First Trials on Hatchery and Larval Development of the Sea Cucumber, Holothuria scabra (Jaeger 1883) In Oman

Holothurians commonly known as sea cucumbers are marine invertebrates of significant importance for marine ecosystems and for a limited number of species as a valuable fishery generally for export. From the early 1990s, the populations of various species of sea cucumbers declined due to an increasing demand of Asian markets, particularly those of China [1]. Among these species, Holothuria scabra is considered as one of the most valuable sea cucumber for bêche-de-mer production and have been widely fished and overfished in the tropics [2]. It is currently valued at more than 1000 US$ per kg dry weight [3], and the demand from the Asian market remains strong [4]. This increasing demand from Asian markets driving high price, an often inadequate management of the fishery and the biological characteristics of the species such as slow growth and easy to access shallow water habitat resulted globally in severe overfishing [5]. To avoid overexploitation of natural populations while maintaining a sustainable level of exports, aquaculture is considered as one of the potential solutions for H. scabraproduction [1,6]. Several countries have progressively developed a viable aquaculture production and in some cases have initiated restocking or sea ranching programs [1,7,8]. Although the sea cucumber Holothuria scabra has always been a small part of the traditional exploitation of the benthos in Mahout Bay of Arabian Sea [9], the foreign demand for the product overseas and its high price have put increased pressure on this resource locally leading rapidly to overfishing [10].

The natural spawning of this species in Oman has been recently documented and showed, as in many other locations, an annual reproductive cycle [11]. Natural maturation occurred in February and March followed by spawning in April and May, which is correlated to high sea surface temperature and precipitation as both may serve as exogenous synchronizing cues for gamete maturation and spawning [11]. Alarming signs of the of the Omani population of H. scabra overfishing have been observed with stock densities decreasing to less than 1 individual per ha [10], a regular decrease in the size of animal processed and a progressive switch to less valuable species in only a few years of exploitation. Therefore, restocking and stock enhancement in addition to fisheries management policies are needed to ensure the sustainability of this resource. This article documents the first attempts made to develop hatchery techniques for the Omani sea cucumber Holothuria scabra population as initial steps towards aquaculture development and stock enhancement programs.

The Aquaculture Research Unit of Sultan Qaboos University located in Al-Hail some 40 km north of the capital of the Sultanate, Muscat, was used to conduct Holothuria scabra hatchery trials. Land based outdoors tanks were used to keep the broodstock of H. scabra. The tanks were continuously supplied with seawater extracted from a marine well at a salinity of 37 PSU and a temperature of 29°C. For the spawning and larval rearing experiments, the water was filtered using sand filter and 5µm particle filter and then passed through a UV sterilization 66 unit [12].

In total 80 healthy animals weighing 200-600g were collected at 4 occasions from Mahout Bay of Al-Wusta Province by local fishermen and through SCUBA diving starting between September 2009 and November 2010 (Figure 1).

 

Figure 1: Location of the Holothuria scabra population used for the spawning experiments. Individuals were collected from various locations within Mahout Bay.

 

Collected animals were carefully handled to avoid evisceration, cleaned by gently washing the body with seawater and packed at low density of 6-8 individuals per bags in transparent plastic bags filled with 1-2L of sea water and inflated with pure oxygen. The bags were placed in cool-boxes containing dry ice to maintain a low temperature in the boxes during the 5 hours trip to Muscat. Upon arrival at the hatchery, the animals were acclimatized while still in the transport bags with ambient seawater and later transferred to clean tanks with running oxygenated sea water until experimentation.

For this study, In vitro maturation and fertilization were conducted using an artificial maturation substance extracted from sea urchin oocytes [13]. It is referred as Maturation Inducing Fractions (MIF) and based on hatchery work in Madagascar [13,14]. Genitors were dissected through a longitudinal incision on the anterior part of the trivium to extract male and female gonads for the in vitro fertilization.

