ABSTRACT
A quantitative study of the growth of Clytia johnstoni is described.
The distance between hydranths is most commonly between 3 and 4 mm. The average internode lengths and their variability frequently differs not only between colonies but also between stolons of a single colony. The differences are therefore unlikely to be genetic ones. Occasionally much longer internodes occur (up to 8 mm).
Hydranths and their stalks differ widely in size but not in morphology. The differences can be caused by the amount of food given.
Very vigorous and very poorly growing colonies tend to have slightly longer internodes than the more common colonies of average vigour.
Most colonies produce both additional (secondary) hydranths and stolon branches. Hydranth branches grow spaced from existing hydranths; in normal colonies the spacing appears to originate from the hydranth away from the growing tip in an internode. Ln vigorous growth the hydranth towards the growing stolon tip is the place of reference.
Stolon branches occur mostly near to hydranths, either primary or secondary, more especially near to the hydranth farther from the growing tip of the main stolon.
There appears to be a limit to the number of branches to an internode. In vigorous growth an optimum of two and a maximum of three is found, either secondary hydranths, or stolons, or both.
Stolons vary in their rate of growth. A stolon slowly increases its rate of growth as it gets longer. In a colony of little vigour, the rate is low and variable and may cease and restart; the coenosarc sometimes breaks, dividing the colony.
Hydranths take 1–2 days to grow, sometimes 3 days in colonies of little vigour. They remain open for a limited period: normally about 5–7 days; sometimes longer -up to 9 or 10 days; and significantly shorter, 1–3 days, in a colony of little vigour.
At the end of their period of existence, hydranths regress. They normally start to regenerate a new hydranth within 1–2 days but some do not do so for longer or very long periods.
The whole cycle of hydranth growth, maturity and regression falls into either a 6-to 7-day cycle or a 10-to 11-day cycle.
Evidence is presented to support the suggestion that there is a situation of competitive interaction between growth of the various parts of the colony. A priority series for the various parts of the colony is constructed.
INTRODUCTION
The typical hydroid consists of three basic structures -stolon, hydranth and reproductive; this paper is concerned with the first two of these. In the hydras a stolon is not normally produced although a mutant is known which produces a structure superficially like a stolon (Brien & Reniers-Decoen, 1952, 1956; Haynes & Burnett, 1964; Haynes, Burnett & Deutschman, 1964). Usually there is present a stolon, growing attached to the substratum, and from this hydranthproducing branches grow away from the substratum. The stolon commonly branches and the branches may or may not anastomose. Hydranth-producing branches may bear a single hydranth or its stalk may branch in various ways to produce many hydranths. In Clytia johnstoni there is a stolon which produces branch stolons and these may or may not anastomose (Hale, 1960b, 1964b); hydranth stalks are initiated at intervals as the stolon grows, giving rise to a basic and conspicuous linear pattern. Often additional hydranths are later produced, and this fact, together with stolon anastomoses and crossing-over, makes the initial regular pattern appear to be blurred.
The study of pattern in hydroids has been almost entirely confined to the hydranth and its stalk, especially in the hydras and in Tubularia. Little attention has been given to the pattern of hydranth initiation by a stolon, although colony growth has been described for Campanularia by Crowell (1957), Crowell & Wyttenbach (1957) and Wyttenbach (1969); for Cordylophora by Fulton (1963); for Tubularia by Mackie (1966); for Podocoryne carnea by Braverman (1963) and Braverman & Shrandt (1966, 1969) and for Clytia attenuata by West & Renshaw (1970). Morphological data concerning certain aspects of stolon or hydranth growth are recorded in studies on Obelia by Child (1923), Berrill (1949a) and Hammett (1943,1946), and on Bougainvillea by Child (1923) and Berrill (1949b).
In this study the pattern of hydranth initiation has been measured and analysed for Clytia johnstoni and in addition the life-cycle of the hydranth and other morphogenetic features measured quantitatively. It will be shown that hydranths do not occur at random but follow a pattern, although considerable variability in growth features of the stolon and hydranth are revealed. The limitation in the life of a hydranth and its pattern of replacement is described. This study follows earlier papers on the morphogenesis of this animal (Hale, 1960b, 1964b) and later papers will be concerned with experimental studies on the pattern of hydranth initiation.
MATERIAL AND METHODS
Culture methods
The material was grown on 11 × 8 cm glass slides immersed in running aerated sea-water at 17 ± 1°C. Newly hatched nauplii of the brine shrimp were given daily as food and the hydroid washed in a vigorous stream of sea-water a few hours after feeding. The water was filtered and returned or alternatively replaced by fresh sea-water in a 2-day cycle; the stock was removed from the sea at about 20-day intervals (Hale, 1957, 1960a, 1964a; Mackie, 1966; West & Renshaw, 1970). Some measurements were made on freshly collected material.
