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Studies of the fissiparous holothurian Holothuria parvula (Selenka) (Echinodermata: Holothuroidea)
195
J. Exp. Mar. Biol. Ecol., 1987, Vol. 111, pp. 195-211
Elsevier
JEM 00931
Studies of the fissiparous holothurian Holothuria parvda (Selenka) (Echinodermata : Holothuroidea) R. H. Emson’ and P. V. Mladenov* ‘Department
of Biology, King’s College, London,U.K.; ‘Depament of Biology. Mount Alltkon University, Sackville, New Brunswick, Canada
(Received 15 January 1987; revision received 18 May 1987; accepted 18 May 1987) Abstract: A Bermudan population of the fissiparous holothurian Holothuriaparvula
(Selenka) was sampled over a 13-month period (1984-1985). Fission was most frequent in the summer when water temperatures were > 25 “C. During fission, the holothurian split into roughly equal parts, and there was little difference in survival of the oral and anal ends. Regeneration of a new gut is a priority and feeding was possible within 2 months of fission. The majority of growth following fission occurred between April and July, just prior to the peak occurrence of fission. Many individuals were tilly regenerated within a year, so fission is possibly an annual event. Individuals showing evidence of multiple fission were found. The capacity for sexual
reproduction was limited and it appeared to occur mainly during the summer, which was also the peak period for asexual reproduction. No small (c 18 mm) individuals were ever found suggesting that larval recruitment to this population had not recently been success&l. The population has probably been maintained recently by fission. Key words: Sea cucumber; Bermuda; Asexual reproduction;
Sexual reproduction; Regeneration
INTRODUCTION
Holothuriaparvula (Selenka) is one of only six species of sea cucumber known to be capable of transverse fission, and one of only three in which fission is suff%ziently common to be potentially an important means of asexual reproduction (Emson & Wilkie, 1980). Very little is known about the phenomenon of fission or the biology of fissiparous holothurians as few workers have had the opportunity to study living populations. H. par&a is known from the Pacific (Benham, 1912). Bermuda, and the Caribbean, and has previously been studied briefly by Crozier (1917) (as H. captiva) and by Deichmann (1921). Crozier (1917) observed fission occurring in the laboratory but did not study it in detail. He stated that fission is a phenomenon associated with young (small) individuals, as larger animals seen by him showed no signs offission. Deichmann (1921) in addition to describing regeneration in H. par&a, demonstrated that fission can be a common Contribution 1120 of the Bermuda Biological Station for Research. Correspondence address: R.H. Emson, Department of Biology, King’s College (KQC), Kensington Campus, Campden Hill Road, London W8 7AH, U.K. 0022-0981/87/%03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)
196
R.H.EMSON
AND P.V.MLADENOV
phenomenon in this species as 65”/, of the preserved specimens studied by her showed evidence of fission. She too believed fission to be a characteristic of young animals. During a visit to Bermuda we were fortunate enough to find a large population of H. parwia and undertook a 13-month study of fissiparity and associated events in that population. The main objectives of the work were: (1) to ascertain the prevalence of fission; (2) to determine whether fission is seasonal; and (3) to determme whether sexual reproduction also occurs and, if so, how the two modes of reproduction interrelate. In addition, we wished to learn more about the basic process of regeneration following fission as this is relevant to the interpretation of some of the data on fissiparity . Specifically, we sought: (1) to learn whether regeneration of an oral or anal end can be achieved with equal facility ; (2) to determine the rate of regeneration ; and (3) to describe the basic sequence of events in the regeneration of internal structures.
MATERIALS
AND METHODS
The population of holothurians sampled was found at Fort St Catherine, Bermuda. The animals were found on the undersides of boulders from OS-l.5 m in length in 1-2 m of water. Our fast collection in August 1984 consisted of 57 animals which were narcotized in MgCl, for 1 h and then preserved in 10% buffered formahn for later dissection and internal ex~nation. Subsequent collections were made from the same population at x 2-month intervals from August 1984 to September 1985, and each collection comprised 30 randomly collected individuals. These were also narcotized for 1 h in MgCl, and then preserved in formalin. The intervals and sample size chosen were intended to provide adequate data without overcollecting the population. No difficulty was ever experienced in collecting 30 individu~s and the popuIation remained apparently numerous when collections terminated in September 1985. The following were noted for preserved specimens. (1) Total length {mm). (2) Wet weight (g; specimens blotted dry with paper towel before weighing to reduce error). (3) External evidence of regeneration. (4) Category of regeneration (i.e., oral or anal end regenerating). (5) Proportion of regenerating tissue to parent tissue (as a length). Individuals were then opened with a mid-dorsaI slit and the internal structures inspected. From this we obtained information on the following. (1) The state of regeneration internally. (2) The regeneration category (to corroborate ~fo~ation obtained from external observation). (3)The sexual status of the animal (whether gonads present and their maturity and size). Surface sea water temperatures for the period of the study (see Fig. 7) were supplied by the Bermuda Biological Station for Research.
FISSION IN BERMUDAN SEA CUCUMBER
197
RESULTS INCIDENCE
AND SEASONALITY OF FISSION
proportion of individuals in each sample in which regeneration of a new oral and anal end was externally obvious varied from 43 to 83% over the year (Fig. 1). Thus, evidence of a high incidence of fission was apparent in this population of H. panda throughout the year. Not all animals, however, showed evidence of regeneration. Whereas some of these may have been individuals in which regeneration was complete, and thus undetectable, some may also have been individuals which had never divided. The
80 %
Regenerating
20
60
4. 20 0
Fig. 1. Histogram showing changes in the proportion of H. parvula in each sample showing evidence of regeneration. Values of N are shown above each column.
The frequency of regenerating individuals varied annually, with the highest incidence of obviously regenerating individuals occurring in late summer (August-September) and the lowest incidence in early summer (Fig. 1). This annual pattern of fission became more evident when the relative proportions of the newly formed tissues (regenerate) and the parent tissue are established and divided into categories (Fig. 2). Newly split animals (i.e., individuals in which fission has obviously occurred recently as there was no sign of a regenerating oral or anal end) were present only in the August 1984 and July 1985 samples. In addition, animals in which splitting had relatively recently occurred (those in which the new oral or anal end formed c 20% of the total body length) were common only in the August 1984 and the July and September 1985 samples (Fig. 2). On the other hand, animals in the later stages of regeneration (i.e., where the length of the regenerate is 20-80% of the length of the “parent” tissue) increased in abundance through the autumn, remained numerous in spring, and only declined in number in the summer (Fig. 2). Fully regenerated animals (those in which there is no obvious division of the body into two halves, as well as those in which the body halves are of equal length) increased in abundance through the winter and spring, reaching a peak in May, and then declined to minimum levels in late summer. It thus appears that fission takes place principally in the summer and that subsequent regeneration takes place during the late summer, autumn and winter.
198
R.H. EMSON
100 LR F
AUG’84 N-57
AND P.V. MLADENOV
NOV N=31
MAR N=29
80 Oh60 40 20 0
100
MAY N-29
JUL N=30
SEP N=28
80 60 40 20 0 Fig. 2. Histograms showing through the year changes in proportion of the population of H. pan&a in various stages of regeneration. LR/LP is the ratio of the length of regenerating tissue to the length of parent tissue. The lines (fitted by eye) show the progression of peaks through time.
MODE
OF FISSION
During our field collections two pairs of animals were seen which we believe had recently split. Each pair consisted of a head and a tail without evidence of regeneration; the two halves were separated by only a few millimetres and were arranged in tandem. In each case, the two halves were similar in size suggesting that division normally divides the animal into two roughly equal parts. None of our animals divided in the laboratory, and thus we have no direct evidence on this point. Indirect support is, however, provided by analysis of data for length of newly split animals and of animals in very early stages of regeneration (new tissue < 20% of body weight). These data (Fig. 3) show that 57% of such animals were between 30 and 39mm in length (mean = 34.5 mm), which is roughly half the size of fully regenerated animals from the same sample (mean = 60.5 mm, n = 22). Furthermore, comparison of the mean lengths of newly split heads and newly split tails revealed them to be very similar (mean length of heads = 28.4 mm, and mean length of tails = 29.1 mm; hypothesis of equality cannot be rejected, Wilcoxon-Mann-Whitney test, P < 0.001). Thus, while it is possible that there are some eccentric divisions, the data indicate that in the majority of cases division is roughly midway between the oral and anal ends.
199
FISSION IN BERMUDAN SEA CUCUMBER
60
I
n=56
z-34-5
50 40 1
%
so 20 10 0
1
1
L 20 40 60 LENGTH mm
Fig. 3. Histogram showing the length of recently split individuals.
