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Synchronous gamete maturation and reliable spawning induction method in holothurians

Jean-François Hamel and Annie Mercier

Society for the Exploration and Valuing of the Environment, Katevale (Québec), Canada

Abstract

Several years of research on the gametic development and spawning of different species of holothurians have produced results that find applications in aquaculture and fisheries management programs. The first set of data shows that sea cucumbers secrete a biologically active chemical which allows gamete synthesis synchrony among conspecifics. Laboratory experiments have revealed that the gametic development was significantly less synchronous among individuals that were maintained separately under natural environmental conditions than it was among similarly treated individuals kept in groups. Furthermore, the presence of mature individuals was found to induce the gametic development of less mature ones. The active substance is present in the mucus secreted by the body wall enabling it to travel fair distances, although transmission is often favoured by pairing and aggregative behaviours. These findings indicate that the lunar cycle, photoperiod, food supply and temperature cannot individually account for the onset and synchronization of reproduction, but rather that environmental cues act synergistically and can be transmitted within and between populations through chemical communication. This has repercussions on both fisheries and aquaculture techniques. Preserving untouched populations while fishing intensively on other grounds should be favoured compared to steadily lessening the biomass, whereas broodstock should be maintained in a way that promotes interactions long before the breeding period.

The other aspect of the study arose from the fact that holothurians are among the most commercially valuable echinoderms for which successful spawning induction is still difficult to obtain on a reliable basis. Recent results show that the transfer of perivisceral coelomic fluid (PCF) can be used as a reliable tool to induce spawning in mature individuals. PCF collected from individuals that had been in the typical spawning posture for about 20 min, without shedding gametes, triggered spawning in 71-100 % of conspecifics. The individuals responded to the injection of a 2-3 ml aliquot by displaying the spawning posture within 30-62 min, followed by massive gamete broadcast 57-83 min later. The results varied according to the time of PCF collection with respect to the spawning activity of the donor and the amount of PCF injected. The triggering substance was found not to be sex-specific since positive responses were observed in individuals of the same or opposite sex as the donor. Thus, PCF collected from early spawners can be used to spread and maximize spawning among the entire broodstock.

Keywords: Chemical ecology, coelomic fluid, gametogenesis, reproduction, sea cucumber

Introduction

Proper gamete maturation and spawning synchrony are key factors in the sustainable development of sea cucumber aquaculture and fisheries programs. Studying and understanding the biological and chemical factors that play a role in the reproductive cycle of sea cucumbers can provide valuable insights for the management of captive-breeding facilities.

Maturation

Photoperiod has been shown experimentally to be the main factor responsible for synchronizing gamete development, or gametogenesis, in shallow-water echinoderms (McClintock and Watts, 1990; Hamel et al., 1993; Hamel and Mercier, 1996a). Increasing temperature and food supply (Chia and Walker, 1991; Hamel et al., 1993) and the lunar cycle (Mercier et al., 2000; Hamel et al., 2001) seem to constitute the other most determinant factors. It is proposed that the environmental fluctuations perceived by the organisms initiate a reaction leading to changes and modifications in the reproductive metabolism, possibly through gene activation or hormone synthesis (Shirai and Walker 1988; Hines et al., 1992; Barker and Xu, 1993). It is not as easy to explain the onset and harmonious inter-individual development of gametogenesis in populations living in total darkness, whether exposed to similar or distinct local environmental conditions. It becomes especially important for freely spawning marine invertebrates, like most holothurians, that may waste a large portion of their gametes if this synchronization is not well orchestrated.

The investigation carried out revealed that environmental factors, coupled with various endogenous reactions, could not adequately explain the synchronous initiation and harmonious development of gamete synthesis in male and female sea cucumbers. This brought up an interesting question: are chemical mediators involved in the harmonious inter-individual development of gametes? This work explores the role of chemical mediators during gametogenesis in populations of sea cucumbers, while providing basic data and explaining a possible synergism with environmental factors. The mucus, one of its constituents or a chemical emitted with it, plays a determinant role in the fine tuning of gametogenesis among entire populations of holothurians.