Body fluid was drained and the gonads of males and females were gently removed using forceps and placed in separate beakers with filtered sea water. Gonads were then cut separately into small pieces using scissors to release oocytes and sperm cells from gonadal tubules. The oocytes were then sieved on 60µm mesh with filtered sea water and placed in petri dishes at concentrations of 100 oocytes/ml. Maturation of the oocytes was induced with the MIF solution for 2 hours, and then rinsed with filtered sea water to remove MIF residuals. Fertilization was achieved by adding few drops of sperm suspension to the mature oocytes. The fertilized eggs were kept in a shallow basin without aeration until hatching of gastruled embryos. Observations and photography of embryonic development stages were carried out on random samples every 30-60 minutes using a light microscope at 40X magnification.

Newly-hatched gastrula larvae were transferred into 100L tanks filled with 85L of 2-µm filtered and UV-treated seawater and provided with aeration through air diffusers. Feeding and regular water exchange commenced with development of Auricularia stage larvae from day-2 Post-Fertilization (PF). The water was changed by siphoning and the larvae collected onto a 60µm wet sieve. Tanks were refilled with 2-µm filtered and UV-treated seawater. This method of water exchange was continued daily for 20 days until larval settlement. The newly settled juveniles were maintained in a flow-thru system with 2-µm filtered and UV-treated seawater.

H. scabra larvae were fed daily from the second day PF with different types of micro-algae until settlement. Three mixed algae were cultured under laboratory conditions with artificial light and aeration. A temperate species Phaeodactylum tricornutum was cultured at low temperature from 19 to 24°C in the laboratory. Chaetoceros sp. and Nanochloropsis sp. were cultured both under laboratory and outdoor conditions (29-30°C) and fertilized with Guillard’s F/2 medium. Initially, the developing larvae were fed twice daily with P. tricornutum at a final concentration of approximately 2500 cells per liter. On day 8 PF, the final concentration of P. tricornutum was increased to 5000 cells per liter. During the Doliolaria stage (a non-feeding stage), the feeding was temporarily suspended and resumed after metamorphosis (typically on day 20 PF). At this stage both microalgae (Chaetoceros, Nanochloropsis and Phaeodactylum) and particulate diet (dried and powdered Sargassum sp.) were added to favor settlement and feed the newly settled juveniles.

All collected animals survived through 5 hours road transportation and were successfully transferred to holding tanks. For the artificial maturation experiments, all female genitors positively responded to the MIF technique and produced more than 90% normal mature oocytes after 2 hours of induction. A fertilization rate of 90% was also achieved on these artificially matured oocytes. A total of more than 400,000 larvae were produced over 4 independent in vitro fertilization trials and developed successfully into normal embryos and larvae (Table 1). Although 40-70% mortality was observed during larval development 9% of the produced larvae settled successfully as pentactula and started feeding.

Larval development was normal and follows the documented stages for H. scabra described in the literature (Figure 2). The mean diameter of fertilized eggs was 150±3.0µm and underwent cell division within 1.5 hours. They developed into blastula and gastrula around 12 and 16 hours Post Fertilization (PF), respectively. Early hatched Auricularia stage was observed around 36 hours PF, and continued to develop to mid-Auricularia larvae at day 6 PF. Late Auricularia larvae was observed by day-13 PF and transformed to non-feeding doliolaria larvae after completing 15 days PF. There was a mass mortality reached 70% during the first 3 days of rearing. Primary microscopic observation of the larvae, revealed an abundance of ciliate protozoan and copepods in the larval tanks. However, from the 4^th day of development, survival rate remained almost stable. After 13 days, the larvae reached late Auriculariastage characterized by the appearance of lipid spheres at the edge of the cilia band. The first settlements were observed on day 20 PF and small juveniles began to appear on most trials on the bottom of the rearing tank 3 to 4 weeks PF. They appeared transparent and only visible using a light torch. Their size was estimated at 0.5 mm.

Figure 2: A. Immature oocyte; B. Mature oocyte; C. Fertilized egg; D. Two cell stage (1 h 30 mn); E. Four-cell stage (2 h); F. Multi- cells (3 h); G. Blastula (12 h); H. Gastrula (16 h); I. Newly hatched Auricularia (36 h); J. Early Auricularia (2.5 d); K. Mid Auricularia (6 d); L. Late Auricularia (13 d); M. Doliolaria (15); N. Early Pentactula with tentacles (20).

