www.elsevier.com/locate/jembe
Journal of Experimental Marine Biology and Ecology
303 (2004) 19–30
A maturity index for holothurians exhibiting
asynchronous development of gonad tubules
Luz M. Fogliettaa, Marıa I. Camejoa,Luis Gallardob, Francisco C. Herrerab,*
aDepartamento de Biologıa de Organismos, Universidad Simon Bolıvar, Caracas, VenezuelabLaboratorio de Ecofisiologıa Animal, Centro de Biofısica y Bioquımica, Instituto Venezolano de Investigaciones
Cientıficas (IVIC), Apartado 21827, Caracas 1020A, Venezuela
Received 22 May 2003; received in revised form 7 September 2003; accepted 28 October 2003
Abstract
The gonad morphology of the sea cucumber Isostichopus badionotus, collected during the
months of May to November 1996 in Morrocoy Bay on the northwestern Venezuelan coast, was
analyzed. The gonadal cycle was characterized by five stages of development: post-spawning,
recovery, growth, advanced growth and maturity. Maturation did not proceed at the same pace in all
gonad tubules of any one animal. Due to this asynchronous gonad development an Individual
Weighted Maturity Index (IWMI) was devised to determine reproductive state. It was calculated
from the proportion of the different tubule stages observed in each specimen. The maximum value
attainable is 5 if all tubules are in the mature stage. Towards the months of July and August, most,
but not all, of the ovarian and testicular tubules had reached maturity as indicated by IWMI values of
4.32 and 4, respectively. IWMI represents a quantitative estimation of gonad maturation in
holothurians exhibiting asynchronous development as it revealed the maturation pattern underlying
gonadal chronological development.
D 2003 Elsevier B.V. All rights reserved.
Keywords: Asynchronous gonad maturation; Holothurian reproduction; Isostichopus; Maturity index
1. Introduction
Considerable evidence indicates that many temperate aspidochirotes show a well-
defined annual reproductive cycle. Male and female Stichopus mollis show generally
0022-0981/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.jembe.2003.10.019
* Corresponding author. Tel.: +58-212-504-1450; fax: +58-212-504-1093.
E-mail address: [email protected] (F.C. Herrera).
L.M. Foglietta et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 19–3020
synchronous development (Sewell, 1992) and as in many aspidochirote species, spawning
occurs during the summer months (Tanaka, 1958; Cameron and Fankboner, 1986; Sewell
and Bergquist, 1990). Hyman (1955) believed that resorption of spawned gonad tubules
was probably the rule in holothurians. This suggests synchronicity of gonad development
in both sexes and of the gonad tubules in each animal and that the gametogenic state of
tubules is similar throughout the entire gonad. Yoshida (1952) proposed a maturity index
based on the number of individuals in each gonad stage present in each sample, implying
homogeneous development of gonad tubules within individuals. Smiley (1988) proposed a
‘‘tubule recruitment model’’ to describe gonad development in the class Holothuroidea.
This model proposes that anterior immature ovary tubules are progressively recruited to a
more posterior position as they develop to maturity. The tubules would develop as a single
cohort or as distinct cohorts that differ in age (Smiley et al., 1991). In species where the
gonad has distinct cohorts of tubules, gametogenesis is synchronous within cohorts but
asynchronous between cohorts (Ramofafia and Byrne, 2002). Tubule recruitment would
be compatible with either synchronous or asynchronous development of tubules in the
gonads of different species. However, Sewell (1992) has indicated that if the gonad is
completely reabsorbed, as occurs in S. mollis from the warmer waters of north–east New
Zealand, progressive recruitment of tubules cannot take place despite homogeneous tubule
development. Sewell et al. (1997) reported that gonad development in many species of the
orders Dendrochirotida, Apoditida and Molpaditida does not conform to the tubule
recruitment model and that it is not applicable to male holothurians. The tubules of the
ovaries of most holothuroids of these orders appear to develop synchronously and not in
successive cohorts. It would appear that whether recruitment takes place or not, tubule
development may be homogeneous in any given individual of species exhibiting
reproductive seasonality.
However, strict seasonality is not the rule in all temperate species. Holothuria edulis
from the deep portions of Heron Reef showed no annual reproductive pattern (Harriott,
1985). In the tropical sea cucumber Actinopyga mauritiana fully developed oocytes and
active sperm were found in some animals year-round (Hopper et al., 1998). Therefore, a
certain degree of asynchronicity or continuous gametogenesis within each tubule within
individuals occurs in some species.