Spawning

Spawning in holothurians has mostly been observed in the field and correlations with environmental factors have been outlined (Cameron and Fankboner, 1986; Smiley et al., 1991, Hamel et al., 1993, Hamel and Mercier, 1995a, 1996a). Some laboratory studies have tested the importance of environmental factors in spawning induction. Hamel and Mercier (1996a) induced gamete release in Cucumaria frondosa by manipulating the temperature and light factors with amaximum success of ca. 30 %. The use of a powerful jet of water on drying individuals also triggered spawning in Holothuria scabra (James, 1994) and Parasitchopus japonicus (Liu et al., 2004; Wang and Yuan, 2004). Recently Battaglene et al. (2002) were able to trigger spawning in roughly 10 % of mature females Holothuria fuscogilva by adding a solution of dried algae Schizochytrium sp. (Algamac). However, apart from spontaneous spawning following the stress of capture recorded in different species (Reichenback, 1999; Hamel et al., 2001), thermal shock, alone or combined with other stresses, remains the most common method used to induce spawning in holothurians (Smiley et al., 1991; Yanagisawa, 1998; Hamel et al., 2001; Pitt and Duy, 2004; Sui, 2004; Wang and Yuan, 2004). All these methods give very inconsistent results that vary with the protocols, the species used and even the different batches of individuals collected. Most of the known physical, chemical or biological treatments used to induce spawning or to activate the final maturation of oocytes in other marine invertebrates act at the cellular level (Smiley et al., 1991) and give essentially species-specific results (Maruyama, 1980). They are not, or only partially, effective in holothurians.

The present work investigates the role of perivisceral coelomic fluid (PCF) in the spawning induction of holothurians, providing evidence of its importance as a transmitter of chemically active substances. The outcomes of this study include a reliable method to induce gamete release and propagate spawning among mature individuals under laboratory conditions.

Methods

The detailed methods used during the various experiments can be found in Hamel and Mercier (1996b, 1999), Mercier et al. (2000) and Mercier and Hamel (2002).

Results and discussion

Gametogenesis and maturation

Influence of environmental factors - Field and laboratory investigations demonstrated that gamete maturation was influenced by several physical and chemical factors, acting either together, successively or independently of one another. This can be observed in numerous species of sea cucumbers and is particularly evident in species that spawn once a year.

As expected from reports pertaining to other marine invertebrates (McClintock and Watts, 1990; Hamel and Mercier, 1995a, 1996a), photoperiod appeared necessary to initiate the gametic cycle of sea cucumbers as only individuals collected after the first increase of day length were able to complete their development when subsequently kept under constant conditions (Figure 1). However, the gonadal index of deep populations living in the absence of light was found to follow the same pattern as that of individuals exposed to light in the shallow photic zone (Figure 2).

Figure 1. Relative frequency of different gametic stages observed in small and large tubules of male and females Cucumaria frondosa collected either before the first increase of day length (December) or after it (February), and kept under naturally varying environmental conditions (from Hamel and Mercier, 1996b).

Figure 2. Seasonal changes of gonadal index in males and females Cucumaria frondosa collected either in the photic (10 m) or aphotic (110 m) zone. Vertical lines indicate the 95 % confidence intervals (from Hamel and Mercier, 1996b).

Figure 3. Relative frequency of different gametic stages in grouped and isolated Cucumaria frondosa kept under natural environmental conditions (from Hamel and Mercier, 1996b).

Figure 4. Number of males and females (± 1 SD) showing mature gonads from April 1992 to October 1993 when maintained either grouped or isolated. Note that only one month out of three is identified on the x-axis (from Hamel and Mercier, 1996b).

Inter-individual fine tuning of gamete development - Another set of experiments was thus carried out to explain the synchrony in the maturation of deep water and shallow water populations of sea cucumbers, despite the fact that individuals in deep water cannot directly perceive the initial environmental cue. Interindividual communication could play a key role, especially since most species of sea cucumbers live in close proximity to one another and often are continuously distributed over a depth range. Waterborne chemicals could be the mode of such communication.

Indeed, interindividual synchrony was obtained only among sea cucumbers allowed some contact with one another, either directly or through the water medium (Figures 3, 4). Furthermore, the presence of more mature individuals of the same sex induced gametic development in less developed sea cucumbers, even when they had not previously been exposed to an increase in day length (Hamel and Mercier, 1996b).