RESULTS
The analyses recorded here are concerned with two sets of observations. In the first, measurements were made of internodes in many colonies to assess the homogeneity of the material. In the second a more detailed set of observations was made on three selected colonies with the object of analysing the relative places of occurrence of hydranths on a stolon, of stolon branches and the lifecycle of hydranths.
Fig. 1 illustrates the essential morphological features of Clytia.
Diagram of part of a Clytia colony to illustrate the primary hydranths and the stolon internodes between them and the arrangement of secondary hydranths and branch stolons from the internodes ( × 6 approximately).
I Colony variations
(a) Materials
Seventeen colonies were grown separately on different glass plates under identical conditions in one aquarium. Each colony was initially prepared as a single organic unit, i.e. all stolons were carefully checked to be confluent. After 5 weeks growth internodes between primary stolons were measured; 445 internodes in 50 stolons were measured in the 17 colonies (between 12 and 36 in each colony): in 3 colonies, 4 stolons; in 11 colonies, 3 stolons; in 2 colonies, 2 stolons; and in 1 colony, 1 stolon. In each case the order of the measurement of the internodes from the growing point was noted.
(b) Internodes
The first obvious observation was that in some colonies an occasional long spacing had occurred. Although the readings mostly ranged, in different stolons, from about 3·0 to 6·0 mm, occasionally some were longer, sometimes very much longer -up to nearly 18 mm -and these changes were sudden, as demonstrated by this typical example (length of internodes in mm in order from the stolon tip).
These very long internodes stand out as different from the normal, more frequent initiation of hydranths. For statistical comparison such long readings might tend to mask other differences between stolons (if they exist) and some means of separating them was necessary. For this analysis, as there was no biological feature known to be associated with the long internodes, the data itself was used. The separation of long internodes was carried out as follows:
Stolons were ear-marked for further analysis if they possessed an internode whose length was greater than the next longest internode by an amount greater than 40% of the total range of lengths in that stolon.
The difference between the length of such an internode and the mean of the remaining internodes in a stolon was compared with the standard deviation of the remaining internode lengths by calculating a t test. Where the probability of a t, equal to or greater than that found, was 0·015 or smaller the internode was considered outside the range to be expected (such observations are about 3·5 and more standard deviations from the mean).
During this analysis, 30 suspected abnormal internode lengths in 26 stolons were tested. Six were found not to be significant, but the remaining 24 (out of a total of 445 measured) readings kept separate. Twenty stolons out of the 50 were affected, 17 by the occurrence of one abnormal internode 2 by 2 abnormal internodes (in 12 measured), and 1 by 3 abnormal internodes (in 16 measured).
The ‘normal length’ readings were then further analysed. Firstly, the variances of the internode lengths in each stolon in a single animal were tested for homogeneity (Bartlet’s test). In two cultures they were found not to be so (χ2 = 9·04, D.F. = 2, 0·025 > P > 0·01, and χ = 7·06, D.F. = 2, 0·05 > P > 0·02). The difference between the means was tested in the remainder and in four cases these were too great to be explained as random variations. In the remaining 11 cultures the data were pooled and the culture variances tested for homogeneity but the probability of this was very small (χ2 = 76·49, D.F. = 10, P < 0·001). Table 1 records the means and standard errors of the 50 stolons in frequency form. The first conclusion from these observations is that hydranths do not occur at random along the stolon. There is a distinct tendency for them to occur at a distance of 3–4 mm from their neighbours.
Secondly, during the growth of the stolon the roughly regular initiation of hydranths is sometimes interrupted (in about 5% of cases) and a much longer internode appears.
Thirdly, the spacing of hydranths appears to be a characteristic of a stolon. A branch may show different characteristics from its ‘parent’ in the variability of the spacing. In general, the variability in hydranth spacing is greater between stolons than would be predicted by the ‘within-stolon’ internode variability.
(c) Hydranths and stalks
Other features which also vary in this animal are the size of the hydranth and the length of the hydranth stalks. Hydranths vary greatly in size. Measurements on some newly collected material gave a range of 0·3–1·0 mm for the distance between the base of the cup and the tip of the buccal region (hydranth extended), the most common size being 0·6–0·8 mm. In cultured material one colony gave a range of readings of 0·5–0·7 mm and another 0·6–1·0, with readings fairly evenly distributed. The larger range in freshly collected material reflects the inclusion of apparently newly growing colonies having smaller hydranths.
Further evidence of the extreme variability in size of the hydranth is shown by regenerating pieces of stolon coenosarc kept without food. During regeneration a stolon is formed first then normally a hydranth grows and differentiates. After some days the hydranth regresses and either immediately or after a quiescent period a new hydranth differentiates; occasionally recession and regeneration occurs a third time. At each regeneration the hydranth becomes smaller and the tentacles fewer in number. The lengths of the hydranths and numbers of tentacles are shown in Table 2.