REPETITION OF DIVISION Six of 233 animals examined showed external evidence of having undergone two fissions. In these specimens, the body was divisible into three areas that differed in diameter and pigmentation. In addition, the internal appearance of several other individuals suggested that they too had probably split more than once. This was indicated by differences in body wall colour and width of the longitudinal muscle bands, as well as the presence of unusually small respiratory trees and cuvierian organs. SURVIVAL OF REGENERATES
In August, shortly after the apparent peak of fission, the numbers of individuals regenerating oral and anal ends was virtually identical (Table I). In subsequent months the proportions varied, possibly as a result of small sample size, but when total records
TABLE I
Comparison of numbers of H. parvulu found regenerating the head or the tail. Totals are not significantly different, 1’ = 2.24, df = 1, P > 0.05. n
Aug. Nov. Jan. Mar. May JOY Sep. Totals
51
31 29 29 29 30 28
Number regenerating Tail
Head
21 7 8 14 5 6 10
27 16 14 4 9 9 11
71
90
200
R.H. EMSON AND P.V. MLADENOV
for the year are compared no significant difference from a 1: I ratio is seen (Table I). The evidence available thus suggests that both halves survive to regenerate and that there is little difference in survival of animals regenerating a head or a tail. LENGTH AND WEIGHT
Examination of the length frequency data shows that the lengths of holothurians in the samples ranged from 22 to 83 mm and, with the exception of the sample for July 1985, the mean was < 50 mm (Fig. 4). Comparison of the size frequency data (Fig. 4) with the numbers of individuals in the various regeneration categories (Fig. 2), demonstrates that the length-frequency distribution of the population was clearly affected by the habit of fission and subsequent regeneration. In August the population consisted of some large indi~du~s which have not split, many recently split ind~~duaIs, and some in an intermediate condition (Fig. 2). The result is a wide size-frequency range (Fig. 4). In November the mean size was lower, the largest animals having disappeared
100
c
9o i
B
B”-
I
70
~
IL
. . . Lb A
A
50 40
.
30 LEi!%TH
20 6o
IF
‘0
-I
* 1 B
A6
A
t I
lb
.
0 AUG.84
NOV
JAN.85
MAR
MAY
IUL
SEP
x
45-7
32.4
31-l
33-9
40 3
s&s
40.0
s D
12.6
45
5.9
81
IO-4
145
to-7
Fig. 4. Histograms to show length-frequency patterns of the population of N. pnrvuh through the year. Sample means are indicated by arrowheads. Analysis of variance revealed siguificant difference between sample means (F ratio = 22.19; df = 6, co;P d 0.001). Distributions with the same letter are not significantly different in mean (Student-Newman-Keuls test P < 0.05). Sample size as Fig. 2.
by this time (presumably having undergone fission) leaving a population in which most were small and in the early stages of regeneration (Fig. 4). Little change in size-frequency pattern occurred during the winter (Fig. 4), although the number of individuals in the higher categories of regeneration was increasing slightly (Fig. 2). Between March and July the mean length of the population increased by 80% (from 34 to 56 mm) (Fig. 4). Moreover, the data for July included a large number of small newly split individuals (Fig. 2). From this it appears that the majority of growth in length in this population was between April and July, immediately before the period when the incidence of fission was highest.
201
FISSION IN BERMUDAN SEA CUCUMBER
Examination of the weight frequency data (Fig. 5) reveals trend. There was a major weight decline between September weight was at its lowest, followed by a slight rise by January. constant until May. Between May and July, however, there
a similar but more dramatic and November, when mean Mean weight then remained was a mean weight increase C
9_
I
8 .
II II I
AB AB WEIGHT GRAMS
I
t
AUG 2.01 s D I-11 i
19B4
NOV l-24 0.53
‘JAN 154 068
’
;.Az 0-72
1985
MAY 157 o-79
.I Jill , 5.21 I.96
B I
SEP 2.53 F45
Fig. 5. Histograms to show weight-frequency changes in the population of H. pwvdu through the year. Sample means indicated by arrowheads. Sample means differ significantly (ANOVA, Fratio = 43.57; df = 6, co; P e 0.001). Distributions with the same letter are not significantly different in mean (S-N-K, P < 0.05). Sample size as Fig. 2.
of > 300% (from 1.6 to 5.2 g). Such a weight increase could be in part due to the presence of heavy gut contents at this time because gut content analysis indicated that H. pnrvula apparently feeds by ingesting coral fragments, sand, and detritus which are heavy. It was found, however, that 63% of the sample had empty guts, and 20% had only a small amount of gut contents. The weight increase was thus totahy the result of massive growth in the summer. The September sample had a si~~~~~y lower mean weight resulting from the period of fission which had just passed. It is notable that the mean weights for August 1984 and September 1985 were not significantly different; this may indicate that the pattern of length and weight change observed here is a regularly repeated phenomenon. Two differences between growth in length and in weight are apparent (Figs. 4,5). First, length began to increase between March and May, whereas weight had not risen at all during this period. Thus, growth in weight began later in the year. Secondly, the
202
R.H. EMSON
AND P.V. MLADENOV
relative magnitude of growth was different, that of weight being much greater than that of length and, in addition, achieved over a shorter time. SEXUAL
REPRODUCTION
Survey qf’gonad presence and maturity*
All individuals dissected were carefuly examined for the presence of gonads. The gonad is found in the anterior part of the body, attached to the right-hand side of the gut in an animal dissected from the dorsal surface (Fig. 6). Individuals with iden~able ta
ab cr
s dv tc
rm
al dl
rt r
Fig. 5. Dorsal view of the internal organs of H. pcmulu. Fission results in separation of the body organs near the point arrowed. Key: ab, aquapharyngeal bulb; al, ascending loop of small intestine; co, cuvierian organ; cr, calcareous ring; dl, descending loop of small intestine; dv, dorsal haemal vessel; g, gonad; m, madreporite; pv, polian vesicle; r, rectum; rm, rete mirabile; rt, respiratory tree; s, stomach; ta, tentacular ampullae; tc, transverse connection of haemal system; vv, ventral haemal vessel.
203
FISSiON IN BERMUDAN SEA CUCUMBER
gonads were present in the population throughout the year in small numbers (Fig. 7B). Only between July and September was it possible to find individuals with large (> 5 % total body weight) gonads containing mature or near-mature sex products and potentially capable of gamete reiease. In July and August such individuals formed only a small propo~on of the whole (Fig. 7B) so potential gamete production is app~ently small. Thus, it appears that if sexual activity occurred, it was during July, August, and September, the period when fission was also taking place (Fig. 7A,B). KEY
80
II
Ia
~
Recently split
509%
< 50 % regenerated
hrlly regenerated
0 AUG 30 6
%
NOV
JAN
MAR
MAY
JAN
MAR
MAY
JUL
regenerated
SEP
.
20. to. 0
AUG
NOV
30,
n JUL
SEP
l /l.
J
MMJ
SNJ
M’M
J
SN
Fig. 7. A, B, Histograms showing the correspondence in time between the peak of asexual reproduction and the time of highest sexual activity in H. parvula at Fort St Catherine, Bermuda. Sample size as Fig. 2. A, proportions in various regeneration categories (asexual reproduction is indicated by the presence of newly split animals in the population). B, numbers of individuals in each sample with detectable gonads (open columns) and with large gonads untying mature or near-mature oocytes (solid columns). C, graph of mean sea surface temperature at Bermuda 1984-1985.
Egg size
Thirty oocytes from an apparently ripe female specimen were measured in a cavity slide. They were very consistent in size, measuring 84-96 pm in diameter (mean = 93 w, SD k 3, n = 30). Assuming that the oocytes were close to their fi.rhsize this holo~~~ is likely to have a pI~kto~ophic mode of development (Thorson, 1946; Tyler & Gage, 1983).
204
R.H. EMSON AND P.V. MLADENOV
Regenerative siate and presence of gonads
Analysis of the regenerative condition of individuals (Table II) reveals that gonads are principally associated with animals in the later stages of regeneration and those which have fully regenerated. There was no significant difference between the number of individuals with gonads regenerating oral and anal ends (x2 = 3.0, df = 1, P > 0.05).
Regenerative category (i.e., proportion of regenerating tissue to overall length) of H. parvuiawith gonads. Regenerative category
Aug. Nov. Jan. Mar. July Sep.
<20
21-49
F&y regenerated
5* 1 0 0 0 0
10 2 0 3 3 2
5 2 5 4 3 0
* Includes regressing gonads present in two newly split individuals.
