a maturity index for holothurians exhibiting a synchronous development of gonad tubules

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.


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.



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.



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.


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.


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.


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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