Stalks were found to vary from about 0·5 to 4·0 mm in length, the most common being about 1–2 mm; occasionally a very long one is found, up to about 5 mm. Again the stalks of apparently young material were shorter than in more mature colonies, from about 0·5–1·0 mm. Stalk lengths in cultured material occur mostly in the range 0·75–2·25 mm, but longer ones, up to 5 mm, again occur.
Apart from size, the morphology of the colonies remain the same. In all the variations which have been observed the differences are in size or magnitude. One may conclude therefore that the hydroid is a single species and that differences between colonies and parts of colonies are due to some other intrinsic factors affecting growth. There is evidence that the amount of food causes changes in size of hydranths; this and other factors at a biochemical and physiological level are likely to be causes of variability in growth.
The similarity in measurements of ‘natural’ and cultured material gives confidence in the culture methods.
II Occurrence of hydranth and stolon branches
(a) Materials
Three colonies were taken, differing in what might be superficially described as ‘vigour’; colony ‘A’ was a vigorous grower (rare), ‘B’ of average vigour (the most common type) and ‘C’ of little vigour (rare). Daily records were taken of the growth and differentiation of the various parts until these were affected by the limitation to normal growth by the confines of the culture plate; the records for the three colonies were kept for 24, 15 and 36 days respectively.
(b) Growth of hydranth and stalk
A stalk is first seen as a tiny knob on the upper (i.e. away from substratum) side of the stolon and at about 0·5 mm from the tip. The stalk lengthens but differs in structure from the parent stolon by its much narrower diameter and the presence of annulations. The annulations occasionally appear along most of the length of the stalk, but normally only at the top and bottom. A hydranth stalk is quite straight.
Sometimes annulations are absent and the stalk looks more like a narrow stolon, indeed occasionally such stalks, and probably only such stalks, become stolons. They grow wider in diameter, and cease their rigid unidirectional growth; the growth (free in the water) is commonly in a wide somewhat irregular helix. In its turn such a stolon buds new hydranths which grow on the outside of the helix at regular intervals. Such a free stolon has an orientation resulting apparently from one side growing a little faster than the other and this is the side which produces hydranth branches. The growth of the stalk and the origins of the cells which give rise to it are described elsewhere (Hale, 1964b).
(c) Size of internode
The size of the internode in the three colonies is indicated in Table 3 and statistical treatment of that data confirms its heterogeneity. Firstly, the proportion of internodes in each colony greater than 5 mm is significantly different (χ2 = 21·03, D.F. = 2, P < 0·001). It is seen that the proportion of these especially long internodes increases markedly as the vigour of the colony decreases, the percentage being 0, 10 and 21 in A, B and C respectively.
Secondly (apart from these extra long internodes), the average internode lengths in A and C do not differ (χ2 = 2·83, D.F. = 3, 0·3 > P > 0·2) but together they differ significantly from B (χ2 = 48·49, D.F. = 3, P < 0·001). Most internodes in the colonies A and C are between 3·0 and 4·4 mm in length whereas in B most are between 2·5 and 3·9 mm.
(d) Secondary hydranths
The hydranths which arise near to the tip of a stolon are called ‘primary’ hydranths, as later additional, ‘secondary’ hydranths often grow between these primaries (Fig. 1). Secondary hydranths only rarely grow near the tip of a growing stolon -that is, in the region where primary hydranths are growing and differentiating; quite commonly they appear between the third to the sixth primary hydranths but they occasionally grow at still greater distances from the tip.
There is no detectable difference in mode of growth and final structure between primary and secondary hydranths.
It is important to know whether or not longer internodes possess more branches (hydranths and stolons) than shorter internodes. In investigating this, the data relating to those internodes which had existed for 10 or more days were used so that there was a high probability that the internodes had produced their branches. The analysis was confined to colonies A and B as colony C only produced one secondary hydranth and few stolon branches. Table 4 records the essential data and it is seen that it provides no support for rejecting the null hypothesis that longer internodes have as many branches as shorter ones.
In an internode up to three secondary hydranths may grow and the incidence of 0, 1, 2 and 3 secondaries is shown in Table 5. The frequency of secondary hydranths per internode is much greater in colony A (1·2) than in colony B (0·66) and there was only one in colony C. This is one of the parameters used to indicate the vigour of the colonies.
Secondary hydranths do not appear at random in relation to primary hydranths and this is demonstrated by measuring the distance of a secondary from its two neighbours. It would be expected that a secondary hydranth would occur equally frequently in every part of an internode if it did so at random. This hypothesis was tested by first converting all measurements so as to relate to a constant internode length. This length was next divided into a number of equal parts and the observed incidence of secondary hydranths in the parts compared with the incidence that would be expected if the hydranths were distributed equally (the number of parts was chosen such that the expected frequency was at least 5). Randomness was not supported: in A, one secondary hydranth, χ2 = 33·0, D.F. = 8, P < 0·001; two secondary hydranths, χ2 = 28·1, D.F. = 7, P < 0·001; in B, one secondary hydranth, χ2 = 37·1, D.F. = 7, P < 0·001. Fig. 2 illustrates the data.
