t / . Embryol. exp. Morph. Vol. 30, l,pp. 1-19, 1973

Printed in Great Britain

h Size determination in Hydra: The rolesK of growth and budding

^

By JOHN W. BISBEE1

From the Department of Biology, University of Pittsburgh

rP S U M M A R Y

\~ Hydra pseudoligactis cultured at 9 °C for 3-4 weeks are one-and-a-half times larger than^ those cultured at 18 °C. The size of Hydra is correlated with the numbers of epithelio-

muscular and digestive cells in the distal port ion of the animal and with the diameters of thek- epithelio-muscular cells in the peduncle.[ Counts of mitot ic figures and tritiated-thymidine-labeled nuclei and determinations ofT increase in mass of Hydra populat ions suggest that the difference caused by these tempera-y. tures does not affect mitosis. At 9 °C buds are initiated at a lower rate and take longer to

develop than at 18 °C. The surface-areas of buds raised at the two temperatures are similar.T Because Hydra raised at the two temperatures have similar growth dynamics, the differencesu in sizes of the animals cannot be due to growth rate. The observed effect of temperature on

bud initiation and development is probably relevant to the increased size of animals raised at9 "C, since these larger animals may be accumulating more cells while losing fewer to buds .

INTRODUCTION

The shape and size of Hydra seems to be a consequence of several dynamicprocesses. Growth, budding, cell sloughing, cell migration, and mesoglealmetabolism 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 deter-mining the dimensions of Hydra pseudoligactis.

Hydra is essentially a cylinder made up of two cell layers, the epidermis andthe gastrodermis, with an acellular mesoglea between them. The cylinder has aring of tentacles and a mouth at its distal end, and an adhesive 'foot' at itsproximal end. The animal is a cellular system with 'input' by cell division, and'output' primarily by budding; Campbell (1965) and Shostak (1968) haveestimated that 60-85 % of cell loss occurs in buds. Additional cell loss occurs viacell sloughing at both ends of the animal, and possibly along its length.

Both Stiven (1965) and Park & Ortmeyer (1972) observed that lowering theambient temperature increased the size of Hydra. This paper confirms Stiven'sobservation 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 uniformthroughout the body column ? Is it due to differential cell size or rate of celldivision ? Is'the change in Hydra size due to differential cell loss ? The approachtaken was to measure body column dimensions and cell numbers and dimensionson serial cross-sections. Since Hydra size was correlated with cell number, aneffort to understand the mechanism through which temperature alters cell jnumbers was made, by determinations of increase in mass of Hydra populationsand counts of mitotic figures and tritiated-thymidine-labeled nuclei. Finally, Jbudding, 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 A

Forrest's (1963) key. The tentacles on buds arose successively in a fixed patternas pictured in fig. 10 of Forrest (1963); adults had tentacles approximately three ^times column length. The holotrichous isorhizas were narrowly oval with trans- A

verse coils. As in Hyman's (1931) original description of Hydra pseudoligactis, ian individual's body column was differentiated into stalk and body (Fig. 1). A

Also some animals raised at 9 °C in the fall were observed to be sexual, havingrather stout testes with nipples.

Stocks of animals were maintained in Pyrex-brand baking dishes kept inincubators at 9 ± 1 and 18 ± 1°C. They were fed to repletion three times aweek on freshly hatched Artemia sp. nauplii at room temperature. The culturesolution (Shostak et al. 1965) was poured off daily (after feeding if they werefed) and replaced with fresh solution already at the appropriate temperature.Animals were transferred to clean dishes every 4-10 days, with the density keptbelow one Hydra per 0-5 ml of solution.

The Hydra used in experiments, drawn from stocks, were raised in fingerbowls. Animals raised for dry-weight determinations and those to be injectedwith tritiated thymidine were maintained at a density of one Hydra per 10 mlof culture solution; all other experimental animals were raised at a density ofone per 20 ml of solution. All groups were incubated for 3 or 4 weeks at theappropriate temperature and starved 48 h before use. With one exception (aHydra raised at 9 °C and used for cell and size determinations), all the Hydraused were asexual budding animals.

