Journal of Experimental Marine Biology and Ecology 303 (2004) 19 30 www.elsevier.

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A maturity index for holothurians exhibiting asynchronous development of gonad tubules

Luz M. Foglietta a, Mara I. Camejo a, b Luis Gallardo , Francisco C. Herrera b,*
b a Departamento de Biologa de Organismos, Universidad Simon Bolvar, Caracas, Venezuela Laboratorio de Ecofisiologa Animal, Centro de Biofsica y Bioqumica, Instituto Venezolano de Investigaciones Cientficas (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 welldefined annual reproductive cycle. Male and female Stichopus mollis show generally
* Corresponding author. Tel.: +58-212-504-1450; fax: +58-212-504-1093. E-mail address: fherrera@cbb.ivic.ve (F.C. Herrera). 0022-0981/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2003.10.019

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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 asynchronous, 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.

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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 Mayers 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 to minimize 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.

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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 1% tubules in stage 1 2%tubules in stage 2 . . . n%tubules in stage n=100: 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

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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.

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(2)

(3)

(4)

(5)

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). 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). 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). 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). Mature. Tubules are dilated with thin walls and are almost completely filled with mature oocytes surrounded by their vitelline membrane. Each oocyte contains a welldefined 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.

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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.

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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 Males M A M J J A S O N Females M A M J J A S O N 6.7 2.1 0.7 0 3.9 10.7 18.3 0.5 0 Recovery 37.1 89.9 99.3 100 2.9 1.0 81.7 94.8 77.8 Growth 30.2 6.6 0 0 3.0 8.9 0 4.7 17.7 Adv. growth 15.1 1.3 0 0 33.9 28.9 0 0 4.5 Mature 10.9 0 0 0 56.4 50.4 0 0 0

37.8 34.9 24.6 0 11.8 18.6 26.1 44.0 2.9

25.0 37.9 5.9 22.8 0 0 38.2 19.0 16.4

0.7 16.3 3.3 39.3 0 0 6.4 1.8 36.2

16.4 0.7 14.2 33.8 0 43.1 12.5 3.2 19.1

26.2 10.1 52.1 4.1 88.2 38.3 16.8 32.0 25.5

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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.

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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 yearround 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

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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]

References
Cameron, J.L., Fankboner, P.V., 1986. Reproductive biology of the commercial sea cucumber Parastichopus californicus (Stimpson) (Echinodermata: Holothuroidea): I. Reproductive periodicity and spawning behavior. Can. J. Zool. 64, 168 175. Costelloe, J., 1985. The annual reproductive cycle of the holothurian Aslia lefevrei (Dendrochirota: Echinodermata). Mar. Biol. 88, 155 165. Engstrom, N.A., 1980. Reproductive cycles of Holothuria (Halodeima) floridana, H. (H.) mexicana and their hybrids (Echinodermata: Holothuroidea) in southern Florida. Int. J. Invertebr. Reprod. 2, 237 244. Giese, A.C., Pearse, J.S., 1974. Reproduction of Marine Invertebrates: Acoelomate and Pseudocoelomate Metazoans, vol. I. Academic Press, New York. Hamel, J.-F., Himmelman, J.H., Dufresne, L., 1993. Gametogenesis and spawning in the sea cucumber Psolus fabricii (Duben and Koren). Biol. Bull. 184, 125 143. Harriott, V.J., 1985. Reproductive biology of three congeneric sea cucumber species, Holothuria atra, H. impatiens and H. edulis, at Heron Reef, Great Barrier Reef. Aust. J. Mar. Freshw. Res. 36, 51 57. Herrero-Perezrul, M.D., Reyes-Bonilla, H., Garca-Domnguez, F., 1998. Casual hermaphroditism in gonochoric Isostichopus fuscus (Ludwig, 1875) (Echinodermata: Holothuroidea) of the southern Gulf of California, Mexico. Bull. Mar. Sci. 63, 611 615.