EVENTS AND SEQUENCE OF R~~EN~R~rION
Position of split in internal organs
Examination of recently split individuals reveals both the position at which the internal organs of N. parvula characteristically divide and the partitioning of the organs between the products of fission. In most individuals fission occurs midway between the oral and anal ends and results internally in the breaking of the gut at or near the junction between the stomach and intestine (Fig. 6). As a result, the posterior half of the splitting animal retains most of the body organs including, in some instances, the gonad. Thus the body cavity of a recently split animal regenerating the oral end appears crammed with organs. The anterior end on the other hand retains only the a~~h~ge~ bulb and sometimes the gonad, so the body cavity of a recently split animal regenerating the anal end appears empty. Consequences
qf jhion
It is plain that the structures needing to be replaced by each of the fission products are radically different. The oral half must regenerate virtually all the viscera (most of the gut, haemal system, cuvierian organs, and respiratory trees) and form a new anus. On the other hand, while the anal half has to regenerate only a small section of gut to reconstitute the viscera, it also has to replace the whole anterior end including the feeding tentacles, the central components of the water vascular system and the nervous
FISSION IN BERMUDAN
SEA CUCUMBER
205
system. Neither half can feed immediately after fission as one lacks a gut, the other the feeding apparatus. Examination of specimens in the early stages of regeneration has revealed the sequence of events in regeneration of the internal organs. Regeneration of a new anal end. The major event internally is the regeneration of a new
gut tube and the sequence of events is as follows. Stage 1. A rod of cells appears along the mesentery to which the remaining gut is attached (Fig. 8, Al). Stage 2. This rod becomes a straight tube (Fig. 8, A2). A sheet of tissue forms across the floor of the body cavity under the tube. Stage 3. The tube continues to grow in length and becomes bent centrally and begins to become differentiated. The tube forms attachments to the body wall near the next adjacent ambulacrum. A haemal vessel becomes visible on the internal margin at this stage (Fig. 8, A3). The anus becomes apparent in most individuals at this stage. Stage 4. Elongation and also widening of the gut (particularly in the intestinal region) continues until the gut has assumed an approximation of its original shape, and ditferentiation of the gut continues. The haemal tissues extend over the junction between the ascending small intestine and the large intestine and the transverse connection of the ventral haemal sinus is established (Fig. 8, A4). Stage 5. Further growth and differentiation of all systems including the longitudinal muscles, cuvierian tubules and respiratory trees occurs. The gut becomes functional at Stage 3 or Stage 4 (i.e., sediment is being passed through it and presumably some digestion is taking place). A functional gut has been found in an animal with a tail regenerate of only 2 mm (total length 32 mm) and most animals with the gut at Stage 3 had some material in their guts. Several had highly distended guts even when there was no evidence of structural differentiation of the gut. The first rudiments of the other visceral organs appear only at or after Stage 4 is reached. This is not surprising as the most prominent of these, the respiratory trees and the cuvierian organs, are derived from the gut. Animals with regenerates as small as 3 mm (9% total body length) may, however, have small but recognizable respiratory trees. Individuals regenerating the anal end with a regenerate in excess of 10% body length are likely to be able to feed actively. Regeneration of a new oraiend. The regeneration of new anterior structures is fast made
obvious both internally and extemahy by the presence of a short length of paler tissue. Stage 1. At this time no tentacle buds are obvious externally, but internally there may be several indications that regeneration may be in progress. The most anterior point of the body cavity may be filled with a mass of tissue and each ambulacrum is extended forward onto the new tissue as a thin line. This ambulacral reorganization is in progress by the time that gut regeneration is first evident. The first evidence of
206
R.H. EMSON
A
1
AND P.V. MLADENOV
tm B 1. Im
rg hv nlm
2.
ng
Fig. 8. Sketches of stages of early regeneration ofH. purvulcr. A, regeneration of a new posterior end: 1, gut at rod of cells stage; 2, gut at straight tube stage; 3, gut at early bending stage; 4, functional gut with gut contents. B, regeneration of a new anterior end: 1, gut at rod of cells stage; 2, gut at early tube stage; 3, gut at later tube stage with developing calcareous ring; 4, early head tentacle regeneration stage, gut potentially functional. Key: bw, body wall; cr, calcareous ring; gb, gut bend; gp, gut primordium; hv, haemal vessel; Im, longitudinal muscle; nlm, new longitudinal muscle; ng, new gut tissue; nt, new tissue; pc, position of sheet of mesentery like cells on floor of cavity; pv, polian vesicle; rg, residual gut; rm, new rete mirabile; ta, tentacular ampulla; tc, transverse connection of ventral haemal vessel; tm, tissue mass.
FISSION
IN BERMUDAN
207
SEA CUCUMBER
gut regeneration is a thickening at the edge of the mesentery to which the remaining part of the gut is attached (Fig. 8, Bl). Stage 2. The thickening becomes a rod and then a narrow tube (Fig. 8, B2) which increases in length and diameter as the oral end enlarges. Stage 3. The gut has become an obvious tube (Fig. 8, B3). Both the rod and the tube are always widest where they join the remaining gut (Fig. 8, B2, B3). Stage 4. The gut is functional. The gut can only become functional in an animal regenerating the oral end when both the gut and the food collecting equipment are sufficiently regenerated. The regeneration of the head tentacles occurs later than the initial stages of gut formation as it is dependent on the reestablishment of the circum-oral water ring and consequently the gut reaches the hollow tube stage before any tentacle buds develop. The tentacles appear as ten tiny stubs which become papillate and branch as they grow. They apparently become able to handle food particles when they have reached x 500 pm in diameter, as food has been found in the gut of an animal with tentacles of this size (Table III, Sept27). Fig. 8, B4 illustrates an animal capable of feeding. The food consisted of very small particles (x 50 pm in diameter) of detritus and coral. This animal had a regenerate which was only 6 y0 of total body length and head tentacles only 20% of the size of a full-sized set. Subsequently, as both the tentacles and the gut aperture (the gut width at the calcareous ring of the aquapharyngeal bulb) increase in TABLE III
Tentacle Animal number
Regeneration category
Aug40 A49 A53 A35 Sept27 A50 A32 S23 s9 A38 s2 s22 S28 Al4 S23 Controls Jul30 s30 523
O/38 o/33 5122 O/28 2132 2134 2132 5127 6/30 4133 513 1 IO/40 5126 5144 5126
dimensions,
gut aperture
and food particle
size.
Tentacle diameter
Gut aperture
Food particle size
(mm)
(mm)
(mm)
0.23 0.26 0.29 0.4-0.58 0.47-0.52 0.47-0.58 0.58 0.88-0.94 1.05-1.17 1.05-1.23 1.05-1.23 1.0-1.35 1.35-1.45 1.47-1.76 1.47-1.76
0.17 0.23 0.29 0.23 0.33 0.29 0.58 0.47 0.47 0.47 0.47 0.29 0.88 0.70 0.82
2.05-2.35 2.35 2.64
0.88 0.76 0.88
0.05 0.05 0.41-0.64 0.52-0.70 0.05 0.47-0.58 0.17-0.29 0.64-1.05 0.11 1.47-1.76
(nonregenerating) 1.76-2.64 0.58-0.91 0.75-1.05
208
R. H. EMSON AND P.V. MLA~ENOV
size (Table III), the holothu~~s become capable of processing larger material. When the tentacles are only 60% of fully regenerated size they are capable of collecting particles as large as those found in the guts of fully regenerated animals (Table III, S28). It should be noted that Table III is made up of data from the animals with the lowest amount of regeneration from the August and September samples. This is significant because it provides some indication of the time scale of important events in regeneration. As the incidence of regeneration is highest in July and August (Fig. 2) it suggests that animals which undergo fission are capable of feeding within two months of splitting. DISCUSSION
The data and observations above provide significant new information about fission and its importance to H. panda. It seems that the population of H. panda at Fort St Catherine, Bermuda, may be dependent upon asexual reproduction for maintenance of numbers. Thus, over a 13-month period, no specimens < 18 mm in length were found and those of small size were principally recently split “half” animals. This kind of size frequency pattern, where there are no obvious juveniles in the population, is common among fissiparous echinoderms (Emson, 1978; Mladenov etal., 1983, 1986). The absence of small animals presumably represents a failure of sexual reproduction to produce enough larvae for any to have survived to recruit as juveniles into the population in the immediate past. Highly variable success in recruitment is common in ech~ode~s with a pl~kto~ophic mode of development both in normal sexually reproducing species (Thorson, 1950) and in other fissiparous species (Emson & Wilkie, 1980; Crump & Barker, 1985). In the case of ff. panda, the egg size of which may indicate planktotrophic development (Tyler & Gage, 1983), the low frequency of detectable gonads in the population for most of the year, and the small numbers of individuals with mature gonads at the apparent season of sexual reproduction, seem to suggest that reproductive output is often small and, consequently, the chance of recruits arising from sexual reproduction is also small. Successful sexual reproduction may, however, occur in particular circumstances. Crozier (1917) reports that some of the H. panda studied by him were 6 mm in length and were described as young. It seems rather likely that these ~~vidu~s were derived from successful sexual reproduction. On the other hand, the high propo~ion of the population which has undergone transverse fission and SuccessfulIy completed regeneration indicates that fission is an important reproductive method for this species, as it appeared to be for H. sutinamensis from Bermuda (Crozier, 1917) and H. atra at Eniwetak (Ebert, 1978,1983). The implication of the large numbers of H. parvula at Fort St Catherine in the absence of evidence of sexual reproduction, is that fission is an important, perhaps dominant, means of recruitment to this population. No direct evidence exists of the proportion of “halves” that survive following fission in holothurians. The few reports of laboratory maintained regenerating individuals (Dalyeli, 1851; Monticelh, 1896) indicate that survival is normai for both halves. Our
FISSION IN BERMUDAN SEA CUCUMBER
209
data suggest that individuals of H. parvda split into more or less equal parts and that there is little difference in survival of the two halves. This is in close correspondence with the conclusions of Crozier (1917) and of Deichmann (1922). In many Bssiparous echinoderms there is an inverse relationship between size and tendency to split (Emson & Wilkie, 1980, for review) and Crazier (1917) thought this to be the case for H. panda. His conclusion was based on the fact that fission was very uncommon in a sample of large individuals of H. panda from a Bermudan population. Tbere is, however, little evidence to support this contention in our data. The absence of large individuals in the autumn when they are prominent in July can only be explained by their emigration, death or division. The presence of large numbers of ~~~du~s in the early stages of regeneration supports the view that division is the likely cause. The large size of some of the “halves” (Fig. 5) provides strong support for the beliefthat all sizes of individual are prone to fission. This also appears true for H; alra (Bonham & Held, 1963; Ebert, 1983). The data presented indicate that fission is concentrated in, and probably confined to, the summer, with regeneration occupying the succeeding late summer, autumn, and winter. Fig. 7C shows that the annual variation in temperature of the sea at Bermuda is considerable, and that fission occurs during the period when the mean sea temperature is > 25 “C. it may be that the trigger for fission is the combination of hi&~temperatures with physical disturbances at low tides, as is possibly the case for H. afru at Rongelap Atoll (Bonham & Held, 1963) and at Guam (Doty, 1977, in Ebert, 1983). This requires further investigation. Examination of individuals in the early stages of regeneration showed that restoration of the ability to feed is a priority. Individuals regenerating the oral end rapidly replace the missing section of gut. This may result from regeneration being by means of morphallaxis, where there is a ready source of appropriate cells in the adjacent remaining gut (Gibson & Burke, 1983). Regeneration of the tentacles is slower, being part of the complex events involved in regeneration of the anterior water vascular system. Once tentacle buds appear, they, however, become functional at a very early stage of development and thus food is found in the gut of animals with minute anterior ends. Regeneration of the anal end requires replacement of the whole of the viscera. Again, the priority is the gut and this is reconstituted rapidly. Full ~erentiation is not necessary before the animal begins to f& again, but whether digestion can be carried out by this “immature” gut has not been established. Although most individuals which split in summer appear to be regenerated sutliciently to be capable of feeding by autumn, it seems that rapid growth only occurs in late spring and summer and results in a high proportion of fully regenerated animals by July. We sug8est that the slow rate of regeneration through the winter results from a combination of low temperature, a reduced food supply, and a lowered rate of food acquisition due to the fact the animal is regenerating. In late spring and summer, temperatures are higher, food is likely to be more available and the animals are more fully formed and able to exploit the food optimally.