Positions of secondary hydranths between primary hydranths. The vertical axis (to the left) represents the node towards the stolon growing tip and the sloping line (to the right) represents the node away from the stolon growing tip. In (a) and (b) are plotted the data from colonies A and B respectively where only one secondary hydranth was present. In (c) is plotted the data from colony A, where two secondary hydranths were present.
Positions of secondary hydranths between primary hydranths. The vertical axis (to the left) represents the node towards the stolon growing tip and the sloping line (to the right) represents the node away from the stolon growing tip. In (a) and (b) are plotted the data from colonies A and B respectively where only one secondary hydranth was present. In (c) is plotted the data from colony A, where two secondary hydranths were present.
Positions of branch stolons between primary hydranths. The vertical axis (to the left) represents the node towards the stolon growing tip and the sloping line (to the right) represents the node away from the stolon growing tip. In (a), (b) and (c) are plotted the data from colonies A, B and C respectively where there was only one branch stolon present in an internode.
Positions of branch stolons between primary hydranths. The vertical axis (to the left) represents the node towards the stolon growing tip and the sloping line (to the right) represents the node away from the stolon growing tip. In (a), (b) and (c) are plotted the data from colonies A, B and C respectively where there was only one branch stolon present in an internode.
Where only one secondary hydranth grows it tends to do so away from the existing primaries towards the middle of the internode. A summary of the lengths of the segments of the internodes so divided is included in Table 6.
In colony A more than one secondary hydranth grew in many internodes. In 46 cases an internode produced initially one secondary hydranth. Later, in 12 of these cases further secondaries grew; in 11 instances a second one appeared, six in the one and five in the other subdivision of the internode, and in the twelfth one section produced two new ones and none in the other section. In a further 12 cases two secondary hydranths appeared together in the internode and in two of these a third hydranth grew later.
The 22 cases where two secondary hydranths develop in an internode have also been analysed (see Table 6). Neither the means nor the variances of the first two segments (i.e. nearer the growing stolon tip) differ significantly; the variance ratio is 1·07 and the means 0·96mm (S.E.±0·075) and 1·13 mm (S.E.±0·078). The lengths of the third segments have a significantly larger variability than the second (F = 3·095, D.F. = 21 and 21, and 0·01 > P > 0·001).
Further clarification is obtained by regression analysis to investigate the position of the secondary hydranths along the length of the internode. In colony A, in those cases where a single secondary appeared, analysis of variance of the regression between the lengths of the parts of the internode towards the growing end of the stolon and the total lengths of the internodes shows no significant relationship; i.e. the lengths of this part of the internode remain constant (within sampling variability). It follows from this that the value of the other half of the internode is larger the longer the internode; the regression analysis confirms this (F = 28·91, D.F. = 44, P < 0·001); the slope of the bestfitting straight line is 1·003 (S.E. ± 0·19). In colony B the same result is obtained but the other way round, i.e. the segments of the internodes towards the node away from the growing tip remain constant in length (within sampling error) and the other segments, towards the growing tip, become longer the longer the total length of the internode (F = 31·14, D.F. = 31, P < 0·001). The slope of the best-fitting straight line to this data is 0·88 with S.E. ±0·16 (Fig. 2). Comparing the data from the two colonies the slopes of the two fitted lines are not significantly different.
Analysis of the data in colony A where two secondary hydranths grew shows that, like the cases in which only one grew, the node towards the growing tip seems to be the reference point. The distances of the secondaries from this node do not vary significantly in relation to variable node length. Again the remaining segments of internodes vary directly with total internode length, the slope of the best-fitting straight line being 0·97 (S.E. ±0·18). The slope of this line is no different from the slopes of the lines fitted in the cases where only one secondary hydranth appears. Neither this nor the other two differ from 1·0. The secondary hydranth nearest the node towards the growing tip is at an average distance of 0·96 mm (S.E. ±0·075) from it and the other is an average 1·13 mm farther on (S.E. ± 0·078). These distances do not differ from each other, taking into account sampling variability.
A summary of the positions of growth of secondary hydranths is given in Table 6.
At a node there may be a growing stalk, a differentiating hydranth, an open hydranth, or a regressing or regressed one. Although the great majority of internodes are bounded by open hydranths other possible combinations clearly occur. The data reveals no evidence to suggest that the secondary hydranths tend to appear more in some nodal situations than in others. The particular structure at a node appears to make no difference to the likelihood of a secondary hydranth appearing.
The occurrence of secondary hydranths in a particular part of the colony may be partly determined by the growth of branch stolons in that part. This point will be taken up later after a discussion of the origin of stolon branches.
(e) Origin of stolon branches
Any internode has the potentiality to produce a branch stolon; even the growing tip segment will occasionally produce a branch or dichotomize. Their frequency in the internodes is given in Table 7.