Synchronization of tubule development within individuals underlies the definition of
maturity indices based on the number of individuals at each reproductive stage present in
each sample (Sewell, 1992; Yoshida, 1952). However, if tubule development is asynchro-
nous, this type of index cannot be defined. In this study histological examination was used
to document the cytological stages of the tubule development. A maturity index was
designed for animals with asynchronous gonad tubule development.
2. Methods
2.1. Collection and field conditions
Specimens of the holothurian Isostichopus badionotus were collected in Morrocoy Bay
on the northwestern Venezuelan coast (10j50VN and 68j15VW) at a depth of 0.5–2.5 m.
L.M. Foglietta et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 19–30 21
The animals tended to collect on the sandy bottom between the mangrove roots and the
Thalassia-covered shoal. Seawater temperature and salinity were measured at a depth of 1
m. Ten specimens, measuring 20 cm or more in length, were collected at monthly intervals
from March to November 1996. In the month of December, no specimens of I. badionotus
were found. A sudden mortality of this and other species in the shallows of the Morrocoy
Bay followed a marked increase in precipitation which caused a decrease in salinity from
36xto 32 xS between October and November (Fig. 1). This natural phenomenon has
been extensively documented by Laboy-Nieves et al. (2001).
2.2. Experimental procedure
At the collection site the gonads of each animal were divided into several fragments.
These were taken at random from each of the 10 animals in each collection and immediately
fixed in 10% formalin in natural seawater. The samples from each animal were numbered
and processed individually. Tissues were dehydrated and embedded in Paraplast. Sections
were cut at 3 Am and stained with Mayer’s hemalum and eosin. Two sections from each
specimen were mounted on each corresponding slide and the tubules of both sections were
classified and counted in duplicate tominimize subjective error. Sections were photographed
on a Zeiss Axioscop 20 microscope provided with an MC 80 photographic camera.
Histologic examination of tubules of individual gonads revealed that the tubules
exhibited different degrees of development. Therefore, each tubule was staged and assigned
to one of five categories: post-spawning, recovery, growth, advanced growth and mature.
Fig. 1. Monthly seawater temperatures and salinities taken at a depth of approximately 1 m at the site of specimen
collection from March to November 1996.
L.M. Foglietta et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 19–3022
The histology of these categories is described in Results. The number of tubules in each
category were counted and expressed as the percentage of the total number of tubules
observed in each slide. A weighted maturity index was calculated for each individual
(Individual Weighted Maturity Index, IWMI). This index was adapted from the maturity
indices published by Yoshida (1952) except that tubules in each slide replace individuals:
IWMI ¼ ½1ð% tubules in stage 1Þ þ 2ð%tubules in stage 2Þ þ . . .
þ nð%tubules in stage nÞ�=100Þ:
3. Results
Monthly temperatures and salinities taken at a depth of approximately 1 m are graphed
in Fig. 1. Seawater temperature increased steadily during the period studied, increasing
from 27 jC in March and April to 31 jC in September, October and November. Measured
salinity was relatively constant throughout the observation period (range: 36–40xS,
mean: 37.6xS) with the exception of the month of November when it dropped to
32xS. The salinity sample for May was inadvertently lost.
Of 90 specimens collected, 52 were male, 36 were female and two appeared to be
hermaphroditic as testicular tissue and oocytes were observed in different tubules of the
same individual as seen previously by Sewell (1990). A chi-square test was performed to
assess the probability that the proportion of male to female individuals corresponded to a
ratio of unity after exclusion of the two hermaphroditic specimens, as Sewell (1992) has
done for individuals of indeterminate sex. As the calculated chi-square value (2.91) was
below the tabulated value of 3.84 for p < 0.05, it was concluded that the sex ratio obtained did
not differ from 1. In 110 specimens collected in 1992, two hermaphroditic individuals were
observed, a proportion similar to that of 1996. In the sections of the gonads of the
hermaphroditic specimens studied, the male and female gametes appeared to occupy
separate tubules.
The color of the gonads ranged from white to ochre. The color did not appear to be
related to sex or season.
3.1. Gametogenesis
The gametogenic cycle of I. badionotus appeared to be a continuous process. However,
it could be characterized by five stages of activity similar to those proposed by Hamel et
al. (1993), Tanaka (1958), Engstrom (1980) and Costelloe (1985). The five stages are
illustrated in Figs. 2 and 3.
