H Size Determination in Hydra: the Roles of Growth and Budding

Home , Hydra (genus)

t /. Embryol. exp. Morph. Vol. 30, l,pp. 1-19, 1973 Printed in Great Britain h Size determination in Hydra: The roles K of growth and budding

By JOHN W. BISBEE1 ^ 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 than ^ those cultured at 18 °C. The size of Hydra is correlated with the numbers of epithelio- muscular and digestive cells in the distal portion of the animal and with the diameters of the k- epithelio-muscular cells in the peduncle. [ Counts of mitotic figures and tritiated-thymidine-labeled nuclei and determinations of T increase in mass of Hydra populations 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 differences u 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 at 9 "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 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 j numbers was made, by determinations of increase in mass of Hydra populations and counts of mitotic figures and tritiated-thymidine-labeled nuclei. Finally, J 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 A Forrest's (1963) key. The tentacles on buds arose successively in a fixed pattern as 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, i an 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, 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 Size determination in Hydra 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 de- paraffinized, 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 r 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 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 four T 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 mesoglea accumulated for corresponding axial positions at both temperatures

Y Source of Degrees of Sum of Mean variation 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. 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 0-78 0-76 0-76 116 1-86 2-12 2-12 2-11 201 1-85 9°C (008) (007) (006) (019) (0-38) (0-50) (0-23) (0-29) (0-29) (0-25)

. —- • • •—- < - ~»- -,, . «- 2 1 3 4 5 6 7 8 9 10

*— . " ^ • 0-68 0-70 0-80 1-25 1-88 2-17 2-33 2-34 215 2-10 18- (008) (Oil) (0-17) (0-22) (016) (0-25) (018) (0-25) (0-29) (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 ... 9°C 18 °C Sample ... 1 r II ir Number of adults 98 99 97 96 Average /tg/adult with buds 265 270 131 163 Average late budsf attached/adult 2-8 2-9 1-4 1-2 Estimated fig of attached late budsj 63 65 23 20 Corrected /tg/adult 202 205 108 143 Average fig of i j adult/temperature 204 126 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

Longitudinal Cross- t 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 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 c o 18 c

i I

4 1 S 3

< 1 i 1—O H I-O— 1 'r

* ** * 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

•* * ** * 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

0 If T B • 9 C 3 .120 o 18 C

0 4 5 6 7 10 Axial position

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 tritium- labeled 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

• 9 C o 18 C

10 H II 3 4 5 6 7 10 Axial position

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.

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 r 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 t 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 Degrees of Sum of Mean s variation freedom squares squares F Treatments 19 1344

Temperature 1 1 1 004 V Axial position 9 1070 119 4-76* Temperature v. axial position 9 273 30 1-20 Error 40 1015 25 J Total 59 — __ 2359 • * Probability less than 1 % that thedifference: is due to random error. J

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

2 18 53 70 60 54 42 30 21 9 LC ° 1 (1) (2) (19) (33) (33) (28) (33) (25) (23) (22)

21 24 22 44 40 37 29 25 18 C (0 (2) .(30) (29) (24) (20) (18) (18) (15) (13)

-—— • _ A 3 4 5 6 7 8. 9 H LN-* - -^^. ______4 o i 4 7 12 9 9 3 5 4 1 c (i) (i) (5) (7) (9) (5) (4) (2) (6) (3)

IS C ° ° 3 5 9 8 7 7 5 18 c (4) (3) (8) (6) (4) 1 (1) (1) (9) (3) (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 gastro- 1 dermal 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 Size determination in Hydra 13

Table 6. Population mass and number of Hydra raised at 9 and 18 °C

Populations i Temperature Determination Average 0 18°C Dry weight (mg) 8-9 8-3 9-2 8-8 Number 65 92 91 83 9°C Dry weight (mg) 80 6-2 7-4 7-2 V Number 33 26 30 30 i Table 7. Analysis of variance of population masses Source of Degrees of Sum of Mean variation freedom squares square F Temperature 1 3-8 3-8 7-60f Error 4 2-1 0-5 y Total 5 5-9 t For these degrees of freedom, F at the 5 % level = 7-71.

*" 3 weeks, after which the total population mass (dry weight) was determined for Y 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, which f 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 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 sub- tracting 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

Surface area Duration of of newly No. of No. of buds bud development detached Temperature feedings/48 h initiated/dayj (days) buds (mm2)§ 9°C 2 0-60 15|| 1 0-20 16«I 2-8 18°C 2 1-78 4|l — 1 1-28 5'; 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 mm2. || 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

Degrees of Sum of Mean Source of variation freedom squares square F Treatment 3 893 . Temperature 1 770 770 59-23* Feeding 1 121 121 9-31* Temperature v. feeding 1 2 2 015 Error 20 264 13 . Total 23 1157 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 15 * * DISCUSSION A. Size and temperature Hydra are well known for their spontaneous movements of the body column 9 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, use k the same methods as mine for Hydra length). The Hydra at the two temperatures were handled identically to minimize histological artifacts. Thus the data k collected are meant to be relative, not absolute. The dry weights of adult Hydra raised at the two temperatures were corrected k 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 T (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 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 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 meaning- less. 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. There- fore, 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 subhypo- stomal 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)