Aggregations - In the field and broodstock tanks, aggregations have been observed to shape the distribution of adult sea cucumbers, mostly in tropical regions, on a regular basis. Since no clusters were observed in the juveniles stage (before the fist sign of sexual maturity), and given the fact that maximum aggregation typically occurred just before spawning events, aggregative habits of some species of sea cucumbers appear to be related to reproduction. Breeding aggregations have been observed in a number of asteroids (Ormond et al., 1973; Hamel and Mercier, 1995b) and echinoids (Levitan et al., 1992; Young et al., 1992), and many authors suggest that aggregations minimize sperm dilution and increase fertilization success. Recently, Rodgers and Bingham (1996) observed aggregations of the sea cucumber Cucumaria lubrica and proposed that they were a result of their subtidal zonation in response to light. Figure 5 illustrates the aggregative behaviour recently observed in Holothuria scabra.

Figure 5. Aggregative behaviour (lines) and spawning (triangles) of adult Holothuria scabra in outdoor tanks. Open circles indicate full moons and darkened circles represent new moons. (from Mercier et al., 2000).

Mucus and chemical communication - This set of data further refines the previously described results by showing that the seasonal production of mucus, secreted by the body wall of sea cucumbers, can play the role of chemical mediator in this interindividual sexual exchange. Synchronous gamete development is apparently related to mucus synthesis which enables the transfer of sexual information between conspecifics (Figure 6). Either a component of the mucus itself or a chemical secreted simultaneously and carried with it acts as the messenger.

Figure 6. Cucumaria frondosa. Seasonal cycle of (A) gonad index of 50 sea cucumbers collected monthly from May to November correlated with (B) the abundance of mucus synthesized by another group of individuals submitted to similar environmental conditions of day length and temperature. The vertical lines indicate the confidence interval (95 %) (from Hamel and Mercier, 1999).

To the best knowledge of the authors, mucus has never before been described to play a role during gametogenesis in any species of marine invertebrate. The data indicate that mucus synthesis follows the gonad cycle, hence it is proportional to the level of maturity of the gonadal tubules, enabling mature individuals to stimulate conspecifics with more efficiency (Figure 7). This suggests that the chemical nature or the quantity of mucus synthesised by an individual changes over time and modulates the intensity of stimulation applied on conspecifics over the seasonal cycle. In fact, the seasonal synthesis of mucus seems to be correlated with the initiation of gametogenesis and with the progressive increase of the gonad index.

Spawning induction

Having shown that sea cucumbers can influence one another during the maturation process by aggregating and transmitting chemical cues, it is only natural to wonder if spawning induction can be achieved by artificially enhancing the transmission of chemical signals. Indeed, the present work demonstrates that a substance or a combination of substances implicated in the initiation and progression of spawning behaviour and gamete broadcasting in holothurians can be transmitted via the perivisceral coelomic fluid (PCF). The injection of PCF collected from a mature individual showing spawning activity, prior to and until gamete release, into a mature non-spawning individual triggered spawning behaviour and subsequent gamete release in up to 100 % of individuals (Figure 8). The same PCF spread in the immediate environment also induced a proportion of spawning behaviour (47-65 %) and gamete release (20-31 %). A broad interpretation of the latter results suggests that messages sent via the PCF could help holothurians synchronize and propagate spawning both in captivity and in the field.

Figure 7. Gametic development in the gonadal tubules of Cucumaria frondosa initially in the recovery stage and submitted to three different sex-specific stimulations: a whole mature individual, mucus and coelomic fluid from a mature individual. The highest stages of development observed are presented as histograms and the number of mature oocytes or the proportion of mature spermatozoa as lines. The gametic stages observed in both sexes were divided into (1) post-spawning; (2) recovery; (3) growth; (4) advanced-growth; and (5) maturity. Control experiments were performed using individuals without the stimulation. The vertical lines represent the confidence interval (95 %) (from Hamel and Mercier, 1999).

Figure 8. Effect of the amount of PCF injected and the time of collection in donor on the proportion of female Bohadschia argus that exhibit spawning behaviour and gamete release, and on the mount of oocytes spawned. The data are presented as mean ± SE (n=4-11). Each experiment was repeated with three different batches of sea cucumbers (from Mercier and Hamel, 2002).