3.1.1. Oogenesis
The five following stages, shown in Fig. 2, were used to quantify the degree of
maturation of each tubule:
(1) Post-spawning. Tubule wall is thin. Elongated empty areas are seen in tubules
suggesting the passage of oocytes along the tubule during spawning. Some residual
Fig. 2. Light micrographs of sections of ovarian tubules. (a) Post-spawning: thin gonadal wall; channels resulting
from the passage of oocytes during spawning (CH); residual oocytes in different stages of deterioration; areas
surrounded by remnants of vitelline membranes, either empty or containing nutritive phagocytes. (b) Recovery:
tubule walls somewhat thicker; some developing oocytes line the tubule wall; abundant nutritive phagocytes
associated with degenerating oocytes (arrows). (c) Growth: tubule wall reaches maximum thickness and clusters
of previtellogenic oocytes line the germinal epithelium (arrow); vitellogenic oocytes occupy the lumen. (d)
Advanced growth: thinner tubular wall; well-defined follicular cells surround previtellogenic and vitellogenic
oocytes with germinal vesicles (arrow); previtellogenic oocytes line the germinal epithelium. (e) Mature: tubules
dilated and thin walled (arrow); the tubules are almost completely filled with mature oocytes surrounded by their
vitelline membrane, a few immature oocytes may be seen. (f) Tubules of any one ovary exhibit different degrees
of maturation. (PS) Post-spawning; (R) recovery with abundant nutritive phagocytes (NP); (G) growth; (AG)
advanced growth. Light bar in black rectangle represents 200 Am.
L.M. Foglietta et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 19–30 23
L.M. Foglietta et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 19–3024
oocytes exhibiting different stages of deterioration are seen; nutritive phagocytes
begin to appear inside the area bounded by the remnants of vitelline membranes that
surrounded the oocytes. Empty spaces surrounded by a wall, probably representing
remnants of the follicular membranes that surrounded the unspawned ovules, are also
seen (Fig. 2a).
(2) Recovery. Gonadal tubule wall begins to thicken. Some developing oocytes are seen
along the tubule wall. Nutritive phagocytes are abundant and closely associated with
degenerating oocytes. Follicular cells are poorly defined (Fig. 2b).
(3) Growth. The thickness of the tubule wall is maximal. Here and there clusters of
abundant previtellogenic oocytes line the germinal epithelium. Vitellogenic oocytes
occupy the lumen (Fig. 2c).
(4) Advanced growth. Tubule wall is thinner. Well-defined follicular cells surround
abundant vitellogenic oocytes with well-defined germinal vesicles. A layer of
previtellogenic oocytes lines the luminal aspect of the germinal epithelium (Fig. 2d).
(5) Mature. Tubules are dilated with thin walls and are almost completely filled with
mature oocytes surrounded by their vitelline membrane. Each oocyte contains a well-
defined germinal vesicle; a few immature oocytes may be seen (Fig. 2e).
3.1.2. Spermiogenesis
Five stages, corresponding to those of oogenesis, have been used to quantify the degree
of maturation of the tubules in spermiogenesis. These are shown in Fig. 3.
(1) Post-spawning. Tubule lumen is practically empty and almost free of residual
spermatozoa. The tubule wall shows some invaginations and is lined by a thin layer of
cells in the early stages of spermiogenesis (Fig. 3a).
(2) Recovery. Tubule wall is quite thick and shows many deep invaginations. The tubules
contain a few spermatozoa and nutritive phagocytes are common (Fig. 3b).
(3) Growth. Abundant spermatogonia and spermatocytes line the invaginations of the
tubular wall; the invaginations reach their maximum thickness and form a well-defined
maze-like pattern in the tubular lumen (Fig. 3c).
(4) Advanced growth. Tubule wall is thinner. Except for a few intraluminal invaginations,
these are practically confined to the outer tubular wall. The lumen is filled with
spermatozoa (Fig. 3d).
(5) Mature. The tubular lumen is packed with mature spermatozoa. The tubule wall is
nearly smooth and stretched to its greatest extent. Early spermatogonial stages are
absent. A central core of densely packed spermatozoa, surrounded by a halo of less
densely packed sperm cells, may be observed in many tubules (Fig. 3e).
It should be noted that in the ovaries the different stages tend to merge into each other;
therefore, they are not as clear-cut as in the testes.