B. Histology

Hydra were placed in 50 x 15 mm Petri dishes with 2-3 ml of approximately25 °C culture solution at 16.00 h, 52 h after feeding. Within 15 min, havingextended to the approximate proportions of Fig. 1, they were quickly floodedwith 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

Size determination in Hydra 3the animals in LiCO3 in 70 % alcohol. They were dehydrated in an alcoholseries, 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 de-paraffinized, hydrated, stained in toluidine blue, dehydrated, and mounted inpermount.

The number of epidermal epithelio-muscular cells and gastrodermal digestivecells in each section were estimated from counts of their nuclei. These cells wereidentified as having cytoplasm extending from the mesogleal surface of therespective epithelium to the surface of the layer, and as having nuclei withprominent 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, 1-0r mCi/ml), was injected through the mouth into the Hydra enteron (Campbell,

1965). The animals were returned to culture solution at the appropriate in-cubation temperature and fixed 6 h later. Serial cross-sections on slides weredipped 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 weremounted and examined at x 200. A nucleus was considered labeled if one halfor 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 beenstarved for 48 h. The animals were lyophilized and weighed on an Oertling R20analytical balance, which can be read to 0-1 mg.

E. Observations of buds

Data on budding were collected by observation often adults at room tempera-ture with a dissecting microscope at x 12, noting the number of buds attachedand 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 animalsraised at the lower temperature were larger than those at the higher temperature.The lengths and diameters of the animals were measured and the circumferencescalculated. The average dimensions at each temperature, their standard devia-tions, 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 thegastric 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 Temperature Peduncle [1-3] Budding [4-5] Gastric [6-10] Total

y 9 C 89(17) 30(14) 135(46) 253(47)L 18 X 58 (5) 21(19) 84(16) 162(17)

t Averages and (standard deviations) of the number of cross-sections, based on fourT animals at each temperatuie.Y % Numbers refer to positions on Hydra body column of Fig. 4.

| Table 2. Analysis of variance for the body column circumferences at the mesogleaaccumulated for corresponding axial positions at both temperatures

Y Source of Degrees of Sum of Meanvariation freedom squares squares F

f Treatments 19 38-26 — —Temperature 1 010 010 186

I Axial position 9 37-92 4-21 84-20*Temperature v. axial position 9 0-24 003 0-50

•" Error 80 4-28 005 —

Total 99 42-54 — —

* Probability less than 1 % that the difference is due to random error.

1. LengthThe 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: thebudding region is the length of the body column that has buds attached; thegastric 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 offour Hydra cultured at each temperature are presented in Table 1. Each regionand the total length of the 9 °C Hydra is approximately one-and-a-half timeslonger 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 similarat representative positions along their length. These circumferences of the bodycolumn at the mesoglea were calculated from measurements made on tenhistological sections at the ten axial positions. Compression during sectioningcaused the sections to be elliptical; the lengths of the major and minor axes weredetermined with an ocular micrometer at x 126. These circumferences at each

9°C

18-

0-78(008)

< - ~»

1

0-68(008)

0-76(007)

-2

0-70(Oil)

0-76(006)

-3

0-80

(0-17)

116(019)

,,

4

1-25(0-22)

J. W.1-86

(0-38)

.

5

^ •

1-88(016)

BISBEE2-12

(0-50)

6

*—

2-17(0-25)

2-12

(0-23). —-

7

2-33(018)

2-11

(0-29)

8

2-34

(0-25)

201

(0-29)

• •

9

215(0-29)

1-85

(0-25)

• — -

10

. "

2-10

(0-40)

«-

Fig. 4. Calculated circumferences of the Hydra pseudoligactis body column at themesoglea in mm. Averages and (standard deviations) at the ten axial positionsshown on the diagram are based on five animals at each temperature.