Although the ability of PCF to induce spawning has been clearly established, including its efficiency when spread in the water column, the path of its transmission and true function in natural spawning events is not fully understood. Theoretically, the PCF and/or the chemically active substance(s) it contains are trapped inside the coelomic cavity and cannot spread outside. However, Doignon et al. (2003) recently observed that the coelomic content of Holothuria tubulosa is purged seasonally. He proposed that the coelomic purge would be an indirect result of the host’s spawning as holothurians make use of powerful muscle contractions to expulse their gametes. This behaviour would increase the pressure on the peri-cloacal end of the individual and set off the discharge of the coelo-cloacal ducts, carrying the coelomic contents away to the environment. This reasoning could even explain how the use of sperm collected from spawning individuals has been found to induce other individuals to spawn: the presence of PCF in the semen may well be the real trigger.

The next results show that the active PCF is neither sex-specific nor species-specific with the holothurians tested (Table 1). However, PCF from echinoids and asteroids did not induce spawning in sea cucumbers.

Interestingly, the time of collection in the donor and the amount of PCF injected have been found to have an impact on the stimulation of the spawning posture and the gamete release. For instance, PCF collected just 1h after gamete release had lost its effectiveness. Thus PCF apparently acts as a carrier of one or more short-lived molecules of a sexual nature during spawning.

Table 1. Effect of PCF from different echinoderms on female Bohadschia argus, using 2 ml aliquots collected from donors having displayed spawning behaviour for 20 min. Data for spawning induction are presented as Mean ± SE (n = 4-11). Spawning success estimated with the gonadal index (GI) and the amount of oocytes spawned (n = 15 males and 15 females) (from Mercier and Hamel, 2002).

DONOR SPECIES

SPAWNING INDUCTION

SPAWNING SUCCESS

Proportion of spawning

GI before spawning

GI after spawning

Oocytes spawned

individuals (%)

(X106)

Sea cucumbers






Bohadschia marmorata





Male

69.1 ± 14.4

19.6 ± 6.5

7.6 ± 3.3

0.7 ± 0.4

Female

83.9 ± 8.4

20.3 ± 4.5

6.5 ± 2.5

0.9 ± 0.6

Holothuria atra





Male

78.3 ± 11.6

18.7 ± 4.7

5.2 ± 4.9

1.2 ± 0.1

Female

86.5 ± 10.3

12.6 ± 6.1

3.1 ± 2.1

1.1 ± 0.8

Holothuria leucospilota





Male

66.7 ± 12.1

20.1 ± 3.6

7.7 ± 3.5

0.9 ± 0.8

Female

54.6 ± 19.9

22.1 ± 7.6

7.2 ± 2.9

0.7 ± 0.1

Sea urchin






Diadema savignyi





Male

0

13.2 ± 5.3

-

-

Female

0

17.7 ± 7.1

-

-

Sea star






Archaster typicus





Male

0

20.0 ± 4.1

-

-

Female

0

14.2 ± 3.5

-

-

The pooled data from studies in conspecific holothurians of both sexes showed that the best spawning success was obtained when 2 or 3 ml of PCF was injected. Furthermore, for a given amount of PCF sampled from a donor 20 min after the beginning of spawning behaviour or anytime later until gamete release was also more effective (Table 2).

The fact that 1 ml of PCF induced a good proportion of individuals to display the spawning posture, but with a smaller amount of oocytes actually being broadcasted, compared to 2 or 3 ml injections, suggests that the chemical plays a double role by triggering gamete release. This could mean that the chemical is a single compound that its concentration in PCF increases from the first sign of spawning to reach a maximum ca. 20 min later and that a threshold concentration is needed to induce a complete response.

Conclusions and applications for aquaculture

Chemical communication during maturation

Summary of results:

Recommendations:

Table 2. Spawning induction in male and female Bohadschia argus. Data as Mean ±SE (n= 4 to 11). The perivisceral coelomic fluid (PCF) was collected from donors having displayed spawning behaviour for 20 min. The same experimental condition was repeated at least three times (from Mercier and Hamel, 2002).

