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Aquaculture of the Galapagos sea cucumber, Isostichopus fuscus

Annie Mercier1, Roberto Ycaza Hidalgo2 and Jean-François Hamel1

1 Society for the Exploration and Valuing of the Environment, Katevale (Québec), Canada;2 Plasfel S.A., Guayaquil, Ecuador

Abstract

This paper presents the results of the first attempt to breed the sea cucumber Isostichopus fuscus in land-based installations on the coast of Ecuador. This species has been intensively fished along the mainland and around the Galapagos Islands, where efforts at management have always met strong opposition from local communities. Ecuadorian populations of I. fuscus have thus been severely depleted over the past decade. The topics presented here include spawning, fertilization, larval rearing, disease control and juvenile growth. Data pooled from monthly trials conducted over three years indicate that, under optimal conditions, juveniles can be grown to a size of ca. 8 cm in length in 3.5 months. The survival rate is typically between 30 and 50 %. Furthermore, preliminary experiments have shown that the growth of young sea cucumbers in abandoned shrimp ponds is a promising option. Overall, this study demonstrates that I. fuscus can be reared in captivity, thus providing an alternative to fisheries, or a way to maintain sustainable harvests and eventually contribute to the restoration of the natural populations.

Keywords: Holothurian, spawning, larvae, development, juvenile, growth, disease

Introduction

Isostichopus fuscus (Figure 1) is a deposit-feeding sea cucumber that is mainly found on reefs and sandy bottoms along the western coast of the Americas, from northern Peru to Baja California, Mexico (Castro, 1993; Toral, 1996; Sonnenholzner, 1997; Gutierrez-Garcia, 1999). Like many other commercial species, I. fuscus has been widely fished over the past decades to meet the growing demand for beche-de-mer on the major Asian markets. As the waters along mainland Ecuador became depleted, the fisheries shifted to the Galápagos Islands, raising international apprehension over the fate of this very unique archipelago, which has been recognized as a national park and marine reserve.

Figure 1. Isostichopus fuscus adults collected along the coast of Ecuador.

In spite of the worldwide concern, the Galapagos sea cucumber populations became the focus of an intensive and poorly managed exploitation in the early 1990s. Since then, governmental attempts at regulating sea cucumber harvests, and banning them in some areas, have met strong opposition from local fishermen in Ecuador. In fact, illegal fisheries have always been a preoccupation and still occur along the mainland, around the Galapagos Islands and elsewhere in the distribution area of I. fuscus. Official information on the fisheries and actual total catches are consequently difficult to obtain and remain sparse (Salgado-Castro, 1993; Castro, 1996; Fajardo-Barajas, 1996; Sonnenholzner, 1997; Gutierrez-Garcia, 1999; Jenkins and Mulliken, 1999). Nevertheless, recent data and reports on average capture sizes (Sonnenhelzner, 1997; Martinez, 2001) indicate that I. fuscus populations have declined drastically and that natural stocks may irreversibly crash in the near future (Toral and Martinez, 2004).

In spite of this alarming situation, a very limited amount of studies have been conducted on the reproductive biology, spatial distribution, population structure, growth and survival rate of this species (Herrero-Perezrul, 1994; Fajardo-Leon et al., 1995; Toral, 1996; Sonnenholzner, 1997; Herrero-Perezrul et al., 1999; Hamel et al., 2003).

Some authors have mentioned that aquaculture and stock enhancement should be investigated as possible solutions to the current I. fuscus crisis (Gutierrez-Garcia, 1995, 1999; Fajardo-Leon and Velez-Barajas, 1996; Jenkins and Mulliken, 1999). However, to the best knowledge of the authors, no results have ever been presented on the captive breeding of the species.

Until recently, aquaculture in Ecuador was largely focused on shrimp. The emergence of viral diseases in 1999-2000 has severely harmed the industry and resulted in the bankruptcy and closing of numerous farms. Consequently, Ecuador now has a lot of shrimp farm infrastructures that could very well be put to use for the development of other species, such as sea cucumbers.