210
R.H. EMSON AND P.V. MLADENOV
The data (Fig. 4) suggest that many individuals may regenerate fully in a year. The presence of some individuals in the later stages of regeneration in the September samples suggests that this is, however, not universal. If regeneration is often complete in a year, then it is possible that fission is an annual event for many individuals. Evidence to show that repeated fission is possible comes from the presence in the population of individuals regenerating both ends. This appears to be the first record of morphological evidence of repeated fission in a natural population of holothurians although it has been seen in the laboratory (Monticelli, 1896) and Ebert (1983) has proved mathematically that it occurs in H. atra. Whether repeated fission is characteristic of H. parvula remains unknown. The restriction of the major proportion of splitting to the summer in H. parvula is in line with the results of Doty (1977, in Ebert, 1983) who detected a higher incidence of regenerating individuals of H. atra in December than in April which suggested a seasonality to fission. It contrasts, however, with the observations of Bonham & Held (1963) and of Harriott (1978, in Ebert, 1983) who could not detect seasonal differences in levels of fission in H. atra. Ebert (1983) comments that if, as Doty (1977, in Ebert, 1983) suggests, fission is stimulated by environmental stress then asexual reproduction should be “less significant” further from the equator. Our results which show that asexual reproduction is restricted to a small part of the year at Bermuda in which environmental stress is likely to be maximal provide indirect support for this contention. The gradual progress of regeneration through the winter results in an advanced state of internal regeneration in the majority of the population by early summer. A slight increase in the proportion of animals with gonads is seen in July and more of these gonads are large and mature. The low frequency of individuals with gonads suggests that the need to regenerate is a constant inhibition on the development of gonads. It is notable that it is at the peak of asexual reproduction that sexual activity is also possible. In this species, therefore, it is evident that sexual and asexual reproduction are temporally related. The presence of large gonads has not been found to deter fission in fissiparous brittlestars (Mladenov et al., 1983) and this is also true for H. parvula, recently split individuals having been found with a large gonad present. Whether asexual and sexual reproduction are linked is not clear but it should be noted that Chadwick (1900) found that fission followed egg release in Ocnusplanci. Although it seems unlikely that this occurs in Holothuria parvula, it is possible that some physiological process associated with gonad development or maturation may also induce a “fission ready” state in H. parvula. Fission is then triggered by appropriate environmental conditions. What causes fission? What restricts the fission plane to a point approximately midway between the oral and anal end? How is fission achieved? These are all questions to which we have as yet no answers. We hope that further investigation of this population will provide answers to these and other questions.
FISSION IN BERMUDAN SEA CUCUMBER
211
ACKNOWLEDGEMENTS
This work was made possible by grants from the National Geographic Society (Grant 2839-84), NSERC of Canada (Grant A7604), and the Donner Foundation. We gratefully acknowledge the field assistance of Sally Carson and the willing help of Sue Jickells with the collection of samples. Kevin Brady assisted with data analysis. We thank the Bermuda Biological Station for Research for provision of facilities for the initial stages of this work and for the water temperature data. We also thank R. Burke for suggestions concerning the manuscript.
REFERENCES BENHAM, W. B., 1912. Report on sundry invertebrates from the Kermadec Islands. Truns. N.Z. Inst. Wellington, Vol. 44, pp. 135-138. BONHAM, K. & E.E. HELD, 1963. Ecological observations on the sea cucumbers Holofhuria atra and H. leucospilota at Rongelap atoll, Marshall Islands. Pac. Sci., Vol. 17, pp. 305-314. CHADWICK,H. C., 1900. Notes on Cucumaria planci. Proc. Trans. Liverpool Biol. Sot., Vol. 5, pp. 81-82. CROZIER,W.J., 1917. Multiplication by fission in holothurians. Am. Nat., Vol. 51, pp. 560-566. CRUMP, R.G. & M. F. BARKER, 1985. Sexual and asexual reproduction in geographically separated population of the llssiparous asteroid Coscinasterius calamariu (Gray). J. Exp. Mar. Biol. Ecol., Vol. 88, pp. 109-127. DALYELL,J. G., 185 1. Thepowers of the Creator displayed in the creation. Vol. 1. Van Voorst, London, 286 pp. DEICHMANN,E., 1921. On some cases of multiplication by fission and coalescence by holothurians; with notes on the synonymy of Actinopyga pan&a (Se]). Vidensk. Medd. Dansk. Naturhist. Foren., Vol. 73, pp. 183-191. EBERT, T. A., 1978. Growth and size of the tropical sea cucumber Holothuria (Halodeima) atru Jager at Enewetak Atoll, Marshall Islands. Pac. Sci., Vol. 32, pp. 183-191. EBERT,T.A., 1983. Recruitment in echinoderms. In, Echinoderm studies. I, edited by M. Jangoux & J.M. Lawrence, A.A. Balkema, Rotterdam, pp. 169-203. EMSON,R. H., 1978. Some aspects of fission in Aliostichasterpolyplax. In, Physiology and behaviour of marine organisms, Proc. 12th Eur. Symp. Mar. Biol., edited by D. S. Mclusky & A.J. Berry, Pergamon Press, Oxford, pp. 321-329. EMSON,R. H. & I. C. WILKIE, 1980. Fission and autotomy in echinoderms. Oceanogr. Mar. Biol. Annu. Rev., Vol. 18, pp. 155-250. GIBSON, A. W. & R. D. BURKE, 1983.Gut regeneration by morphallaxis in the sea cucumber Leptosynapta clarki (Heding, 1928). Can. J. Zoel., Vol. 61, pp. 2720-2732. MLADENOV,P.V., S. F. CARSON & C. W. WALKER,1986. Reproductive ecology of an obligately lissiparous population ofthe sea-star Stephanasterias albula (Stimpson). J. Exp. Mar. Biol. Ecol., Vol. 96, pp. 155-175. MLADENOV,P. V., R. H. EMSON,L. V. COLP~TS & I. C. WILKIE, 1983. Asexual reproduction in the West Indian brittle star Ophiocomella ophiactoides (H. L. Clark) (Echinodermata : Ophiuroidea). J. Exp. Mar. Biol. Ecol., Vol. 72, pp. l-23. MONTICELLI,F. S., 1896. Sull’autotomia delle Cucumaria plunci. (Br). Atti Accad. Nuz. Lincei Cl. Sci. Fir. Mat. Nat. Rendi. Ser. 5, Vol. 5, pp. 231-239. THORSON,G., 1950. Reproduction and larval ecology of marine bottom invertebrates. Biol. Rev., Vol. 25, pp. l-45. TYLER, P.A. & J.D. GAGE, 1983. The reproductive biology of Ypsiothuna talismani (Holothuroidea: Dendrochirota) from the N. E. Atlantic. J. Mar. Biol. Assoc. U.K., Vol. 63, pp. 609-616.
