncle are indistinguishable
statistically at the two temperatures. But generally larger Hydra have more cells.
An increased rate of cell division does not seem to play a role in producing
these larger Hydra, since in Hydra raised at 9 and 18 °C the numbers of mitotic
figures per axial position in the epidermis, and of 3H-thymidine-labeled nuclei
in both cell layers were indistinguishable statistically.
The number of dividing cells observed at any one time is assumed to be a
function of the duration of mitotis and of the length of the cell cycle. Since the
number of dividing cells at the two temperatures did not differ, one would
Size determination in Hydra
17
ordinarily assume that temperature, within the limits tested, does not affect
either of these variables.
To test this conclusion, namely that growth at the two temperatures is similar,
the change in total mass of Hydra populations was determined. Although the
average individual mass was 62 % less for the 18 °C Hydra than for the 9 °C
Hydra (Table 3), the total population mass of the 18°C Hydra was 18 % greater
at the end of the incubation period (Table 6). For this reason, and since this
18 % difference in population masses was not significant, differences in growth
rate can not play a major role in determining Hydra size.
On the other hand, Brien and Burnett, working with constant temperature,
argue that growth does have a role in Hydra morphogenesis. Also, Berrill (1961)
has partly interpreted the structure and polymorphic variations of colonial
hydroids as results of ordered cell division. According to Brien's view (see Brien
& Reniers-Decoen, 1949) Hydra's epithelial cells are produced in a subhypostomal growth zone, which they gradually leave to differentiate and finally slough
off at either the foot or at the tips of the tentacles. The length of the animals*
body and of the tentacles are thus thought to depend on the equilibrium reached
between growth and sloughing. Burnett (1961, 1962, 1966) and Burnett and
Garofalo (1960) have supported this view; indeed, Burnett (1966) has proposed
a model of growth and cell differentiation in Hydra that elevates growth to the
essential element in development of form. Burnett also considers the growth
pattern to be intimately related to the maintenance of form.
2. Budding and Hydra size
A second possibility to explain the increase in cell numbers of Hydra incubated at 9 °C could involve cell loss via budding. Campbell (1965) estimated
that the bud is the site of cell exit for 85 % of the cells lost from the body of
Hydra. Shostak's (1968) calculations showed that about 60 % of gastrodermal
cell loss occurs by movement on to developing buds. Larger animals may be
accumulating more cells because they lose fewer to buds.
Hydra raised at 9 °C initiate fewer buds and these buds take longer to develop
and to detach than at 18 °C. Newly detached buds at the two temperatures have
similar surface areas. From this observation it can be assumed that newly
detached buds at the two temperatures have similar cell numbers. However, as
noted above, both Stiven (1965) and Park & Ortmeyer (1972) have found that
newly detached buds at cooler temperatures have a larger dry weight than those
at warmer temperatures, which may mean more cells. Whether my assumption
and Stiven's and Park & Ortmeyer's observations are actually reconcilable can
not be determined without data on cell numbers in buds. Even if the buds at the
cooler temperature had twice as many cells (Park & Ortmeyer found that Hydra
littoralis buds detached from animals raised at 10 °C had dry weights approximately twice those of buds at 21 °C, while Stiven observed differences of only
about one-third of this with Hydra pseudoligactis), fewer cells would be lost by
2
E M B 30
18
J. W. BISBEE
budding in Hydra pseudoligactis raised at 9 °C because one-third as many buds
are initiated and these buds take three times as long to develop and detach
(Table 8).
Actually, bud cells are derived not only by the movement of parental cells on
to buds (Shostak & Kankel, 1967; Shostak, 1968), but partly by intrinsic growth
on the bud (Shostak, 1968). I assumed the rate of cell division on buds at
different temperatures is smaller, as the parents' rate seems to be. This leads to
the conclusion that since fewer buds detached from Hydra raised at 9 °C, fewer
cells were lost by the parent.
Shostak (1968), following the movement of graft borders in green and white
Hydra viridis, has concluded that the population of gastrodermal cells lying
approximately between (and exclusive of) axial positions 4 and 10 (see Fig. 4)
provided most of the parental gastrodermal contribution to developing buds,
while the population lying approximately between axial positions 1 and 5
provided gastrodermis to the feet of developing buds. Also, Tammariello (1969)
has shown that cells in the distal portion of Hydra viridis are important for their
contribution to buds. From both these reports, one would predict that cells not
lost to buds would accumulate in the distal portion of the animal. Indeed, the
increased cell numbers of Hydra raised at 9 °C in the present study were localized
primarily in the distal portion (Figs. 5, 6).
Thus, because Hydra raised at the two temperatures have similar growth
dynamics and because of the observed effect of temperature on bud initiation
and development, a probable explanation for the increased size of animals
raised at 9 °C is that these Hydra are accumulating more cells because they lose
fewer to buds.
This research was supported by an Institutional Grant from the American Cancer Society,
by an NSF Predoctoral Fellowship, and by the University of Pittsburgh.
The author thanks Dr Stanley Shostak for advice during this research in his laboratory,
and during the preparation of the manuscript.
This paper represents a portion of a dissertation submitted to the Graduate Faculty of
Arts and Sciences of the University of Pittsburgh in paitial fulfillment of the requirements
for the degree of Doctor of Philosophy.
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(Received 6 July 1972, revised 12 January 1973)