Injection

Sex
(receiver)

Spawning behaviour
(%)

Time before first sign of behaviour
(min)

Gamete release
(%)

Total duration of response
(min)

Spawning success

GI before spawning

GI after spawning

Oocytes spawned
(X 106)

PCF from spawning male

0.5 ml

Male

7.2 ± 1.5

67.5 ± 5.9

5.7 ± 2.3

80.2 ± 12.3

11.6 ± 2.3

9.7 ± 1.9

-

1 ml

Male

81.1 ± 3.3

60.3 ± 7.7

75.4 ± 7.2

82.3 ± 8.4

15.9 ± 5.7

12.4 ± 3.4

-

2 ml

Male

94.3 ± 3.1

59.5 ± 2.6

92.1 ± 9.4

72.1 ± 7.3

21.3 ± 6.1

5.2 ± 3.9

-

3 ml

Male

92.7 ± 4.4

50.1 ± 1.7

90.4 ± 4.7

70.7 ± 9.6

14.6 ± 4.6

2.4 ± 1.1

-

PCF from spawning female

0.5 ml

Female

49.5 ± 5.5

74.6 ± 12.3

35.3 ± 9.1

83.5 ± 7.9

13.5 ± 3.5

11.3 ± 0.7

0.3 ± 0.1

1 ml

Female

71.7 ± 9.3

65.1 ± 20.2

44.1 ± 6.3

79.3 ± 6.6

18.3 ± 4.8

12.6 ± 4.7

0.7 ± 0.1

2 ml

Female

84.5 ± 6.4

59.3 ± 5.8

79.2 ± 5.4

69.5 ± 5.4

14.9 ± 4.4

3.2 ± 1.9

1.1 ± 0.2

3 ml

Female

74.5 ± 3.1

61.3 ± 7.7

71.9 ± 6.3

71.3 ± 7.6

12.5 ± 6.7

2.9 ± 1.5

1.4 ± 0.5

PCF from spawning male

0.5 ml

Female

36.3 ± 11.2

72.2 ± 9.4

22.7 ± 6.4

91.3 ± 8.1

20.4 ± 5.5

18.1 ± 3.5

0.1 ± 0.1

1 ml

Female

65.2 ± 3.9

75.2 ± 10.1

56.5 ± 8.4

81.2 ± 7.3

15.1 ± 3.6

11.5 ± 2.7

0.5 ± 0.2

2 ml

Female

80.8 ± 1.2

61.1 ± 5.3

71.3 ± 7.1

83.2 ± 6.4

18.5 ± 7.3

1.7 ± 1.3

1.6 ± 0.4

3 ml

Female

92.4 ± 2.8

62.1 ± 8.7

84.7 ± 5.2

78.3 ± 5.5

17.4 ± 5.1

2.1 ± 1.0

1.3 ± 0.2

PCF from spawning female

0.5 ml

Male

21.9 ± 9.9

74.3 ± 4.1

9.3 ± 3.2

83.2 ± 9.3

10.5 ± 6.4

9.1 ± 1.7

-

1 ml

Male

62.4 ± 3.4

69.2 ± 3.1

48.0 ± 7.2

83.3 ± 6.7

15.3 ± 4.1

11.6 ± 4.2

-

2 ml

Male

89.4 ± 6.7

31.4 ± 3.3

87.3 ± 3.6

59.1 ± 9.2

13.7 ± 6.4

4.4 ± 2.8

-

3 ml

Male

100 ± 0

30.3 ± 2.0

100 ± 0

57.4 ± 4.7

19.3 ± 7.5

5.7 ± 2.1

-

Spawning induction

Summary of results:

Applications:

Acknowledgments

We would like to thank the staff of Plasfel S.A. (Ecuador), the Marshall Islands Science Station (CMI, Majuro), and the former Coastal Aquaculture Centre of ICLARM (now WorldFish Center) in the Solomon Islands for their assistance during the various stages of the research. We are also grateful to the numerous colleagues and friends who have helped us in one way or another during the different studies. The research was partly supported by grants from the United States Department of Agriculture (USDA) under the College of Micronesia Land Grant programme and by the Canadian International Development Agency (CIDA) under the CGIAR-Canada Linkage Fund Programme.

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