Recycling of material from unspawned gametes appeared to take place. Nutritive
phagocytes were abundant in the recovery phase of oogenesis and spermiogenesis. In the
ovary they are seen as cell clusters within spaces surrounded by follicular cells. In the
testis the clusters of nutritive phagocytes may be seen in the lumen and their eosinophilic
cytoplasm contains basophilic bodies which may represent phagocytized sperm nuclei.
Fig. 3. Light micrographs of testicular sections. (a) Post-spawning: lumen practically empty and almost free of
residual spermatozoa (L), tubule wall lined by germinal epithelium. (b) Recovery: tubule wall is thick and shows
invaginations; few spermatozoa; abundant nutritive phagocytes are common (NP). (c) Growth: tubule wall
invaginations lined with abundant spermatogonia and spermatocytes; the invaginations reach their maximum
thickness and form a well-defined maze-like pattern in the tubular lumen. (d) Advanced growth: tubule wall is
thinner; invaginations are practically confined to the outer tubular wall; lumen filled with spermatozoa (SP). (e)
Mature: tubule lumen packed with mature spermatozoa; tubule wall is smooth and stretched to its greatest
extent; early stages of spermiogenesis are absent; a central core of densely packed spermatozoa may be
surrounded by a halo of less densely packed sperm cells (H). (f) Tubules of any one testis exhibit different
degrees of maturation. (PS) Post-spawning; (G) growth; (AG) advanced growth; (M) mature. Light bar in black
rectangle represents 200 Am.
L.M. Foglietta et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 19–30 25
L.M. Foglietta et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 19–3026
Spherule cells, which represent ova in various stages of disintegration and resorption, as
reported by Costelloe (1985), were also common.
3.2. The individual weighted maturity index
The monthly percentage of each tubule stage is shown in Table 1. The data from this
table has been used to calculate the individual monthly IWMI values.
The Fmax test was applied to the monthly mean IWMI values for males and they were
found to be heteroscedastic. Therefore the non-parametric Kruskal–Wallis test was
applied. The monthly IWMI values for females and for pooled ovaries and testes were
found to be homoscedastic and have been subjected to analysis of variance. The Student–
Newman–Keuls a posteriori test has been applied to detect differences among the monthly
means (Sokal and Rohlf, 1969). As each IWMI was obtained from one individual
specimen, statistical independence was insured and pseudoreplication was avoided
(Hurlbert, 1984). The time course of the pooled IWMI values for ovaries and testes is
shown in Fig. 4.
The monthly mean IWMI values for ovaries are graphed in Fig. 4. The lowest value
corresponds to the month of April and the highest to July. These extreme values are
statistically different from each other but they do not differ from the other months, which
do not differ among themselves.
Table 1
Percent tubule stages in each month
Months Percent tubule stages
Post spawn Recovery Growth Adv. growth Mature
Males
M 6.7 37.1 30.2 15.1 10.9
A 2.1 89.9 6.6 1.3 0
M 0.7 99.3 0 0 0
J 0 100 0 0 0
J 3.9 2.9 3.0 33.9 56.4
A 10.7 1.0 8.9 28.9 50.4
S 18.3 81.7 0 0 0
O 0.5 94.8 4.7 0 0
N 0 77.8 17.7 4.5 0
Females
M 37.8 25.0 0.7 16.4 26.2
A 34.9 37.9 16.3 0.7 10.1
M 24.6 5.9 3.3 14.2 52.1
J 0 22.8 39.3 33.8 4.1
J 11.8 0 0 0 88.2
A 18.6 0 0 43.1 38.3
S 26.1 38.2 6.4 12.5 16.8
O 44.0 19.0 1.8 3.2 32.0
N 2.9 16.4 36.2 19.1 25.5
Fig. 4. Mean monthly Individual Weighted Maturity Index (IWMI) values for ovaries and testes. For statistical
analysis and further details, see text.
L.M. Foglietta et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 19–30 27
The monthly mean IWMI for testes are graphed in Fig. 4. The Fmax test indicated that
the means were not homoscedastic. The non-parametric Kruskall–Wallis test showed that
the values differed among themselves at the p< 0.05 level. However, no further statistical
analysis is applicable.