The positions of origin of branch stolons were recorded in the three colonies. If they originated from the parental stolon at random equal numbers would be expected (within sampling variation) at all distances between the adjacent hydranths, and this hypothesis has been tested. The lengths of parental stolon between hydranths was divided into equal parts and the number of stolon branches occurring on these segments compared with the number expected if they had arisen equally in these segments. The size of the segments was chosen to give an average of at least five branch stolons per segment so that the; χ2 test could be safely applied.
A further point introduced into the analysis was to determine whether or not the position of hydranths had any relation to the position of origin of branch stolons. For this purpose the counts of branch stolons were made on equal segments of parental stolon at known distances from them.
Considering firstly those cases where branch stolons grew in internodes undivided by secondary hydranths (see Table 8) the frequencies were counted in equal segments of parental stolon from the hydranth towards the growing tip H 1(first column for each colony in Table 8) and also in the reverse direction from the hydranth at the other end of the internode H2(second column for each colony). In addition, since internodes vary in length, they were adjusted to a common length of 100 units; this length was divided into a series of equal parts and the incidence of branch stolons in them between H1 and H2 counted (third column for each colony). Also, the occurrence of branch stolons at increasing distance from H1 and H2, up to the centre of internodes, were added together (fourth column for each colony). In these ways any possible relation to the two hydranths could be judged.
It will be seen in Table 8 that, for colony A, the evidence gives no reason to dispute the hypothesis of random origin of branch stolons. In colonies B and C this hypothesis has to be discarded as it is clear that branch stolons tend to originate nearer to the hydranth away from the growing tip of the parental stolon.
Turning now to those cases where branch stolons originated at the same time as, and after, the growth of secondary hydranths there is a complication (which was not large enough to affect the comparable analyses of undivided internodes) that the lengths of parent stolon between hydranths vary considerably, from about 0·5–3·5 mm. This feature required a somewhat modified approach to analysing the data when in its original unit (mm) since the number of possible observations at longer distances from the hydranths was reduced in proportion to the frequency at which these longer lengths of parental stolon occurred. The expected frequencies of branch stolons, E in Table 9, have been weighted to correct the situation and are recorded, together with the observed frequencies O, in the first two pairs of columns in each colony in Table 9. The data in the first pair of columns refer to the distribution of branch stolons between H1 and H2 and the data in the second pair of columns sum the data according to the distance from H1 and H2 together. The third and fourth pairs of columns comprise the data adjusted to a common length between H1 and H2 of 100 units and give the expected (E) and observed (O) distributions of branch stolons between H1 and H2 separately and together respectively. Colony C is not included as no stolons grew in these circumstances; there was only one secondary hydranth.
Table 9 provides evidence that branch stolons do not originate at random but tend to grow from positions on the parent stolon near to hydranths, about equally from both hydranths in colony A but especially near to the hydranth away from the growing tip in colony B.
To summarize the results on all this data on branch stolon initiation it may be said that they tend to grow near to hydranths, especially in the colonies of medium and low vigour. In the latter colonies it is the hydranth farther from the parental stolon growing tip which is favoured over the hydranth towards the growing tip.
So far the analysis has not included a test to determine whether the number of hydranths and of stolons in the same internode are independent or whether the growth of the two kinds of branch interact. In Table 10 are recorded the number of internodes possessing various numbers of hydranths and stolons. These data are restricted to those internodes that had existed for 10 or more days; whereas younger internodes do not necessarily possess their full compliment of branches, internodes older than this rarely add further branches.
Tests of association between the numbers of hydranths and stolons in internodes in the two colonies gives, on the null hypothesis, a low probability in A (χ2 = 14·61, D.F. = 2,0·001 > P > 0·0005) and high in B χ2 = 0·22, D.F. = 1, 0·7 > P > 0·6). A similar result is obtained from analysis of covariance (A: t = 3·42, D.F. = 71,0·005 > P > 0·001; B: t = 0·17, D.F. = 48,0·9 > P > 0·8).
In colony A Table 10 indicates that internodes most commonly have two branches (42%) but one (32%) and three (22%) are also common; the average number is 1·88 (S.E. ±0·03). Where no stolon exists, one or two secondary hydranths commonly grow; if there is one stolon, only one secondary hydranth normally grows, or none at all.
In Colony B the average number of branches (1·46; s.E. ± 0·14) is significantly less. Commonly an internode has either a secondary hydranth or a stolon, less commonly both or neither.
(f) Rate of stolon growth
The rate of growth of stolons was measured daily in the three colonies. In every stolon there was considerable variation from day to day.
In colonies A and B the rate of stolon growth slowly increased during the period of measurement. (Regression analyses gave in A: t = 7·354, D.F. = 35, P < 0·001; and in B: t = 8·114, D.F. = 65, P < 0·001.) No increase was detectable in colony C but there was also greater variation. In colonies A and B, as might be expected from previous analyses, there were significant differences between the average growth rates of the various stolons in a colony (analyses of variance gave, in A: F = 7·52, D.F. = 4, P < 0·001, and in B: F = 3·03, D.F. = 4, P ≏ 0·025). There was a greater growth rate in A than in B (t = 4·84, D.F. = 100, P < 0·001). In colony C growth was on average slower than the other two. It was also erratic, stolons sometimes ceasing to grow altogether for a time, as stated above. Essential quantitative data on the three colonies are indicated in Table 11.