J. Exp. Mar. Biol. Ecol., 1987, Vol. 111, pp. 195-211
Elsevier
JEM 00931
Studies of the fissiparous holothurian Holothuria parvda (Selenka) (Echinodermata : Holothuroidea) R. H. Emson’ and P. V. Mladenov* ‘Department
of Biology, King’s College, London,U.K.; ‘Depament of Biology. Mount Alltkon University, Sackville, New Brunswick, Canada
(Received 15 January 1987; revision received 18 May 1987; accepted 18 May 1987) Abstract: A Bermudan population of the fissiparous holothurian Holothuriaparvula
(Selenka) was sampled over a 13-month period (1984-1985). Fission was most frequent in the summer when water temperatures were > 25 “C. During fission, the holothurian split into roughly equal parts, and there was little difference in survival of the oral and anal ends. Regeneration of a new gut is a priority and feeding was possible within 2 months of fission. The majority of growth following fission occurred between April and July, just prior to the peak occurrence of fission. Many individuals were tilly regenerated within a year, so fission is possibly an annual event. Individuals showing evidence of multiple fission were found. The capacity for sexual
reproduction was limited and it appeared to occur mainly during the summer, which was also the peak period for asexual reproduction. No small (c 18 mm) individuals were ever found suggesting that larval recruitment to this population had not recently been success&l. The population has probably been maintained recently by fission. Key words: Sea cucumber; Bermuda; Asexual reproduction;
Sexual reproduction; Regeneration
INTRODUCTION
Holothuriaparvula (Selenka) is one of only six species of sea cucumber known to be capable of transverse fission, and one of only three in which fission is suff%ziently common to be potentially an important means of asexual reproduction (Emson & Wilkie, 1980). Very little is known about the phenomenon of fission or the biology of fissiparous holothurians as few workers have had the opportunity to study living populations. H. par&a is known from the Pacific (Benham, 1912). Bermuda, and the Caribbean, and has previously been studied briefly by Crozier (1917) (as H. captiva) and by Deichmann (1921). Crozier (1917) observed fission occurring in the laboratory but did not study it in detail. He stated that fission is a phenomenon associated with young (small) individuals, as larger animals seen by him showed no signs offission. Deichmann (1921) in addition to describing regeneration in H. par&a, demonstrated that fission can be a common Contribution 1120 of the Bermuda Biological Station for Research. Correspondence address: R.H. Emson, Department of Biology, King’s College (KQC), Kensington Campus, Campden Hill Road, London W8 7AH, U.K. 0022-0981/87/%03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)
196
R.H.EMSON
AND P.V.MLADENOV
phenomenon in this species as 65”/, of the preserved specimens studied by her showed evidence of fission. She too believed fission to be a characteristic of young animals. During a visit to Bermuda we were fortunate enough to find a large population of H. parwia and undertook a 13-month study of fissiparity and associated events in that population. The main objectives of the work were: (1) to ascertain the prevalence of fission; (2) to determine whether fission is seasonal; and (3) to determme whether sexual reproduction also occurs and, if so, how the two modes of reproduction interrelate. In addition, we wished to learn more about the basic process of regeneration following fission as this is relevant to the interpretation of some of the data on fissiparity . Specifically, we sought: (1) to learn whether regeneration of an oral or anal end can be achieved with equal facility ; (2) to determine the rate of regeneration ; and (3) to describe the basic sequence of events in the regeneration of internal structures.
MATERIALS
AND METHODS
The population of holothurians sampled was found at Fort St Catherine, Bermuda. The animals were found on the undersides of boulders from OS-l.5 m in length in 1-2 m of water. Our fast collection in August 1984 consisted of 57 animals which were narcotized in MgCl, for 1 h and then preserved in 10% buffered formahn for later dissection and internal ex~nation. Subsequent collections were made from the same population at x 2-month intervals from August 1984 to September 1985, and each collection comprised 30 randomly collected individuals. These were also narcotized for 1 h in MgCl, and then preserved in formalin. The intervals and sample size chosen were intended to provide adequate data without overcollecting the population. No difficulty was ever experienced in collecting 30 individu~s and the popuIation remained apparently numerous when collections terminated in September 1985. The following were noted for preserved specimens. (1) Total length {mm). (2) Wet weight (g; specimens blotted dry with paper towel before weighing to reduce error). (3) External evidence of regeneration. (4) Category of regeneration (i.e., oral or anal end regenerating). (5) Proportion of regenerating tissue to parent tissue (as a length). Individuals were then opened with a mid-dorsaI slit and the internal structures inspected. From this we obtained information on the following. (1) The state of regeneration internally. (2) The regeneration category (to corroborate ~fo~ation obtained from external observation). (3)The sexual status of the animal (whether gonads present and their maturity and size). Surface sea water temperatures for the period of the study (see Fig. 7) were supplied by the Bermuda Biological Station for Research.
FISSION IN BERMUDAN SEA CUCUMBER
197
RESULTS INCIDENCE
AND SEASONALITY OF FISSION
proportion of individuals in each sample in which regeneration of a new oral and anal end was externally obvious varied from 43 to 83% over the year (Fig. 1). Thus, evidence of a high incidence of fission was apparent in this population of H. panda throughout the year. Not all animals, however, showed evidence of regeneration. Whereas some of these may have been individuals in which regeneration was complete, and thus undetectable, some may also have been individuals which had never divided. The
80 %
Regenerating
20
60
4. 20 0
Fig. 1. Histogram showing changes in the proportion of H. parvula in each sample showing evidence of regeneration. Values of N are shown above each column.
The frequency of regenerating individuals varied annually, with the highest incidence of obviously regenerating individuals occurring in late summer (August-September) and the lowest incidence in early summer (Fig. 1). This annual pattern of fission became more evident when the relative proportions of the newly formed tissues (regenerate) and the parent tissue are established and divided into categories (Fig. 2). Newly split animals (i.e., individuals in which fission has obviously occurred recently as there was no sign of a regenerating oral or anal end) were present only in the August 1984 and July 1985 samples. In addition, animals in which splitting had relatively recently occurred (those in which the new oral or anal end formed c 20% of the total body length) were common only in the August 1984 and the July and September 1985 samples (Fig. 2). On the other hand, animals in the later stages of regeneration (i.e., where the length of the regenerate is 20-80% of the length of the “parent” tissue) increased in abundance through the autumn, remained numerous in spring, and only declined in number in the summer (Fig. 2). Fully regenerated animals (those in which there is no obvious division of the body into two halves, as well as those in which the body halves are of equal length) increased in abundance through the winter and spring, reaching a peak in May, and then declined to minimum levels in late summer. It thus appears that fission takes place principally in the summer and that subsequent regeneration takes place during the late summer, autumn and winter.
198
R.H. EMSON
100 LR F
AUG’84 N-57
AND P.V. MLADENOV
NOV N=31
MAR N=29
80 Oh60 40 20 0
100
MAY N-29
JUL N=30
SEP N=28
80 60 40 20 0 Fig. 2. Histograms showing through the year changes in proportion of the population of H. pan&a in various stages of regeneration. LR/LP is the ratio of the length of regenerating tissue to the length of parent tissue. The lines (fitted by eye) show the progression of peaks through time.
MODE
OF FISSION
During our field collections two pairs of animals were seen which we believe had recently split. Each pair consisted of a head and a tail without evidence of regeneration; the two halves were separated by only a few millimetres and were arranged in tandem. In each case, the two halves were similar in size suggesting that division normally divides the animal into two roughly equal parts. None of our animals divided in the laboratory, and thus we have no direct evidence on this point. Indirect support is, however, provided by analysis of data for length of newly split animals and of animals in very early stages of regeneration (new tissue < 20% of body weight). These data (Fig. 3) show that 57% of such animals were between 30 and 39mm in length (mean = 34.5 mm), which is roughly half the size of fully regenerated animals from the same sample (mean = 60.5 mm, n = 22). Furthermore, comparison of the mean lengths of newly split heads and newly split tails revealed them to be very similar (mean length of heads = 28.4 mm, and mean length of tails = 29.1 mm; hypothesis of equality cannot be rejected, Wilcoxon-Mann-Whitney test, P < 0.001). Thus, while it is possible that there are some eccentric divisions, the data indicate that in the majority of cases division is roughly midway between the oral and anal ends.
199
FISSION IN BERMUDAN SEA CUCUMBER
60
I
n=56
z-34-5
50 40 1
%
so 20 10 0
1
1
L 20 40 60 LENGTH mm
Fig. 3. Histogram showing the length of recently split individuals.