Table 3. Dry weight of adult Hydra

Temperature ...

Sample ...

Number of adultsAverage /tg/adult with budsAverage late budsf

attached/adultEstimated fig of attached late budsjCorrected /tg/adultAverage fig of

adult/temperature

9°C

1

98265

2-863

202

204

r99

270

2-965

205i

18 °C

II

97131

1-423

108

126

i r

96163

1-220

143j

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 signifi-cantly when tested with the F statistic (Table 2). The averages and standarddeviations for each temperature are presented in Fig. 4.

3. Mass of Hydra

The dry weights of adult Hydra were determined by lyophilizing duplicatecultures of approximately 100 adults previously raised for 18 days at either 18or 9 °C. The measurements were corrected for the weight of attached buds asfollows: the numbers of late-stage buds attached (stages 4-6 of Shostak, Bisbee,Ashkin & Tammariello, 1968) were estimated from observations of ten Hydrain each culture the day the adults were lyophilized; these buds were considereda 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 204figand those at 18 °C, 126/*g; this is a 62 % increase in mass, which is significant atthe 5% level.

t

Size determination in Hydra

Table 4. Cells per unit distance and cell diametersin longitudinal and cross-sections of Hydraf

Longitudinal Cross-sections sections

Y Epithelio-muscular cells Cells/mm 28 (5) 35 (9)K Cell diameter (/tm) 36 (6) 31 (10)

Digestive cells Cells/mm 51 (7) 60 (10)Cell diameter Qim) 20 (3) 17 (3)

f Averages and (standard deviations) for thiee of each kind of section in the gastric region.

B. Hydra cell number and size

L Cell numbers and cell sizes were determined to learn whether the larger Hydracultured at 9°C have more cells, larger cells, or both.

First, the dimensions of the epithelio-muscular cells and the digestive cellswere compared along each of their longitudinal and circumferential axes. Ifthese cells were cylindrically shaped, cell diameters could be calculated fromeither longitudinal or cross-sections. The numbers of epithelial cells per unit oflength and per unit of circumference in each of three cross and longitudinalsections in the gastric region were calculated. Their reciprocals (distance at themesoglea divided by the number of nuclei counted over that distance) are celldiameters. These data are presented in Table 4 as means and standard deviations.The numbers of epithelio-muscular cells per mm (and, of course, their recipro-cals, epithelio-muscular cell diameters) in the two types of sections were notsignificantly different, nor were the number of digestive cells per mm (and theirreciprocals, digestive cell diameters).

The epithelio-muscular cells are considered to be contiguous, as are the diges-tive cells, as illustrated in Fig. 3 (except that in the epidermis, interstitial cellsmay be inserted between the epithelio-muscular cells at their basal ends, whilein the gastrodermis, gland and mucous cells intervene occasionally). Thus theepithelio-muscular cells and the digestive cells are essentially equi-dimensionalalong the longitudinal and circumferential axes and can be considered asroughtly compact cylinders contiguous with each other.

In what follows, the numbers and diameters of epithelial cells in the two layersare calculated from data from cross-sections.

1. Cell number

The numbers of epidermal epithelio-muscular cells and gastrodermal digestivecells in each of ten cross-sections at the axial positions were counted. Thesenumbers of cells per axial positions multiplied by the numbers of sections ineach 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

S 3

i

i

• 9

o 18

I

4 1

< 1

I-O—

1 'r

cc

1—O

H

* * * *

10

Fig. 5. Epithelio-muscular cell number in Hydra pseudoligactis. Means and rangesof estimated cell numbers per axial tenth in five animals at each temperature arepresented. • 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 numbersare plotted as a function of the axial tenths. These estimates of cell numberswere compared statistically, considering the estimates for each tenth from allthe Hydra as a block. The first question asked was, 'Is there an interactionbetween the numbers of cells per axial tenth and temperature ?' The 'interaction *term calculated here represents the differences between the means for tempera-tures with different axial tenths and differences between means for axial tenthswith different temperatures. Since this term is significant for both cell types, allfurther comparisons had to be performed either for different axial tenths withonly one temperature or different temperatures at the same axial tenth.