The present paper brings forward preliminary results on the larval development and juvenile growth of I. fuscus in land-base nursery systems on the coast of Ecuador. The data show that aquaculture of this species is feasible and that it could potentially be developed as an alternative or complement to fisheries. Then again, it could be used to maintain sustainable harvests and eventually contribute to the restoration of the natural populations. Further research to complement the present work is being conducted on the commercial-scale aquaculture of this highly prized sea cucumber, which is also a dominant feature of the Ecuadorian marine ecosystem. In time, aquaculture and stock enhancement of I. fuscus might provide part of the solution to the Galapagos sea cucumber crisis.

Methods and results

Spawning and fertilization

Adult sea cucumbers were routinely collected from nearby coastal areas to serve as broodstock. The adults were conditioned for a few days prior to spawning. Various methods of spawning induction have been tried at different periods of the month. However, close monitoring of the broodstock and spawning experiments over several months have revealed that the species follows a predictable spawning periodicity, even in captivity (Figure 2).

Figure 2. Example of the typical spawning periodicity observed in captive Isostichopus fuscus.

It has thus been possible to obtain male and female gametes on a monthly basis. Only a very limited number of spawning trials have been unsuccessful, mostly due to poor environmental conditions. Typically, between 300 and 400 adults were maintained in large 30-tonne tanks. Males and females were isolated in plastic buckets as soon as they showed signs of imminent spawning. Each female was then placed separately in a 300 litre spawning tank and maintained there until it had released its oocytes. Once the female had been removed from the tank, a dry sperm solution, prepared using the isolated spawning males, was added to the oocytes. The best fertilization rates and lowest occurrence of polyspermy were obtained with a concentration of 500 spermatozoa/ml.

After fertilization, the eggs were rinsed to remove excess sperm. A few hours later, the developing larvae were transferred to the hatchery tanks where their development was closely monitored. The routine protocol included daily cleaning of the tanks during the first days, followed by installation of a flow-through system. The larvae were fed every day using a mix of live microalgae (dominated by Rhodomonas and Dunaliella) at a frequency and concentration dictated by the daily observation of the digestive tract contents. Although several million oocytes could be obtained almost every month, space constraints have kept the size of the cultures between 1 000 000 and 1 500 000 eggs. With the improvement of the rearing techniques over the past year, a 50 % survival rate has often been achieved but the average success remains approximately 30 % of juveniles developed from every larval culture.

Larval development

Isostichopus fuscus possess oligotrophic transparent larvae that follow an indirect development, meaning that the larvae will need to feed during their pelagic phase and will undergo a series of transformations to reach the juvenile stage (Table 1, Figures 3 and 4).

In most trials, the development, settlement and early growth of the juveniles were somewhat asynchronous, and different stages and sizes could be found simultaneously in the culture. Extreme examples were observed in a few tanks where residual auriculariae neighboured 4 mm long juveniles. However, Table 1 provides a developmental kinetic that is based on the observation of the bulk of the culture, discarding the asynchronous animals.

Table 1. Development of Isostichopus fuscus, from fertilization to 35 mm long juvenile, at a salinity of 34-35, a temperature between 22 and 29 °C, a pH of 8.4-8.5 and a dissolved oxygen level between 5.4 and 6.1 mg/l.

Stage

Time

Stage

Time

Fertilization

0

Doliolaria

19-24 d

Elevation of the fertilization envelope

4 min

Early pentactula

21-26 d

Expulsion of the first polar body

7 min

Settlement (metamorphosis completed)

22-27 d

Expulsion of the second polar body

9 min

Juvenile, 1 mm

28 d*

2-cell

52 min

Juvenile, 2 mm

30 d

4-cell

70 min

Juvenile, 3 mm

32 d

8-cell

95 min

Juvenile, 4 mm

38 d

16-cell

124 min

Juvenile, 5 mm

40 d

32-cell

140 min

Juvenile, 8 mm

44 d

Blastula

3h

Juvenile, 10 mm

47 d

Early gastrula

6h

Juvenile, 15 mm

51 d

Hatching

10 h

Juvenile, 20 mm

56 d

Late gastrula (elongation)

14 h

Juvenile, 25 mm

63 d

Early auricularia

1-2 d

Juvenile, 30 mm

69 d

Auricularia

3-15 d

Juvenile, 35 mm

72 d

Late auricularia (early metamorphosis)

16-18 d



* For the juvenile stages, the time indicated corresponds to the first noteworthy observations of a particular size in the tanks.