It may be seen in Fig. 4 that the highest IWMI values for ovaries and testes were
obtained in the months of July and August. This has been analyzed statistically by pooling
the monthly ovary and testis IWMI values to obtain an overall maturity index. These
values are homoscedastic. Therefore, analysis of variance and the Student–Newman–
Keuls a posteriori test were applied. As shown in Table 2, the maximum gonadal maturity
of the species coincided with the months of July and August. The months of March, May
and June, on one side of the maximal maturity peak and October and November, on the
other side, exhibited the next lower values. April, on one side of the peak, and September
and October on the other, exhibited the lowest values. Therefore, IWMI revealed that
males and females showed a certain degree of synchronicity of gonadal development
between sexes. Maximal IWMI values were observed during the summer months of the
North Temperate Zone.
Table 2
Mean pooled ovary and testis IWMI values
Student–Newman–Keuls a posteriori test. Underlined means do not differ statistically at the p< 0.05 level.
L.M. Foglietta et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 19–3028
4. Discussion
Histological examination of the gonads of the population of I. badionotus investigated
in the present study indicated that tubule development was not uniform throughout the
organ. In any gonad, different tubules exhibit different degrees of maturation (Figs. 2f and
3f). Consequently a single maturation stage cannot be ascribed to any one animal. This
contrasts with the observations on the gametogenesis in other species where synchronous
tubule development among and within animals has been reported (Tanaka, 1958;
Costelloe, 1985; Sewell, 1992).
According to Sewell et al. (1997) the tubule recruitment model would predict that in each
year the gonad should exhibit three cohorts of tubules, which would constitute the year-
round gonad morphology (YGM), composed of primary, secondary and fecund tubules,
approximately corresponding to growth, advanced growth and mature tubules of the present
study. As shown in Table 1, the percentages of tubules in each stage varies considerably
from month to month. In June 100% of tubules are in the recovery stage and mature stages
are seen only in March, July and August. Therefore, there is no consistent YGM. Moreover,
the recruitment model would not apply where immature previtellogenic, vitellogenic and
mature oocytes are found in the same ovarian tubule as may be seen in the growth, advanced
growth and mature stages of I. badionotus (Fig. 2c, 2d and 2e). Therefore, its gonadal
development in I. badionotus does not appear to conform to the tubule recruitment model.
Considerable overlapping of the stages occurred in I. badionotus and synchronous
maturation of both sexes was not easily observed. Therefore, the Individual Weighted
Maturation Index (IWMI) has been proposed. This index revealed the maturation pattern
underlying gonadal chronological development. In both males and females of I. badio-
notus IWMI attains its peak value during the month of July. Herrero-Perezrul et al. (1999),
also studied gonad maturation, as determined by the gonad index, GI=(gonad weight/
drained weight)� 100 (Giese and Pearse, 1974), of Isostichopus fuscus. In this species,
maximum GI was attained during the summer (July to September), coinciding with the
maximum IWMI value of I. badionotus. This has also been observed in other tropical
holothurians such as Holothuria impatiens (Harriott, 1985).
An even distribution of the sexes was observed in I. badionotus as has been reported in
other holothurian species (Cameron and Fankboner, 1986; Tuwo and Conand, 1992;
Hopper et al., 1998; Herrero-Perezrul et al., 1999).
Two apparently hermaphroditic individuals were observed in the present study; one
specimen (92% male tubules, 8% female tubules) was collected in May and a second
hermaphrodite (45% male tubules, 55% female tubules) was collected in July. A section of
the latter gonad is shown in Fig. 5. Herrero-Perezrul et al. (1998) have observed casual
hermaphroditism in the closely related gonochoric species I. fuscus. As in I. badionotus,
male and female gametes developed in separate tubules in the hermaphroditic gonad of I.
fuscus. A low proportion of hermaphroditic individuals has also been observed in
Holothuria atra (Harriott, 1985), Peniagone azorica and P. diaphana (Tyler et al.,
1985) and S. mollis (Sewell, 1990). Two synallactid species, Paroriza pallens and P.
prouhoi, have been reported as hermaphroditic (Tyler et al., 1992).
In conclusion, the gametogenesis stages of I. badionotus were similar to those observed
in other holothurians. However, tubular maturation did not proceed at the same pace in all
Fig. 5. Section of a gonad of a hermaphroditic individual. Tubules show either testicular (T) or ovarian (O)
structure. Light bar represents 200 Am.
L.M. Foglietta et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 19–30 29
tubules of any one animal and considerable overlapping of the stages was observed.
Therefore, an individual weighted maturity index, IWMI, based on the fraction of gonad
tubules in each stage in a given gonad has been proposed as an estimate of gonad maturity.
The gonads reached maximal IWMI values approximately simultaneously during the
months of July and August.
Acknowledgements
[SS]
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