Comparisons of the estimated cell numbers for the two temperatures at eachaxial tenth show that in the upper budding region and gastric region, theestimated cell numbers per axial tenth differ significantly, with more in theanimals raised at 9 °C. That is, as shown in the abscissa of Fig. 5, the Hydraraised at 9 °C have significantly more epithelio-muscular cells in the upperbudding region and lower gastric region (axial tenths 5, 6, and 7) than the Hydracultured at 18 °C; there are significantly more digestive cells (Fig. 6) in the upper

Size determination in Hydra

9 C18 C

i

• *5

Axial6

tenths7

*

8

* *

9

*

10

Fig. 6. Digestive cell number in Hydra pseudoligactis. Means and ranges of estimatedcell numbers per axial tenth in five animals at each temperature are presented.• Probability less than 5% that the difference is due to random error ; • • probabilityless 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 approxi-mately the distal one-half of the Hydra, between the tentacles and the stalk ofthe peduncle. The budding region is the proximal one-fourth of the gastricregion, distinguished functionally by the presence of buds. Thus the Hydraraised 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 themesoglea for each cross-section were calculated by dividing the circumferencesin mm at the mesoglea by the number of nuclei counted in that section. Theaverages and ranges of the cell diameters at each axial position of Hydra raisedat the two temperatures are presented in Fig. 7. Comparisons of the cell dia-meters accumulated for corresponding axial positions at both temperaturesshowed that the interaction term was significant. Therefore comparisons of thecell diameters for the two temperatures are required at each axial position. Therewere significant differences only in the epithelio-muscular cell diameters of the

10 J. W. BISBEE

40

20

0

If3 .120

<u

I 100

I 80

° 60

40

20

0

T B • 9 Co 18 C

4 5 6 7Axial position

10

Fig. 7. Epithelio-muscular cell diameter (A) and digestive cell diameter (B) in Hydrapseudoligactis. Means and ranges of cell diameters per axial position in five animalsat each temperature are presented.

peduncle (positions 1-3, Fig. 7 A). These on Hydra cultured at 9°C were signifi-cantly larger than the cells in the corresponding axial positions of the 18 °CHydra. The diameters of the digestive cells at the axial positions (Fig. 7B) weresimilar 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 andgastric regions (positions 4-10) of the Hydra cultured at the two temperatureswere similar.

Thus, the increased size of the Hydra raised at 9 °C is correlated with increasednumber of epithelio-muscular and digestive cells in the distal portion of the bodycolumn, and increased size of the epithelio-muscular cells in the proximalportion.

C. Cell division

Three ways of estimating growth in Hydra were used: (1) the number ofmitotic figures was counted in histological sections: (2) the number of tritium-labeled nuclei was counted in autoradiographs of sections of Hydra injectedwith tritiated thymidine; and (3) the increase in mass of the Hydra populationswas determined.

^

r

t

Size determination in Hydra 11

30

20

10

• 9 Co 18 C

IIH

3 4 5 6 7Axial position

10

Fig. 8. Mitotic figures in the epidermis of Hydra pseudoligactis. Means and rangesof mitotic figures per axial position in three animals at each temperature arepresented.

1. Mitotic figures

The numbers of mitotic figures in the epidermis and gastrodermis werecounted in a section at each of the ten axial positions on three Hydra at eachtemperature. The means and ranges for the epidermis are presented graphicallyin Fig. 8. The numbers of mitotic figures per axial position in the epidermis ofHydra raised at the two temperatures were compared statistically. The differ-ences were not significant (Table 5).