Ovulation in I. fuscus occurs in the gonadal tubule and gonoduct as the oocytes are released (Figure 3a). Thus, fully mature oocytes (ca. 120 mm in diameter) are expulsed directly in the water column at the metaphase-I of meiosis, after the germinal vesicle breakdown.

The development of I. fuscus is initiated with the elevation of the fertilization envelope, roughly 4 min after fertilization. The expulsion of the first polar body occurs ca. 3 min later (Figure 3b). The second polar body follows rapidly within ca. 2 min. The first cleavage is equal, radial and holoblastic and divides the cell into two equal hemispheric blastomeres (Figure 3c). The second cleavage again occurs along the animal-vegetal axis, yielding more spherical blastomeres. Embryos hatch from the fertilization envelope as early gastrulae, ca. 10 h after fertilization (Figure 3d). These early gastrulae swim with the help of cilia covering their entire surface; they elongate into full-size gastrulae after ca. 14 h (Figure 3e). Auricularia larvae begin to appear ca. 24 h after fertilization; they constitute the first feeding stage. Growing auriculariae can be observed during the next two weeks of culture (Figure 3f, Table 1). At this stage, they begin to accumulate hyaline spheres. The oesophagus, the sphincter, the digestive tract, the cloaca as well as the anus are clearly visible. After 16-18 days, the auricularia reaches its maximum size of 1.1-1.3 mm; it has left and right somatocoels, as well as an axohydrocoel (Figure 3g).

Figure 3. Early development of the sea cucumber Isostichopus fuscus. The bars represent 200 m. A. Oocytes collected surgically from a mature gonad. The germinal vesicle (GV) is clearly visible. The insert shows a close-up of an ovulating oocyte with the follicular cells (FC) still attached to it. B. Fully mature, newly fertilized eggs with clear germinal vesicle breakdown. The insert shows the expulsion of the two polar bodies (PB). C. 2-cell stage. D. Newly hatched gastrula. E. Elongated gastrula with visible blastopores (BP). F. Early auricularia on which the ciliary bands (CB), hyaline spheres (HS), buccal cavity (BC), oesophagus (E), intestine (I), cloaca (C) and anus (A) are identifiable. Food items (F) are present in the buccal cavity. G. Ventral view of a fully developed auricularia showing the left somatocoel (LS), axohydrocoel (A), hyaline spheres (HS), ciliary bands (CB), buccal cavity (BC), oesophagus (E), sphincter (S), intestine (I) and the right somatocoel (RS). H. Dorsal view of a metamorphosing auricularia. With a noticeable decrease in size, the buccal cavity disappears and the hyaline spheres (HS) are pulled closer together. The mouth (M), intestine (I), oesophagus (E), left somatocoel (LS) and axohydrocoel (A) are clearly visible.

In the following hours, many auriculariae initiate the transformation that will lead to the doliolaria stage (Figure 3h). In the course of this process, the larvae shrink down to nearly 50 % of their initial size, the buccal ciliated cavity disappears and the hyaline spheres are pressed closer together (Figure 4a). The doliolaria stage is reached ca. 19-24 days after fertilization (Figure 4b, Table 1) as the larvae stop feeding and the cilia are aligned in five distinct crowns along their cylindrical body. At this time, the movement of the primary tentacles can be observed through the translucent body wall. The somatocoel is also visible. A few days later, the doliolaria transforms into an early pentactula possessing five buccal tentacles (Figure 4c). At this stage, the larvae remain close to the substrate, successively going through swimming and settling phases. Definitive settlement, with the complete loss of cilia, completion of metamorphosis and emergence of the two first ambulacral podia, occurs about 22 to 27 days after fertilization (Figure 4d, e).