III Life-cycles of hydranths
The daily records of the three colonies included the state of the hydranths. After hydranths are initiated either as primary or secondary, they grow, first by a lengthening of the stalk, then by the swelling of the tip of the stalk to a ‘bulb’, which proceeds to differentiate into a hydranth polyp. The distal end of the enclosing perisarc is dissolved away and the hydranth opens out and, after a short period, catches food. Some days later the hydranth regresses. Regeneration normally follows with growth of a further short length of stalk followed by hydranth differentiation. Regeneration is usually repeated, up to five hydranths have been recorded in succession from one node. In colony A a few hydranths grew as branches of other hydranth stalks instead of the usual branching from a stolon; such branched stalks are rare.
(a) Growth of hydranth
Table 12 records the lengths of time for hydranth growth.
The first hydranths take a little longer to grow than their regenerates because of the extra time required to manufacture a full-length stalk.
(b) The open hydranth
Table 13 gives the records for the time a hydranth remains open.
Table 13 shows that the hydranths had a limited life. The pattern of the lifespan in the colonies was different. In colony A there is a distinct difference between the pattern shown by the first hydranth and subsequent regenerations. The first hydranths have a most likely life-span of 3 days and nearly 80% lived for 2–4 days. The second generation of hydranths lived either 3–4 days (50%) or went on to about 8 days (25%). The third generation showed a similar bimodal pattern of length of life with about 30% living for 3–4 days and 35% for 8 days.
In colony B the life of the first hydranth lasted mostly 5–7 days (70%) -quite different from Colony A.
In colony C the most likely life-span of hydranths was 1–3 days (85%), with no detectable difference between initial and regenerated hydranths.
(c) The degenerated hydranth
Table 14 gives the data for the length of periods of time nodes bear degenerated hydranths.
Like Tables 12 and 13, Table 14 only includes data on regressed hydranths the beginning and end of whose state of degeneration was seen. The majority (80%) of those observed regenerated within 2 days of hydranth regression.
Many more hydranths had not begun to regenerate by the end of the period of observations. Some of these had only recently regressed but others had remained in a regressed state for a much longer time; the stalks of the latter became empty of coenosarc. An indication of the frequency of these nonregenerating hydranths is given by counting those which had failed to regenerate after 6 or more days in a state of regression: in the colonies A, B and C there were 3%, 10% and 20% respectively.
(d) Length of the whole hydranth cycle
Table 15 shows the lengths of the hydranth cycle, growth, open hydranth and regressed hydranth, in frequency form.
The noticeable feature of Table 15 is the preponderance of 6-to 7-day cycles (in A and C) and 10-to 11-day cycles (in A and B). This data does of course reflect the time the hydranth is open (Table 13) but it does show also that, in spite of the variability in the lengths of the different phases, the life-cycle exhibits a clear 6-to 7-or 10-to 11-day repetitive pattern. (The fewer observations in B and C is due to increased numbers of hydranths which failed to regenerate during the period of observation.)
DISCUSSION
1 Variability of internodes
A feature of this material has been the variability of the quantitative morphological data extracted; it might be genetic in origin. Wyttenbach (1969) has discussed genetic variations in Campanularia flexuosa, finding differences between 18 of 19 stolons; these stolons came from wild colonies growing on separate pieces of substrate which he assumed would be derived from different planulae even when obtained from, the same general collecting site. Differences between colonies of Hydractina echinata scored by stolonic fusion or stolonic incompatability have been reported by Hauenschild (1954, 1956).
Whereas features used by Wyttenbach and Hauenschild might provide evidence of genetic differences, that of size appears not to be one. For example, it has long been known that the tentacle number in Hydra is variable (Rand, 1898; Park, 1900; King, 1901; Morgan, 1902, 1903); Mackie (1966) reports an increase in tentacle number during hydranth growth in Tubularia and he suggests this to be a characteristic of gymnoblasts. The number of tentacles in calypto-blasts is also variable, as recorded by Berrill (1949 b) in Obelia and here in Clytia johnstoni-, the number, once established in a hydranth, does not change in that hydranth, as Mackie (1966) suggests.
In deciding whether the material used as Clytia johnstoni was in fact a single genus, the two more important observations are (i) that stolons differ significantly from each other when scored by the length of their internodes and (ii) such stolons can be part of a single colony and must therefore be genetically identical. When comparing different colonies as regards internode length, the differences are of the same size as those between stolons of a single colony. This evidence thus gives no reason to believe otherwise than that all the colonies are genetically identical. Indeed the consensus of one’s knowledge of the animal is that there is a lot of variability but no evidence of genetic multiplicity.