The numbers of mitotic figures in the gastrodermis averaged 0-21 per axialposition, 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 incubationtemperature and fixed 6 h later. The tritiated-thymidine-labeled nuclei werecounted in cross-section autoradiographs at the ten axial positions of five Hydraraised at each of the temperatures. The means of labeled nuclei and their stan-dard deviations per axial position in the epidermal and gastrodermal layers arepresented in Fig. 9.

The numbers of labeled nuclei per axial position in the epidermis of Hydraincubated 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 accumulatedfor corresponding axial positions at both temperatures

Source ofvariation

TreatmentsTemperatureAxial positionTemperature v. axial

ErrorTotal

position

* Probability less than 1 % that the

Degrees offreedom

19199

4059

difference: is due

Sum ofsquares

13441

1070273

10152359

to random

Temp-erature Number of labeled nuclei in the epidermis per axial position

9 C ° 2L (1) (2)

18 C (0 (2)

o ic ( i ) ( i )

IS C ° °18 c ( 1 ) (1 )

18(19)

21.(30)

3

L4

(5)

3(4)

53(33)

24(29)

4

N - * -

7(7)

(3)

70(33)

22(24)

- — —

5

- ^ ^ .

12(9)

5(8)

60(28)

44(20)

6

9(5)

9(6)

54(33)

40(18)

7

9(4)

8(9)

42(25)

37(18)

8.

3(2)

7(4)

Meansquares

11193025—

error.

30(23)

29(15)

• _

9

______

5(6)

7(3)

F

0044-76*1-20

__

21(22)

25(13)

4(3)

5(1)

s

V

J

•

J

i

1

A

H

4

1

11j

Number of labeled nuclei in the gastrodermis per axial position

Fig. 9. Numbers of 3H-thymidine-labeled nuclei in the epidermis and gastrodermisof Hydrapseudoligactis. Averages and (standard deviations) at the ten axial positionsshown 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 gastro-dermal labeled nuclei for both temperatures at each axial position, the numbersfor 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 wereused to initiate six populations of Hydra. All parents and detached buds wereretained 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

i0

V

i

y

Temperature Determination

18°C Dry weight (mg)Number

9°C Dry weight (mg)Number

Populations

8-965

8033

8-392

6-226

9-2917-4

30

Table 7. Analysis of variance of population masses

Source of Degrees ofvariation freedom

Temperature 1Error 4

Total 5

t For these degrees of freedom, F at

Sum ofsquares

3-82-1

5-9

the 5 % level

Meansquare

3-80-5

= 7-71.

Average

8-883

7-230

F

7-60f

*" 3 weeks, after which the total population mass (dry weight) was determined forY each of the six groups. These data are presented in Table 6. The numbers of

f individual Hydra per population were highly significantly different, averaging

83 animals at 18 °C and 30 animals at 9 °C. The total population masses, whichf 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 distri-buted differently.

D. Bud initiation, development and sizeThree aspects of Hydra budding were studied: (1) rate of bud initiation, (2)

duration of bud development, and (3) size of newly detached buds. Two groupsof Hydra were raised at each temperature; one group was fed once a day, andthe other every other day.

The rate of bud initiation is the average number of new buds appearing on aparent per day; the number of buds initiated on day n is determined by sub-tracting the number of buds attached on day n— 1 from the sum of the attachedand freshly detached buds on day n. The duration of bud development is thelength of time between bud initiation and establishment of the bud as anindependent individual.

Table 8 presents the data on bud initiation. Hydra cultured at 9 °C initiatesignificantly fewer buds than animals raised at 18 °C. Feeding schedule, aswell 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 the18 °C animals when they were fed once a day (twice per 48 h). When the parentswere 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

9°C

18°C

No. offeedings/48 h

2121

No. of budsinitiated/dayj

0-600-201-781-28

Duration ofbud development

(days)

15||16«I4|l5';

Surface areaof newlydetached

buds (mm2)§

2-8—2-6

t Averages for variables.% Observations made for the first 6 days after Hydra had been fed three times on appro-

priate schedule.§ Averages for four buds at each temperature; average for 14 additional buds at 18°C =

2-7 mm2.|| Parents observed for 7 days.if Parents observed for 14 days.