REPETITION OF DIVISION Six of 233 animals examined showed external evidence of having undergone two fissions. In these specimens, the body was divisible into three areas that differed in diameter and pigmentation. In addition, the internal appearance of several other individuals suggested that they too had probably split more than once. This was indicated by differences in body wall colour and width of the longitudinal muscle bands, as well as the presence of unusually small respiratory trees and cuvierian organs. SURVIVAL OF REGENERATES
In August, shortly after the apparent peak of fission, the numbers of individuals regenerating oral and anal ends was virtually identical (Table I). In subsequent months the proportions varied, possibly as a result of small sample size, but when total records
TABLE I
Comparison of numbers of H. parvulu found regenerating the head or the tail. Totals are not significantly different, 1’ = 2.24, df = 1, P > 0.05. n
Aug. Nov. Jan. Mar. May JOY Sep. Totals
51
31 29 29 29 30 28
Number regenerating Tail
Head
21 7 8 14 5 6 10
27 16 14 4 9 9 11
71
90
200
R.H. EMSON AND P.V. MLADENOV
for the year are compared no significant difference from a 1: I ratio is seen (Table I). The evidence available thus suggests that both halves survive to regenerate and that there is little difference in survival of animals regenerating a head or a tail. LENGTH AND WEIGHT
Examination of the length frequency data shows that the lengths of holothurians in the samples ranged from 22 to 83 mm and, with the exception of the sample for July 1985, the mean was < 50 mm (Fig. 4). Comparison of the size frequency data (Fig. 4) with the numbers of individuals in the various regeneration categories (Fig. 2), demonstrates that the length-frequency distribution of the population was clearly affected by the habit of fission and subsequent regeneration. In August the population consisted of some large indi~du~s which have not split, many recently split ind~~duaIs, and some in an intermediate condition (Fig. 2). The result is a wide size-frequency range (Fig. 4). In November the mean size was lower, the largest animals having disappeared
100
c
9o i
B
B”-
I
70
~
IL
. . . Lb A
A
50 40
.
30 LEi!%TH
20 6o
IF
‘0
-I
* 1 B
A6
A
t I
lb
.
0 AUG.84
NOV
JAN.85
MAR
MAY
IUL
SEP
x
45-7
32.4
31-l
33-9
40 3
s&s
40.0
s D
12.6
45
5.9
81
IO-4
145
to-7
Fig. 4. Histograms to show length-frequency patterns of the population of N. pnrvuh through the year. Sample means are indicated by arrowheads. Analysis of variance revealed siguificant difference between sample means (F ratio = 22.19; df = 6, co;P d 0.001). Distributions with the same letter are not significantly different in mean (Student-Newman-Keuls test P < 0.05). Sample size as Fig. 2.
by this time (presumably having undergone fission) leaving a population in which most were small and in the early stages of regeneration (Fig. 4). Little change in size-frequency pattern occurred during the winter (Fig. 4), although the number of individuals in the higher categories of regeneration was increasing slightly (Fig. 2). Between March and July the mean length of the population increased by 80% (from 34 to 56 mm) (Fig. 4). Moreover, the data for July included a large number of small newly split individuals (Fig. 2). From this it appears that the majority of growth in length in this population was between April and July, immediately before the period when the incidence of fission was highest.
201
FISSION IN BERMUDAN SEA CUCUMBER
Examination of the weight frequency data (Fig. 5) reveals trend. There was a major weight decline between September weight was at its lowest, followed by a slight rise by January. constant until May. Between May and July, however, there
a similar but more dramatic and November, when mean Mean weight then remained was a mean weight increase C
9_
I
8 .
II II I
AB AB WEIGHT GRAMS
I
t
AUG 2.01 s D I-11 i
19B4
NOV l-24 0.53
‘JAN 154 068
’
;.Az 0-72
1985
MAY 157 o-79
.I Jill , 5.21 I.96
B I
SEP 2.53 F45
Fig. 5. Histograms to show weight-frequency changes in the population of H. pwvdu through the year. Sample means indicated by arrowheads. Sample means differ significantly (ANOVA, Fratio = 43.57; df = 6, co; P e 0.001). Distributions with the same letter are not significantly different in mean (S-N-K, P < 0.05). Sample size as Fig. 2.
of > 300% (from 1.6 to 5.2 g). Such a weight increase could be in part due to the presence of heavy gut contents at this time because gut content analysis indicated that H. pnrvula apparently feeds by ingesting coral fragments, sand, and detritus which are heavy. It was found, however, that 63% of the sample had empty guts, and 20% had only a small amount of gut contents. The weight increase was thus totahy the result of massive growth in the summer. The September sample had a si~~~~~y lower mean weight resulting from the period of fission which had just passed. It is notable that the mean weights for August 1984 and September 1985 were not significantly different; this may indicate that the pattern of length and weight change observed here is a regularly repeated phenomenon. Two differences between growth in length and in weight are apparent (Figs. 4,5). First, length began to increase between March and May, whereas weight had not risen at all during this period. Thus, growth in weight began later in the year. Secondly, the
202
R.H. EMSON
AND P.V. MLADENOV
relative magnitude of growth was different, that of weight being much greater than that of length and, in addition, achieved over a shorter time. SEXUAL
REPRODUCTION
Survey qf’gonad presence and maturity*
All individuals dissected were carefuly examined for the presence of gonads. The gonad is found in the anterior part of the body, attached to the right-hand side of the gut in an animal dissected from the dorsal surface (Fig. 6). Individuals with iden~able ta
ab cr
s dv tc
rm
al dl
rt r
Fig. 5. Dorsal view of the internal organs of H. pcmulu. Fission results in separation of the body organs near the point arrowed. Key: ab, aquapharyngeal bulb; al, ascending loop of small intestine; co, cuvierian organ; cr, calcareous ring; dl, descending loop of small intestine; dv, dorsal haemal vessel; g, gonad; m, madreporite; pv, polian vesicle; r, rectum; rm, rete mirabile; rt, respiratory tree; s, stomach; ta, tentacular ampullae; tc, transverse connection of haemal system; vv, ventral haemal vessel.
203
FISSiON IN BERMUDAN SEA CUCUMBER
gonads were present in the population throughout the year in small numbers (Fig. 7B). Only between July and September was it possible to find individuals with large (> 5 % total body weight) gonads containing mature or near-mature sex products and potentially capable of gamete reiease. In July and August such individuals formed only a small propo~on of the whole (Fig. 7B) so potential gamete production is app~ently small. Thus, it appears that if sexual activity occurred, it was during July, August, and September, the period when fission was also taking place (Fig. 7A,B). KEY
80
II
Ia
~
Recently split
509%
< 50 % regenerated
hrlly regenerated
0 AUG 30 6
%
NOV
JAN
MAR
MAY
JAN
MAR
MAY
JUL
regenerated
SEP
.
20. to. 0
AUG
NOV
30,
n JUL
SEP
l /l.
J
MMJ
SNJ
M’M
J
SN
Fig. 7. A, B, Histograms showing the correspondence in time between the peak of asexual reproduction and the time of highest sexual activity in H. parvula at Fort St Catherine, Bermuda. Sample size as Fig. 2. A, proportions in various regeneration categories (asexual reproduction is indicated by the presence of newly split animals in the population). B, numbers of individuals in each sample with detectable gonads (open columns) and with large gonads untying mature or near-mature oocytes (solid columns). C, graph of mean sea surface temperature at Bermuda 1984-1985.
Egg size
Thirty oocytes from an apparently ripe female specimen were measured in a cavity slide. They were very consistent in size, measuring 84-96 pm in diameter (mean = 93 w, SD k 3, n = 30). Assuming that the oocytes were close to their fi.rhsize this holo~~~ is likely to have a pI~kto~ophic mode of development (Thorson, 1946; Tyler & Gage, 1983).
204
R.H. EMSON AND P.V. MLADENOV
Regenerative siate and presence of gonads
Analysis of the regenerative condition of individuals (Table II) reveals that gonads are principally associated with animals in the later stages of regeneration and those which have fully regenerated. There was no significant difference between the number of individuals with gonads regenerating oral and anal ends (x2 = 3.0, df = 1, P > 0.05).
Regenerative category (i.e., proportion of regenerating tissue to overall length) of H. parvuiawith gonads. Regenerative category
Aug. Nov. Jan. Mar. July Sep.
<20
21-49
F&y regenerated
5* 1 0 0 0 0
10 2 0 3 3 2
5 2 5 4 3 0
* Includes regressing gonads present in two newly split individuals.