2 Pattern of hydranths along internode
The analyses of internode lengths has established that there is a pattern in the arrangement of hydranths along a stolon; hydranths do not occur at random. The pattern is a twofold one. The primary pattern is normally laid down by the regular initiation of new hydranths just behind the advancing tip of a growing stolon. Most often this occurs every 3–4 mm. Later a second pattern is often laid down by the growth of secondary hydranths between existing, normally full grown, hydranths. The secondary hydranths do not grow at random but at a distance from an existing hydranth; further, within a colony they all tend to space themselves either to the primary hydranth towards the growing end or all to the primary hydranth away from the growing tip. In this way commonly one, less often two, and occasionally three secondaries may grow in an internode, sometimes together and sometimes separately. A pattern is also found in the position of branch stolons. These are initiated more commonly near to a hydranth, primary or secondary, the opposite situation to that of secondary hydranth branches; their origin probably is not random except perhaps in extremely vigorous colonies.
These observations recall the work initiated by Child (1941) on inhibitor gradients in hydroids and their influence on, especially, the patterns of regeneration. These ideas have been considered and developed by Barth (1940), Burnett (1961 and 1966), Tardent (1963), Clarkson (1969) and Webster (1971).
The causes of the spacing between the primary hydranths and the pattern of secondary hydranths and branch stolons will be considered in another paper after further relevant observations have been described.
3 Vigour
The term ‘vigour’ as applied to different colonies has been used as a descriptive term earlier in this report without definition. The three colonies A, B and C can now be distinguished more precisely and the meaning of vigour more clearly described.
In general, more vigour means more hydranth and stolon branches, faster growth rate of stolon and more rapid regeneration of hydranths after regression. Long internodes become more frequent in colonies of decreasing vigour.
Two features which fail to conform to this pattern are the normal length of the internode and the length of life of a hydranth. Although colony B has a significantly shorter internode than A or C, the difference is a relatively small one (15% less). Another feature observed in colony C was the occasional break in the stolon coenosarc (autotomy?) separating one part of a colony from the rest. Breaks of this sort do not occur naturally in a normal or vigorous colony and artificially made breaks repair within a few hours. In colony C the gaps became wider, the impression being gained that as the stolon tip continued to grow, advance and bud hydranths the colony was not creating enough new cellular material to maintain the extra parts of the colony so established; cells from older parts needed to be used.
4 Pattern and competitive growth
Part of the explanation of ‘vigour’ may be found in ideas first recorded by Downing (1905) and later by Spiegelman (1945). Downing suggested that regeneration and other growth features in Hydra might be due to competition for available materials in an animal. Spiegelman called this ‘competitive interaction’ between the cells, meaning a competition for limited materials by separate morphogenetically active centres.
The observations on Clytia have shown that colonies can differ widely in their so-called vigour. There is evidence that even different stolons forming a single colony differ in vigour. Also, since stolons tend to increase their rate of growth as they get longer, stolons and perhaps colonies of increased vigour might be formed.
The most common type of colony is like colony B. All of its stolons grow in length and hydranths are budded off fairly regularly; occasionally a longer interval ensues which might be due to a temporarily raised demand for materials for the growth of secondary hydranths and/or stolon branches elsewhere in the colony. The hydranths have a relatively long life-span and regeneration usually occurs soon after regression. Some secondary hydranths and branch stolons are produced; again insufficient materials may be the limiting factor to greater numbers.
At a lower level of ‘vigour’ the stolon grows more slowly. Hydranths are slightly more distant from one another and long internodes become more frequent. The life-span of the hydranths becomes shorter and regressed periods longer. Secondary hydranths and branch stolons are infrequently produced.
At a lower stage still, stolons may grow very slowly or stop growing or grow erratically. They tend to break and grow only at one end or at neither end. New hydranths are formed if the stolon grows but they remain open for relatively short periods and remain regressed for longer periods. Long internodes are more common. Secondary hydranths are not produced and branch stolons rare.
The low-vigour state may tend to perpetuate itself since, as the number of open hydranths is relatively small, it may be unable to catch sufficient food to raise its vigour.
A similar situation is seen by observing an isolated length of stolon from a normal colony. After an initial small contraction the stolon grows at both ends and then one or both bud a hydranth. Usually stolon growth ceases as the hydranths grow. In the absence of food the hydranths regress and regenerate two or three times, getting smaller each time. Eventually regeneration ceases and the stolon gets thinner and thinner and eventually it dies.
If food is given to the regenerate the stolons grow actively and hydranths of increasing size grow, and a normal animal is soon formed.
At the ‘most vigour’ end of the scale the stolons grow rapidly and hydranths are budded regularly; long internodes cease to occur. Hydranths have a relatively long life-span; surprisingly, regenerated hydranths live longer than the initial ones. Secondary hydranths and branch stolons are freely produced but not in unlimited numbers. This limitation is unlikely to be one of materials shortage since there are a great many open and feeding hydranths per ‘unit’ of colony. The spacing of secondary hydranths from primaries and the tendency of stolons to branch near to existing hydranths indicate a morphogenetic limitation of sites available for branches. These inhibitions to further growth also determine the basic morphological pattern of the animal.