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L.M. Foglietta et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 1930

Herrero-Perezrul, M.D., Reyes Bonilla, H., Garca-Domnguez, F., Cintra-Buenrostro, C.E., 1999. Reproduction and growth of Isostichopus fuscus (Echinodermata: Holothuroidea) in the southern Gulf of California, Mexico. Mar. Biol. 135, 521 532. Hopper, D.R., Hunter, C.L., Richmond, R.H., 1998. Sexual reproduction of the tropical sea cucumber Actinopyga mauritiana (Echinodermata: Holothuroidea) Guam. Bull. Mar. Sci. 63, 1 9. Hurlbert, S.H., 1984. Pseudoreplication and the design of ecological field experiments. Ecol. Monogr. 54, 187 211. Hyman, L.H., 1955. The Invertebrates, Vol. IV, Echinodermata. The Coelomate Bilateria. McGraw-Hill, New York. Laboy-Nieves, E., Klein, E., Conde, J.E., Losada, F., Cruz, J.J., Bone, D., 2001. Mass mortality of tropical marine communities in Morrocoy, Venezuela. Bull. Mar. Sci. 68, 163 179. ` Ramofafia, C., Byrne, M., 2002. Evaluation du modele de recrutment des tubules chez trois holothuries tropicales aspidochirotes. La beche-de-merBulletin de la CPS. 15, 13 16. Octobre. Sewell, M.A., 1990. Aspects of the ecology of Stichopus mollis (Echinodermata: Holothuroidea) in Northeastern New Zealand. N.Z. J. Mar. Freshw. Res. 24, 97 103. Sewell, M.A., 1992. Reproduction of the temperate Aspidochirote Stichopus mollis (Echinodermata: Holothuroidea) in New Zealand. Ophelia 35, 103 121. Sewell, M.A., Bergquist, P.R., 1990. Variability in the reproductive cycle of Stichopus mollis (Echinodermata: Holothuroidea). Invertebr. Reprod. Dev. 17, 1 7. Sewell, M.A., Tyler, P.A., Young, C.M., Conand, C., 1997. Ovarian development in the class holothuroidea: a reassessment of the tubule recruitment model. Biol. Bull. 192, 17 26. Smiley, S., 1988. The dynamics of oogenesis and annual ovarian cycle in Stichopus californicus (Echinodermata: Holothuroidea). Biol. Bull. 175, 79 93. Smiley, S., McEuen, F.S., Chaffee, C., Krishnan, S., 1991. Echinodermata: holothuroidea. In: Giese, A.C, Pearse, J.S., Pearse, V.B. (Eds.), Reproduction of Marine Invertebrates. Echinoderms and Lophophorates, vol. VI. Boxwood, Pacific Grove, CA, pp. 663 750. Sokal, R.R., Rohlf, F.F., 1969. Biometry. Freeman, San Francisco. Tanaka, Y., 1958. Seasonal changes occurring in the gonad of Stichopus japonicus. Bull. Fac. Fish., Hokkaido Univ. 9, 29 36. Tuwo, A., Conand, C., 1992. Reproductive biology of the holothurian Holothuria forskali (Echinodermata). J. Mar. Biol. Assoc. U.K. 72, 745 758. Tyler, P.A., Muirhead, A., Billet, D.S.M., Gage, J.D., 1985. Reproductive biology of the deep-sea holothurians Laetmogone violacea and Benthogone rosea, (Elasipoda: Holothroidea). Mar. Ecol. Prog. Ser. 23, 269 277. Tyler, P.A., Young, C.M., Billett, D.S.M., Giles, L.A., 1992. Pairing behaviour, reproduction and diet in the deepsea holothurian genus Paroriza (Holothuroidea: Synallactidae). J. Mar. Biol. Assoc. U.K. 72, 447 462. Yoshida, M., 1952. Some observations on the maturation of the sea urchin, Diadema setosum. Annot. Zool. Jpn. 25, 265 271.