——

Table 9. Analysis of variance for initiated buds accumulatedfor corresponding feedings at both temperatures

Source of variation

TreatmentTemperatureFeedingTemperature v. feeding

Error

Total 23 1157

Degrees offreedom

3111

20

Sum ofsquares

893770121

2264

Meansquare

770121

213

F

.

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 detachedbuds are also presented in Table 8. Buds at 9°C took approximately three timesas long to develop and detach as buds at 18 °C. The feeding schedule did notseem to alter this variable.

The surface areas of newly detached buds were calculated from measurementsof length and width made on 2 x 2 transparencies. The surface areas of thesebuds at the two temperatures (Table 8) were not significantly different. ThusHydra raised at 9 °C initiated fewer buds than Hydra cultured at 18 °C; thesebuds took longer to develop, but were similar in size to buds on animals raisedat l8°C.

* Size determination in Hydra 15** DISCUSSION

A. Size and temperature

Hydra are well known for their spontaneous movements of the body column9 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 photo-

graphs 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, usek the same methods as mine for Hydra length). The Hydra at the two temperatures

were handled identically to minimize histological artifacts. Thus the datak collected are meant to be relative, not absolute.

The dry weights of adult Hydra raised at the two temperatures were correctedk 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 °CT (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, Hydraraised at the lower temperature did not differ in representative circumferences

i along their length from Hydra raised at the warmer temperature. Thus, tempera-^ ture 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 Hydralittoralis. He expressed size in terms of calories per 1-day-old bud. The mass ofbuds rather than of adults was measured, since the presence of different numbersof buds on adults would have led to considerable variation in mass measure-ments. He assumed that bud size was proportional to the relative sizes of thespecies, and concluded that a lowering of the temperature from 25 to 15 °Cincreased the mass of three species. For H. pseudoligactis, Stiven observed a 39,33 and 35 % increase, respectively, of calories, protein and dry weight, whenlowering the culture temperature from 25 to 15 °C.

Also, Park & Ortmeyer (1972) have reported on the size of Hydra littoralisadults and newly detached buds raised at several different temperatures. Adultsraised at 10 ° C have a dry weight 66 % greater than those at 21 °C, while buds atthis lower temperature were 113 % larger than buds at the higher temperature.

Thus my observations on the size of Hydra at two temperatures add anotherexample to the literature implicating temperature among normal size-regulatingmechanisms of organisms. It is well known that some animals (notably homeo-therms) are larger at the more polar extremes of their ranges (Ray, 1960). Theapplicability 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 whengrown at a lower temperature. He surveyed 36 species and found that 27 ofthem were larger when they were cultured or lived at lower compared to highertemperatures. On the other hand, Rensch (1959) suggests that poikilotherm'sbody size will decrease towards colder climates. He describes examples that bothsupport and refute his generalization.

Representatives of homeotherms may be larger in colder climates becausemaintenance energy per unit of body weight is usually smaller in a large animalthan in a small one. This difference in metabolic rate can be explained by the factthat surface-area in relation to body weight decreases with increasing size of theanimal (Davson, 1964). Ray notes the arguments of some studies that anattempt to generalize about poikilotherm body size and temperature is meaning-less. Their suggested explanation for this, that these animals produce no signifi-cant metabolic heat, is not entirely true. Certainly Hydra have a negligibletemperature difference from their environment. Because any heat produced israpidly conducted to the environment, they stay at ambient temperature. There-fore, ascribing some adaptive significance to observations that Hydra are largerwhen 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 atdifferent temperatures. The possibility of an overall increase in cell size has beenruled 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 mightinvolve 'output' in the form of cell loss during budding. Also Stiven (1965) hassuggested 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 numberof epithelio-muscular and digestive cells. Only in the peduncle of 9 °C Hydra dothe epithelio-muscular cells of the epidermis differ in diameter. The increasedsize of these cells in the peduncle is correlated with the increased size of thisregion, since the numbers of epithelial cells in the peduncle are indistinguishablestatistically 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 producingthese larger Hydra, since in Hydra raised at 9 and 18 °C the numbers of mitoticfigures per axial position in the epidermis, and of 3H-thymidine-labeled nucleiin both cell layers were indistinguishable statistically.