Figure 4. Late development of the sea cucumber Isostichopus fuscus. The bars represent 200 m. A. Late metamorphosing auricularia, showing the hyaline spheres (HS), oesophagus (E), intestine (I), somatocoel (S) and axohydrocoel (A). B. Fully developed doliolaria with hyaline spheres (HS), primary tentacles (PT), ciliary bands (CB) and somatocoel (S). C. Early pentactula with 5 tentacles (T) and the still visible ciliary bands (CB). D. Dorsal view of newly settled pentactula with tentacles (T) and hyaline spheres (HS). E. Ventral view of newly settled pentactula showing the first ambulacral podia (AP) and the 5 buccal tentacles (T). F. Early juvenile, measuring 1.5 mm in length, with tentacles (T), ambulacral podia (AP) and ossicles (O). The hyaline spheres have disappeared. G. A 2 mm long juvenile with 5 tentacles (T) and 3 pairs of ambulacral podia (AP). The intestine (I) and ossicles (O) are visible. H. A 3 mm long juvenile showing the tentacles (T), papillae (PA), intestine (I), anus (A) and the ring canal and aquapharyngeal bulb (RC + APB).

Juvenile growth

Although the first settled juveniles can be observed as early as on day 22, a majority of juveniles measuring 1 to 1.5 mm in length are generally recorded in the tanks after 28 days of culture (Figure 4f, Table 1). They reach ca. 2-3 mm only a few days later (Figure 4g, h), and 5 mm after ca. 40 days. The juveniles continue to grow at a rate of ca. 0.5-1.0 mm per day for the next 3 to 4 weeks. When they are ca. 5 mm in length, the juveniles start to accumulate reddish-brown pigments. In 8 mm long juveniles, the tip of the tentacles becomes ramified. After 52 days of culture, the juveniles are 1.5-1.8 cm long and 4 mm wide (Figure 5). They possess several papillae and an elongated intestine that already exhibits strong peristaltic movements. The body wall becomes more opaque as the ossicle density and the tegument thickness increase. When the juveniles reach ca. 2 cm in length, the whitish colouration that characterises the early stages of life is gradually replaced by a brownish tinge similar to the one observed in adults. After approximately 72 days of culture, the juveniles are ca. 3.5 cm long and 1 cm wide and are nearly ready to be released in outdoor ponds, or in the field, to complete their growth.

Figure 5. Juvenile sea cucumber Isostichopus fuscus measuring 1.5 cm in length and showing the tentacles (T), early body wall pigments (P), intestine (I), ambulacral podia (AP), anus (A) and papillae (PA).

The typical growth of I. fuscus larvae and juveniles is shown in Figure 6. Recent cultures have yielded significantly faster growth rates with juveniles measuring 1.1 cm after 28 days, 3.1 cm after 56 days and 5.6 cm after 77 days. However, the average growth of juveniles during the second month (Figure 6b) roughly follows the second-order polynomial equation below:

f(x) = 0.77 - 0.29(x) + 0.01(x2)

where f(x) is the size in mm and x is the time in days (r2=0.99).

Figure 6. Average growth of the larvae (A) and juveniles (B) of the sea cucumber Isostichopus fuscus. Note that the x axis in B is a prolongation of the one in A, with a slightly different scale, and that size is expressed in mm in A and in mm in B.

Diseases and other problems

Intestinal parasites in larvae - The most common problem observed during the culture of I. fuscus is the development of a disease in the digestive system of early larvae. The first stage is the appearance of opaque cells around the digestive tract. The second visible symptom is the contraction of the intestine and stomach. In the worst cases, the digestive tract completely shrivels up and disappears. When it becomes visible, the condition is usually fatal to the larvae.

Upon close examination of the affected larvae under the microscope, the authors have determined that the disease is probably caused by parasites (Figures 7, 8). A protozoan displaying jerky movements has been observed outside the larvae, whereas a slower moving, smoother form appears inside the larvae. The authors have not yet been able to establish whether they are dealing with the same organism at two different stages of development, or two completely different causal agents.

During the first stage of the disease, the parasites can be seen entering through the body wall and the digestive tract, probably inducing the observed contraction. Later on in the development of the disease, the parasites become larger and are present everywhere around the intestine, both inside and outside. The parasites appear to feed on the intestinal contents or tissues, slowly making it shrink and disappear, typically causing the death of the larvae.