EVENTS AND SEQUENCE OF R~~EN~R~rION
Position of split in internal organs
Examination of recently split individuals reveals both the position at which the internal organs of N. parvula characteristically divide and the partitioning of the organs between the products of fission. In most individuals fission occurs midway between the oral and anal ends and results internally in the breaking of the gut at or near the junction between the stomach and intestine (Fig. 6). As a result, the posterior half of the splitting animal retains most of the body organs including, in some instances, the gonad. Thus the body cavity of a recently split animal regenerating the oral end appears crammed with organs. The anterior end on the other hand retains only the a~~h~ge~ bulb and sometimes the gonad, so the body cavity of a recently split animal regenerating the anal end appears empty. Consequences
qf jhion
It is plain that the structures needing to be replaced by each of the fission products are radically different. The oral half must regenerate virtually all the viscera (most of the gut, haemal system, cuvierian organs, and respiratory trees) and form a new anus. On the other hand, while the anal half has to regenerate only a small section of gut to reconstitute the viscera, it also has to replace the whole anterior end including the feeding tentacles, the central components of the water vascular system and the nervous
FISSION IN BERMUDAN
SEA CUCUMBER
205
system. Neither half can feed immediately after fission as one lacks a gut, the other the feeding apparatus. Examination of specimens in the early stages of regeneration has revealed the sequence of events in regeneration of the internal organs. Regeneration of a new anal end. The major event internally is the regeneration of a new
gut tube and the sequence of events is as follows. Stage 1. A rod of cells appears along the mesentery to which the remaining gut is attached (Fig. 8, Al). Stage 2. This rod becomes a straight tube (Fig. 8, A2). A sheet of tissue forms across the floor of the body cavity under the tube. Stage 3. The tube continues to grow in length and becomes bent centrally and begins to become differentiated. The tube forms attachments to the body wall near the next adjacent ambulacrum. A haemal vessel becomes visible on the internal margin at this stage (Fig. 8, A3). The anus becomes apparent in most individuals at this stage. Stage 4. Elongation and also widening of the gut (particularly in the intestinal region) continues until the gut has assumed an approximation of its original shape, and ditferentiation of the gut continues. The haemal tissues extend over the junction between the ascending small intestine and the large intestine and the transverse connection of the ventral haemal sinus is established (Fig. 8, A4). Stage 5. Further growth and differentiation of all systems including the longitudinal muscles, cuvierian tubules and respiratory trees occurs. The gut becomes functional at Stage 3 or Stage 4 (i.e., sediment is being passed through it and presumably some digestion is taking place). A functional gut has been found in an animal with a tail regenerate of only 2 mm (total length 32 mm) and most animals with the gut at Stage 3 had some material in their guts. Several had highly distended guts even when there was no evidence of structural differentiation of the gut. The first rudiments of the other visceral organs appear only at or after Stage 4 is reached. This is not surprising as the most prominent of these, the respiratory trees and the cuvierian organs, are derived from the gut. Animals with regenerates as small as 3 mm (9% total body length) may, however, have small but recognizable respiratory trees. Individuals regenerating the anal end with a regenerate in excess of 10% body length are likely to be able to feed actively. Regeneration of a new oraiend. The regeneration of new anterior structures is fast made
obvious both internally and extemahy by the presence of a short length of paler tissue. Stage 1. At this time no tentacle buds are obvious externally, but internally there may be several indications that regeneration may be in progress. The most anterior point of the body cavity may be filled with a mass of tissue and each ambulacrum is extended forward onto the new tissue as a thin line. This ambulacral reorganization is in progress by the time that gut regeneration is first evident. The first evidence of
206
R.H. EMSON
A
1
AND P.V. MLADENOV
tm B 1. Im
rg hv nlm
2.
ng
Fig. 8. Sketches of stages of early regeneration ofH. purvulcr. A, regeneration of a new posterior end: 1, gut at rod of cells stage; 2, gut at straight tube stage; 3, gut at early bending stage; 4, functional gut with gut contents. B, regeneration of a new anterior end: 1, gut at rod of cells stage; 2, gut at early tube stage; 3, gut at later tube stage with developing calcareous ring; 4, early head tentacle regeneration stage, gut potentially functional. Key: bw, body wall; cr, calcareous ring; gb, gut bend; gp, gut primordium; hv, haemal vessel; Im, longitudinal muscle; nlm, new longitudinal muscle; ng, new gut tissue; nt, new tissue; pc, position of sheet of mesentery like cells on floor of cavity; pv, polian vesicle; rg, residual gut; rm, new rete mirabile; ta, tentacular ampulla; tc, transverse connection of ventral haemal vessel; tm, tissue mass.
FISSION
IN BERMUDAN
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SEA CUCUMBER
gut regeneration is a thickening at the edge of the mesentery to which the remaining part of the gut is attached (Fig. 8, Bl). Stage 2. The thickening becomes a rod and then a narrow tube (Fig. 8, B2) which increases in length and diameter as the oral end enlarges. Stage 3. The gut has become an obvious tube (Fig. 8, B3). Both the rod and the tube are always widest where they join the remaining gut (Fig. 8, B2, B3). Stage 4. The gut is functional. The gut can only become functional in an animal regenerating the oral end when both the gut and the food collecting equipment are sufficiently regenerated. The regeneration of the head tentacles occurs later than the initial stages of gut formation as it is dependent on the reestablishment of the circum-oral water ring and consequently the gut reaches the hollow tube stage before any tentacle buds develop. The tentacles appear as ten tiny stubs which become papillate and branch as they grow. They apparently become able to handle food particles when they have reached x 500 pm in diameter, as food has been found in the gut of an animal with tentacles of this size (Table III, Sept27). Fig. 8, B4 illustrates an animal capable of feeding. The food consisted of very small particles (x 50 pm in diameter) of detritus and coral. This animal had a regenerate which was only 6 y0 of total body length and head tentacles only 20% of the size of a full-sized set. Subsequently, as both the tentacles and the gut aperture (the gut width at the calcareous ring of the aquapharyngeal bulb) increase in TABLE III
Tentacle Animal number
Regeneration category
Aug40 A49 A53 A35 Sept27 A50 A32 S23 s9 A38 s2 s22 S28 Al4 S23 Controls Jul30 s30 523
O/38 o/33 5122 O/28 2132 2134 2132 5127 6/30 4133 513 1 IO/40 5126 5144 5126
dimensions,
gut aperture
and food particle
size.
Tentacle diameter
Gut aperture
Food particle size
(mm)
(mm)
(mm)
0.23 0.26 0.29 0.4-0.58 0.47-0.52 0.47-0.58 0.58 0.88-0.94 1.05-1.17 1.05-1.23 1.05-1.23 1.0-1.35 1.35-1.45 1.47-1.76 1.47-1.76
0.17 0.23 0.29 0.23 0.33 0.29 0.58 0.47 0.47 0.47 0.47 0.29 0.88 0.70 0.82
2.05-2.35 2.35 2.64
0.88 0.76 0.88
0.05 0.05 0.41-0.64 0.52-0.70 0.05 0.47-0.58 0.17-0.29 0.64-1.05 0.11 1.47-1.76
(nonregenerating) 1.76-2.64 0.58-0.91 0.75-1.05
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R. H. EMSON AND P.V. MLA~ENOV
size (Table III), the holothu~~s become capable of processing larger material. When the tentacles are only 60% of fully regenerated size they are capable of collecting particles as large as those found in the guts of fully regenerated animals (Table III, S28). It should be noted that Table III is made up of data from the animals with the lowest amount of regeneration from the August and September samples. This is significant because it provides some indication of the time scale of important events in regeneration. As the incidence of regeneration is highest in July and August (Fig. 2) it suggests that animals which undergo fission are capable of feeding within two months of splitting. DISCUSSION
The data and observations above provide significant new information about fission and its importance to H. panda. It seems that the population of H. panda at Fort St Catherine, Bermuda, may be dependent upon asexual reproduction for maintenance of numbers. Thus, over a 13-month period, no specimens < 18 mm in length were found and those of small size were principally recently split “half” animals. This kind of size frequency pattern, where there are no obvious juveniles in the population, is common among fissiparous echinoderms (Emson, 1978; Mladenov etal., 1983, 1986). The absence of small animals presumably represents a failure of sexual reproduction to produce enough larvae for any to have survived to recruit as juveniles into the population in the immediate past. Highly variable success in recruitment is common in ech~ode~s with a pl~kto~ophic mode of development both in normal sexually reproducing species (Thorson, 1950) and in other fissiparous species (Emson & Wilkie, 1980; Crump & Barker, 1985). In the case of ff. panda, the egg size of which may indicate planktotrophic development (Tyler & Gage, 1983), the low frequency of detectable gonads in the population for most of the year, and the small numbers of individuals with mature gonads at the apparent season of sexual reproduction, seem to suggest that reproductive output is often small and, consequently, the chance of recruits arising from sexual reproduction is also small. Successful sexual reproduction may, however, occur in particular circumstances. Crozier (1917) reports that some of the H. panda studied by him were 6 mm in length and were described as young. It seems rather likely that these ~~vidu~s were derived from successful sexual reproduction. On the other hand, the high propo~ion of the population which has undergone transverse fission and SuccessfulIy completed regeneration indicates that fission is an important reproductive method for this species, as it appeared to be for H. sutinamensis from Bermuda (Crozier, 1917) and H. atra at Eniwetak (Ebert, 1978,1983). The implication of the large numbers of H. parvula at Fort St Catherine in the absence of evidence of sexual reproduction, is that fission is an important, perhaps dominant, means of recruitment to this population. No direct evidence exists of the proportion of “halves” that survive following fission in holothurians. The few reports of laboratory maintained regenerating individuals (Dalyeli, 1851; Monticelh, 1896) indicate that survival is normai for both halves. Our
FISSION IN BERMUDAN SEA CUCUMBER
209
data suggest that individuals of H. parvda split into more or less equal parts and that there is little difference in survival of the two halves. This is in close correspondence with the conclusions of Crozier (1917) and of Deichmann (1922). In many Bssiparous echinoderms there is an inverse relationship between size and tendency to split (Emson & Wilkie, 1980, for review) and Crazier (1917) thought this to be the case for H. panda. His conclusion was based on the fact that fission was very uncommon in a sample of large individuals of H. panda from a Bermudan population. Tbere is, however, little evidence to support this contention in our data. The absence of large individuals in the autumn when they are prominent in July can only be explained by their emigration, death or division. The presence of large numbers of ~~~du~s in the early stages of regeneration supports the view that division is the likely cause. The large size of some of the “halves” (Fig. 5) provides strong support for the beliefthat all sizes of individual are prone to fission. This also appears true for H; alra (Bonham & Held, 1963; Ebert, 1983). The data presented indicate that fission is concentrated in, and probably confined to, the summer, with regeneration occupying the succeeding late summer, autumn, and winter. Fig. 7C shows that the annual variation in temperature of the sea at Bermuda is considerable, and that fission occurs during the period when the mean sea temperature is > 25 “C. it may be that the trigger for fission is the combination of hi&~temperatures with physical disturbances at low tides, as is possibly the case for H. afru at Rongelap Atoll (Bonham & Held, 1963) and at Guam (Doty, 1977, in Ebert, 1983). This requires further investigation. Examination of individuals in the early stages of regeneration showed that restoration of the ability to feed is a priority. Individuals regenerating the oral end rapidly replace the missing section of gut. This may result from regeneration being by means of morphallaxis, where there is a ready source of appropriate cells in the adjacent remaining gut (Gibson & Burke, 1983). Regeneration of the tentacles is slower, being part of the complex events involved in regeneration of the anterior water vascular system. Once tentacle buds appear, they, however, become functional at a very early stage of development and thus food is found in the gut of animals with minute anterior ends. Regeneration of the anal end requires replacement of the whole of the viscera. Again, the priority is the gut and this is reconstituted rapidly. Full ~erentiation is not necessary before the animal begins to f& again, but whether digestion can be carried out by this “immature” gut has not been established. Although most individuals which split in summer appear to be regenerated sutliciently to be capable of feeding by autumn, it seems that rapid growth only occurs in late spring and summer and results in a high proportion of fully regenerated animals by July. We sug8est that the slow rate of regeneration through the winter results from a combination of low temperature, a reduced food supply, and a lowered rate of food acquisition due to the fact the animal is regenerating. In late spring and summer, temperatures are higher, food is likely to be more available and the animals are more fully formed and able to exploit the food optimally.