The number of dividing cells observed at any one time is assumed to be afunction of the duration of mitotis and of the length of the cell cycle. Since thenumber 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 affecteither 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 theaverage individual mass was 62 % less for the 18 °C Hydra than for the 9 °CHydra (Table 3), the total population mass of the 18°C Hydra was 18 % greaterat the end of the incubation period (Table 6). For this reason, and since this18 % difference in population masses was not significant, differences in growthrate 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 colonialhydroids as results of ordered cell division. According to Brien's view (see Brien& Reniers-Decoen, 1949) Hydra's epithelial cells are produced in a subhypo-stomal growth zone, which they gradually leave to differentiate and finally sloughoff 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 reachedbetween growth and sloughing. Burnett (1961, 1962, 1966) and Burnett andGarofalo (1960) have supported this view; indeed, Burnett (1966) has proposeda model of growth and cell differentiation in Hydra that elevates growth to theessential element in development of form. Burnett also considers the growthpattern 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 incu-bated at 9 °C could involve cell loss via budding. Campbell (1965) estimatedthat the bud is the site of cell exit for 85 % of the cells lost from the body ofHydra. Shostak's (1968) calculations showed that about 60 % of gastrodermalcell loss occurs by movement on to developing buds. Larger animals may beaccumulating more cells because they lose fewer to buds.

Hydra raised at 9 °C initiate fewer buds and these buds take longer to developand to detach than at 18 °C. Newly detached buds at the two temperatures havesimilar surface areas. From this observation it can be assumed that newlydetached buds at the two temperatures have similar cell numbers. However, asnoted above, both Stiven (1965) and Park & Ortmeyer (1972) have found thatnewly detached buds at cooler temperatures have a larger dry weight than thoseat warmer temperatures, which may mean more cells. Whether my assumptionand Stiven's and Park & Ortmeyer's observations are actually reconcilable cannot be determined without data on cell numbers in buds. Even if the buds at thecooler temperature had twice as many cells (Park & Ortmeyer found that Hydralittoralis buds detached from animals raised at 10 °C had dry weights approxi-mately twice those of buds at 21 °C, while Stiven observed differences of onlyabout 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 budsare 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 onto buds (Shostak & Kankel, 1967; Shostak, 1968), but partly by intrinsic growthon the bud (Shostak, 1968). I assumed the rate of cell division on buds atdifferent temperatures is smaller, as the parents' rate seems to be. This leads tothe conclusion that since fewer buds detached from Hydra raised at 9 °C, fewercells were lost by the parent.

Shostak (1968), following the movement of graft borders in green and whiteHydra viridis, has concluded that the population of gastrodermal cells lyingapproximately 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 5provided gastrodermis to the feet of developing buds. Also, Tammariello (1969)has shown that cells in the distal portion of Hydra viridis are important for theircontribution to buds. From both these reports, one would predict that cells notlost to buds would accumulate in the distal portion of the animal. Indeed, theincreased cell numbers of Hydra raised at 9 °C in the present study were localizedprimarily in the distal portion (Figs. 5, 6).

Thus, because Hydra raised at the two temperatures have similar growthdynamics and because of the observed effect of temperature on bud initiationand development, a probable explanation for the increased size of animalsraised at 9 °C is that these Hydra are accumulating more cells because they losefewer 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 ofArts and Sciences of the University of Pittsburgh in paitial fulfillment of the requirementsfor the degree of Doctor of Philosophy.

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(Received 6 July 1972, revised 12 January 1973)