Figure 7. 1000 x enlargement of an infested intestine of I. fuscus.

Figure 8. Close-up view of the intestinal parasite of I. fuscus.

The parasites have never been observed in the larvae before hatching. However, the condition develops rapidly shortly thereafter, suggesting that the causal agents are present in the surrounding environment and that they enter the larvae at the first opportunity. They seem to remain inactive until the larvae start to feed. Afterwards, they can be seen to develop in different areas of the mouth and, most commonly, the digestive tract (stomach and intestine). A form with thin appendices can be found attached all over the larvae, but the amoeboid form is mostly observed around the digestive organs; it has the ability to move in and out of in what appears to be a trophosoit form. The parasites penetrate the intestine and feed on the intestinal contents or tissues, sometimes rupturing the intestinal wall.

The authors have tried different methods of collecting the gametes to establish whether the parasites were coming from the seawater itself or from the spawning adults. It has proven impossible to develop a culture without the presence of the parasites at one stage or another, even when using artificial seawater from the start of the trial. It would seem that the parasites are either present around the gametes and/or develop spontaneously in the culture.

Fortunately, the authors have found that a close monitoring of the early larval stages allows the detection of the first occurrence of the parasites. This enables to control the further development of the disease by using different environmental parameters. If the disease is not contained in its earliest phase, the whole culture usually crashes.

Another less virulent problem has been noted to affect mainly the body wall of cultured larvae. It appears as dense agglomerations and furrows at the surface of the affected individuals. This condition may degenerate and cause the larvae to shrink and eventually die. Whether the same parasite or another agent is involved is still not clear. Bacteria of the Vibrio genus are suspected to be involved, either as primary or secondary pathogens.

Water and food quality - Due to variable and often poor environmental conditions along the coast where the water was being pumped, a very complete filtration system, including UV treatment, had to be installed to provide the best possible water quality throughout the trials. The conventional treatment used for prawn culture was not dependable enough to grow sea cucumber larvae with optimum success. Strict sanitary measures were adopted in the handling of gametes and larvae to maximize survival rates and minimize incidence of infections and diseases. Bacterial counts are routinely made from water samples to monitor the efficiency of the sanitary and filtration procedures.

Bacterial contamination of algae cultures was another common problem that had to be overcome. Growing larvae need large quantities of healthy live algae to develop steadily, especially during the crucial step of metamorphosis into pentactulae. The inability to provide a healthy mix of algae can significantly delay growth and metamorphosis of larvae for extended periods. Thus, it has proven crucial to develop a system of algae production that is reliable and efficient.

As the size of the culture batches grew from a few tens of thousands to over a million larvae per month, rearing conditions had to be maintained and eventually improved to avoid mass mortalities.

Grow-out experiments

From settlement (0.5-1.0 mm) onward, the juveniles are usually transferred to larger 18 m2 pre-conditioned flow-through tanks, with or without settlement plates. After about 110 days, some of the juveniles have reached sizes up to 8 cm (ca. 26 g; Figure 9). The authors are presently trying to assess at which size they would be fit to be transferred into growout ponds or eventually released in the wild.

Isostichopus fuscus juvenile can survive and grow in abandoned shrimp ponds. A preliminary experiment has been conducted early in the study to find out if small sea cucumbers collected from the wild would fare well in ponds from different locations. Enclosures of 1 m2 were used to facilitate recapture. As it turned out, the sea cucumbers grew an average of 17 g/week and presented a 98 % survival rate, suggesting that shrimp ponds along the coast can provide a good environment to grow I. fuscus juveniles to adult size in a reasonable delay. Further research on pond grow-out techniques is currently being conducted.

Figure 9. Two 110-day old Isostichopus fuscus juveniles.

Conclusions and future prospects

So far, after three years of research and development:

Current and future research efforts are focused on:

Acknowledgements

We would like to acknowledge the hard work and technical assistance of Jorge Jaramillo, Jose Pico, Pedro Gonsaby, Maricela Garcia, Vicente Cruz and Cesar Pico, during the course of this work.

References

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