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The data (Fig. 4) suggest that many individuals may regenerate fully in a year. The presence of some individuals in the later stages of regeneration in the September samples suggests that this is, however, not universal. If regeneration is often complete in a year, then it is possible that fission is an annual event for many individuals. Evidence to show that repeated fission is possible comes from the presence in the population of individuals regenerating both ends. This appears to be the first record of morphological evidence of repeated fission in a natural population of holothurians although it has been seen in the laboratory (Monticelli, 1896) and Ebert (1983) has proved mathematically that it occurs in H. atra. Whether repeated fission is characteristic of H. parvula remains unknown. The restriction of the major proportion of splitting to the summer in H. parvula is in line with the results of Doty (1977, in Ebert, 1983) who detected a higher incidence of regenerating individuals of H. atra in December than in April which suggested a seasonality to fission. It contrasts, however, with the observations of Bonham & Held (1963) and of Harriott (1978, in Ebert, 1983) who could not detect seasonal differences in levels of fission in H. atra. Ebert (1983) comments that if, as Doty (1977, in Ebert, 1983) suggests, fission is stimulated by environmental stress then asexual reproduction should be “less significant” further from the equator. Our results which show that asexual reproduction is restricted to a small part of the year at Bermuda in which environmental stress is likely to be maximal provide indirect support for this contention. The gradual progress of regeneration through the winter results in an advanced state of internal regeneration in the majority of the population by early summer. A slight increase in the proportion of animals with gonads is seen in July and more of these gonads are large and mature. The low frequency of individuals with gonads suggests that the need to regenerate is a constant inhibition on the development of gonads. It is notable that it is at the peak of asexual reproduction that sexual activity is also possible. In this species, therefore, it is evident that sexual and asexual reproduction are temporally related. The presence of large gonads has not been found to deter fission in fissiparous brittlestars (Mladenov et al., 1983) and this is also true for H. parvula, recently split individuals having been found with a large gonad present. Whether asexual and sexual reproduction are linked is not clear but it should be noted that Chadwick (1900) found that fission followed egg release in Ocnusplanci. Although it seems unlikely that this occurs in Holothuria parvula, it is possible that some physiological process associated with gonad development or maturation may also induce a “fission ready” state in H. parvula. Fission is then triggered by appropriate environmental conditions. What causes fission? What restricts the fission plane to a point approximately midway between the oral and anal end? How is fission achieved? These are all questions to which we have as yet no answers. We hope that further investigation of this population will provide answers to these and other questions.
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ACKNOWLEDGEMENTS
This work was made possible by grants from the National Geographic Society (Grant 2839-84), NSERC of Canada (Grant A7604), and the Donner Foundation. We gratefully acknowledge the field assistance of Sally Carson and the willing help of Sue Jickells with the collection of samples. Kevin Brady assisted with data analysis. We thank the Bermuda Biological Station for Research for provision of facilities for the initial stages of this work and for the water temperature data. We also thank R. Burke for suggestions concerning the manuscript.
REFERENCES BENHAM, W. B., 1912. Report on sundry invertebrates from the Kermadec Islands. Truns. N.Z. Inst. Wellington, Vol. 44, pp. 135-138. BONHAM, K. & E.E. HELD, 1963. Ecological observations on the sea cucumbers Holofhuria atra and H. leucospilota at Rongelap atoll, Marshall Islands. Pac. Sci., Vol. 17, pp. 305-314. CHADWICK,H. C., 1900. Notes on Cucumaria planci. Proc. Trans. Liverpool Biol. Sot., Vol. 5, pp. 81-82. CROZIER,W.J., 1917. Multiplication by fission in holothurians. Am. Nat., Vol. 51, pp. 560-566. CRUMP, R.G. & M. F. BARKER, 1985. Sexual and asexual reproduction in geographically separated population of the llssiparous asteroid Coscinasterius calamariu (Gray). J. Exp. Mar. Biol. Ecol., Vol. 88, pp. 109-127. DALYELL,J. G., 185 1. Thepowers of the Creator displayed in the creation. Vol. 1. Van Voorst, London, 286 pp. DEICHMANN,E., 1921. On some cases of multiplication by fission and coalescence by holothurians; with notes on the synonymy of Actinopyga pan&a (Se]). Vidensk. Medd. Dansk. Naturhist. Foren., Vol. 73, pp. 183-191. EBERT, T. A., 1978. Growth and size of the tropical sea cucumber Holothuria (Halodeima) atru Jager at Enewetak Atoll, Marshall Islands. Pac. Sci., Vol. 32, pp. 183-191. EBERT,T.A., 1983. Recruitment in echinoderms. In, Echinoderm studies. I, edited by M. Jangoux & J.M. Lawrence, A.A. Balkema, Rotterdam, pp. 169-203. EMSON,R. H., 1978. Some aspects of fission in Aliostichasterpolyplax. In, Physiology and behaviour of marine organisms, Proc. 12th Eur. Symp. Mar. Biol., edited by D. S. Mclusky & A.J. Berry, Pergamon Press, Oxford, pp. 321-329. EMSON,R. H. & I. C. WILKIE, 1980. Fission and autotomy in echinoderms. Oceanogr. Mar. Biol. Annu. Rev., Vol. 18, pp. 155-250. GIBSON, A. W. & R. D. BURKE, 1983.Gut regeneration by morphallaxis in the sea cucumber Leptosynapta clarki (Heding, 1928). Can. J. Zoel., Vol. 61, pp. 2720-2732. MLADENOV,P.V., S. F. CARSON & C. W. WALKER,1986. Reproductive ecology of an obligately lissiparous population ofthe sea-star Stephanasterias albula (Stimpson). J. Exp. Mar. Biol. Ecol., Vol. 96, pp. 155-175. MLADENOV,P. V., R. H. EMSON,L. V. COLP~TS & I. C. WILKIE, 1983. Asexual reproduction in the West Indian brittle star Ophiocomella ophiactoides (H. L. Clark) (Echinodermata : Ophiuroidea). J. Exp. Mar. Biol. Ecol., Vol. 72, pp. l-23. MONTICELLI,F. S., 1896. Sull’autotomia delle Cucumaria plunci. (Br). Atti Accad. Nuz. Lincei Cl. Sci. Fir. Mat. Nat. Rendi. Ser. 5, Vol. 5, pp. 231-239. THORSON,G., 1950. Reproduction and larval ecology of marine bottom invertebrates. Biol. Rev., Vol. 25, pp. l-45. TYLER, P.A. & J.D. GAGE, 1983. The reproductive biology of Ypsiothuna talismani (Holothuroidea: Dendrochirota) from the N. E. Atlantic. J. Mar. Biol. Assoc. U.K., Vol. 63, pp. 609-616.