To summarize, the animal may only demonstrate the full pattern of growth under the most vigorous of growth conditions. Most commonly the pattern is incomplete due probably to a competition for available materials or the inability of particular stolons to utilize it.
5 Hydranth regression
A feature of Clytia is the cyclic regression and regeneration of hydranths. That hydranths regress under unfavourable conditions has been known for a long time (Huxley & De Beer (1923) in Obelia and Campanularia, Child (1923) in Bougainvillea, Obelia and Gonothyraea).
It is also reported to occur in naturally occurring material of Obelia(Hammett, 1946) and in apparently vigorously growing material in cultures of Obelia and Campanularia(Crowell, 1953). Regression of hydranths was described by Gast & Godlewski (1903) in Pennaria and Thacher (1903) in Campanularia, Eudendrium and Pennaria. The re-use of cells in regressing hydranths in Clytia has been demonstrated by Hale (1964b). Mackie (1966) grew cultures of Tubularia in which there was neither regression nor autotomy and questioned Tardent’s (1963) conclusion that autotomy was a normal part of the life-cycle of these hydranths. That the calyptoblastic hydranth is the part of the colony most sensitive to adverse conditions there can be no doubt. But empty hydrothecae are found in freshly collected Clytia(and Obelia -Hammett, 1946) although this could be the result of predation. None of this conclusively proves that hydranth regression is a naturally occurring phenomenon. Nevertheless there seems to be strong grounds for believing it to be so.
It must be admitted that natural regression is a most surprising phenomenon and appears to be quite wasteful of energy, reminding one of cell death in vertebrate development (Glücksmann, 1951). Nevertheless, the re-use of its cellular material provides a considerable flexibility of growth potential. Tardent (1963) believes that in Hydra and Tabularia the hydranth cells sencsce and require replacement. Crowell (1953) sees an advantage in the feeding members being ‘youthful’. Apart from Mackie’s observation that in Tubularia autotomy and regression need not occur there is the observation (Hale, 1964b) that in Clytia the cells are used again in the growth of other structures and are thus unlikely to be senescent.
6 Stolon fusion
A further variable feature is that stolons, both in culture conditions and in naturally occurring colonies, may meet in their growth. When the tip of a growing stolon meets another stolon it either grows over it or fuses with it. In the latter case a hole is dissolved in the side of the perisarc tube by the tip of the advancing stolon and the coenosarc grows into it and fuses, the hydroplasms becoming confluent. Why some contacts result in fusion and others do not may only be due to the physical shapes of the juxtaposed faces. If the advancing tip is halted at the perisarc tube the chitin producing reaction is reversed to dissolve the hole in the opposing stolon. Fusion of the coenosarcs ensues and pulsations cease. Production of an anastomosing network does not alter the ratio of hydranths to stolon length, but fusion does reduce the number of growing points and thus reduces the pressure for growth materials (if one exists). Apparently fusion does not occur in Tubularia(Mackie, 1966).
7 Growth models
Loomis (1954) found that his cultures of Hydra grew logarithmically, presumably only in the earlier stages, as he later stated growth to follow a sigmoid curve (Loomis & Lenhoff, 1956). Fulton (1962) found the same for Cordylophora hydranths, the doubling time being about 3 days, but exponential growth was not predicted by his model based on the development of a single stolon (Fulton, 1963). Braverman (1962, 1963) described exponential growth of nutritative polyps in Podocorynae carnea and Braverman & Schrandt (1966) attempted to simulate growth with an electronic computer although they later described the hydroid as producing distinctly irregular colonies (Braverman & Schrandt, 1969).
In Clytia johnstoni it is seen that the stolonic growth rate increases with time and a linear regression is highly significant in ‘normal’ and ‘vigorous’ colonies. A similar test with the logarithms of the stolonic growth rate also gives a highly significant result. It is clear therefore that the total size of a colony increases in a curved form, but whether growth is exponential, quadratic or some higher order cannot be deduced because of the variability in the rate of growth.
The number of open hydranths produced in unit time depends on the rate of growth of its stolon. The number of open hydranths in a colony depends on the length of the life-cycle of the hydranth which is seen to be largely either 6–7 days or 10–11 days. It depends, thirdly, on the time taken for hydranths to regenerate -generally brief, but an increasing number fail to regenerate in colonies of decreasing vigour. Anastomoses will decrease the growth rate, perhaps in a fairly constant manner in a colony.
To summarize, although some important features of the pattern of growth have been defined, considerable variability remains. Further investigations might account for some of this variation. Alternatively, such variation might be intrinsic to this primitive animal; possibly it does not possess the mechanisms to grow with any greater precision.