There was a high mortality during the first few days PF during all four trials. With the exception of trial 1 for which there were no microalgae to feed the larvae, from day 4 to day 15 mortality remained quite low. A second mortality was observed between day 15 and day 21 in all three remaining trials (Figure 3). During the daily microscopic observations of the larvae, abundant ciliate protozoans and copepods were observed in the larval tanks. However, after the 4^th day PF, survival was stable.

Figure 3: Survival rates of H. scabra over 4 hatchery runs and the mean of the 3 runs during 21 days of embryonic and larval development and rearing.

Overfishing of Holothuria scabra went beyond just an ecological issue, it became a socio-economic problem as livelihoods and income of many coastal dwellers rely on its fishery.

There are several national management measures applied on this fishery, including total ban as the current case here in Oman; however, these seem to be insufficient for a sustainable use of the resources [6]. A sustainable and efficient solution so far to enhance production as well as depleted natural stock of this species is through developing aquaculture [5]. To ensure H. scabra aquaculture sustainability and affordability, the production of fertilizable eggs and larvae in large quantities and at any time is a key element [14]. Several methods have been described to induce H. scabra spawning, mainly thermal shock [15-17] alone or combination with water pressure and drying treatments [18]. Spawning can also be induced using chemical such as spirulina. However, these methods may not be reliable to produce mature fertilizable oocytes outside the natural reproductive season of the organism [14,19]. Such critical point has been addressed through a new methodology based on in vitro maturation using artificial inducers [13,14] which recently have been identified [20]. The use of such maturation inducers allows holothurians farmers to obtain clean, fertilized eggs year-round. This method was tested in Oman H. scabra population and achieved more than 90% oocyte maturation and fertilization, which is consistent with Léonet et al. [13] and Eeckhaut et al. [14], results on Madagascar H. scabra populations. The method requires the sacrifice of several large adult specimens which may be difficult to justify for a locally endangered species, although these few specimens will produce 1000s of juveniles [21]. Despite larval developmental timing variations among various H. scabra populations, the embryos underwent normal development processes up to the pentactula stage over 21 days PF which is consistent with published values for larval development in H. scabra as it is reviewed by Junus et al. [3].

The high mortality observed during the first few days PF took place before feeding and is thus unlikely the result of a contamination by the feeding solutions. However, the abundant copepods and ciliates may be responsible for this temporary mortality [22]. Copepods attack larvae either directly or by repeated collisions causing bodily damage to the larvae [15]. Asha & Muthia [23] observed 65% mortality due to ciliates on the 9^th day of rearing H. spinifera larvae. Since the water used for larval rearing was filtered and UV-treated, it is unlikely the source of the copepods and ciliates, but some of the equipment used to siphon the water, transfer the larvae from one container to another, the container themselves may have been contaminated by a concomitant fish hatchery and emphasize the need for high levels of training in sterilization and decontamination and a sepsis. Unfortunately, separating copepods from sea cucumber larvae is difficult as their sizes are similar [24]. To control copepod population, Pitt & Duy [15] recommended the application of trichlorofon (Dipterex), an insecticide which may be effective against some arthropod infestation. Alternative solutions using either naturally occurring substances or better management practices are needed [25]. In addition, optimization of the diet using locally available species is also necessary [26].

The result presented here is a first insight on the artificial production of sea cucumbers in Oman. The potential to develop sea cucumber aquaculture in Oman is high because (i) H. scabra, a high commercial value, is still present in Oman particularly in shallow waters (ii) There are some natural locations ideally suited for sea ranching such as the Bay of Mahout, (iii) The human and the basic technological potential are already in place. For example, less than 1% of the surface of the Bay of Mahout Bay could support the sea ranching of 4 M of sea cucumbers H. scabra if it is supplied by an efficient farm having 50,000 m2 of ponds and an efficient hatchery with 600,000 l of adequate tanks.