PART II
SEA CUCUMBER (STICHOPUS JAPONICUS) CULTURE IN CHINA

CHAPTER I BIOLOGY OF THE SEA CUCUMBER

Sea cucumbers, which have been collected as food organisms for a long time, belong to class Holothuroidea of phylum Echinodermata. There are over 1,100 species of sea cucumbers under six orders.

1.1 Taxonomy of Holothuroidea

There are more than 100 species of Holothuroidea in China. They are distributed mostly in the South China Sea and Xisha Archipelago. There are about 20 edible species and the most preferred is Stichopus japonicus. The following 5 species are also commercially valuable sea cucumbers.

• Thelenota ananas (Jaeger): Large body, reaching a maximum length of 1 m. Its dorsal caruncles connected to each other like petal, so that it is known as “Plum blossom cucumber”, or “Pineapple cucumber”. Alive, its body colour is orange-yellow or orange-red on its dorsal side and red on its ventral side. Inhabits coral reef and co-exists with pearl fish in its cloaca. Only distributed in Xisha, Zhongsha and Dongsha Archipelago of China.

• Stichopus variegatus: Square shaped body, dorsal caruncles lining irregular. Little variation in body colour, usually dark yellow with olive dots. Found in intertidal coral reefs. The maximum length can be 40–50 cm, usually found in deeper waters. Distribution: Hainan Island, Leizhou Peninsula and along the coast of Xisha Archipelago.

• Holothuria scabra (Jaeger): Rough body surface, few tube feet. Dorsal side is dark brown-green, ventral side is white. Usually living along coastal reefs or in sandy areas where seaweeds are lush and tidal current is strong. Can reach a maximum length of 70 cm. Distribution: Hainan Island and Dongxing off Guangxi Province.

• Holothuria nobilis (Selenka): Also known as “Wuyun Shen”. Usually there are several breast-like caruncles on its body. Found under the sand with seaweeds in fort reef. Sometimes on seaweed in coral reef. Only distributed in Xisha Archipelago and Xinchun of Hainan Island.

• Actinopyga mauritiana (Quoy et Gaimard): 25–27 tentacles, lined irregularly. Around the anus there are five small calcareous teeth. Dorsum is olive-brown when it is alive. Each tube foot has a white ring. Distribution: Xisha Archipelago and Hainan Island.

1.2 Nutritional and Medicinal Values of S. japonicus

1.2.1 Nutritional Value

China is one of the earliest countries where sea cucumbers were eaten. Sea cucumbers are known their high protein content and absence of cholestrol. They are also considered as a tonic food. According to laboratory analysis, soaked Stichopus japonicus contains 76 % water, 21.5 % protein, 0.3 % fat, 1 % carbohydrate, 1.1 % ash, and 118 mg of calcium, 22 mg of phosphorus and 1.4 mg of iron per 100 g body weight. Dried S. japonicus contains 6 mg of iodine per kg body weight. Its intestine contains 72.49 % water, 8.836 % crude protein, 2.687 % crude fat and 15.987 % ash.

1.2.2 Medicinal Value

In China, sea cucumbers have been used as a beneficial drug since the Ming Dynasty. The body wall of S. japonicus is known to cure kidney diseases, constipation, lung tuberculosis, anaemia, diabetes, etc. Its viscera is said to be a cure for epilepsy and the intestine has some curative effects on stomach and duodenal ulcers.

According to current medical research, the dermal connective tissues, body cavity membrane and corium inner gland tube of S. japonicus contain many kinds of acidic mucopolysaccharide, that can have special effect on growth, recovery from illness, anti-inflammation, bone formation and prevention of tissue ageing and arteriosclerosis. Mucopolysaccharide is also an extensive anti-tumour drug. Through abdominal or intravenous injection, it was seen to have apparently suppressed the transplanted experimental tumours, S-180, S-37, etc. At the same time, it also has intensive effect on contravariant. Holotoxin extracted and purified from sea cucumber is an effective antimycin. It can suppress many kinds of molds at the density of 6.25–25 μg/ml.

So far, the species that have been known to have similar medicinal effects as S. japonicus are Stichopus variegatus Semper, Stichopus chloronotus Bramde, Thelenota ananas Jaeger and Bohadschia argus Jaeger.

1.3 Morphology

1.3.1 External Characteristics

Tube-shaped body, 20–40 cm in length and 3–6 cm in width. Quadrilateral in transverse section. Flat ventral side, on which tube feet are lined in three irregular longitudinal rows. Dorsum slightly raised with irregular caruncles in 4–6 rows. Mouth anterior and inclined to dorsum. There is a convex area behind the mouth, known as the gonophore.

1.3.2 Internal Structure (Fig. 1)

a. Body Wall

The cuticle is the external protective layer, under which there is the dermal cortex in which the ossicles are embedded. The ossicles within the body wall are table-like in shapes. The bases of table-like ossicles are either low or not completely developed and only have perforated trays. The muscular layer consists of transverse and longitudinal muscles. Under the muscular layer, there is a thin membrane, known as body cavity or mesentery, which is connected with the intestine. This membrane is divided into three segments: dorsal mesentery, left mesentery and right mesentery.

b. Digestive System

The digestive tract is a longitudinal tube and winds two times in the body cavity. It includes the following organs: tentacles, mouth, pharynx, oesophagus, stomach, intestine, general cloacal cavity and anus. The mouth is anterior and lacks masticatory organs. Twenty tentacles, which help in feeding, surround the mouth of S. japonicus. There is a calcarious ring around the pharynx, which is followed by a short oesophagus and an elastic stomach. The part where the oesophagus joins the stomach contains a lot of red pigments. The digestive tract gets easily broken at this point. Next to the stomach is the first small intestine (descending intestine), which abounds with yellow pigment and accompanies a dorsal blood vessel network. The end of the first small intestine extends forward to the left and appears “U”- shaped. This part is called the second small intestine (ascending intestine). The dorsal blood vessel network attaches to the second small intestine and closely connects with the left respiratory tree. The end of the second small intestine connects with the large intestine. The large intestine lies along the centre of the longitudinal muscle and directly reaches the general cloacal cavity, around which are muscles that enable its expansion and contraction. The external opening of the general cloacal cavity is the anus.

c. Respiratory System

With a thin lateral wall, the general cloacal cavity is a wide and short tube which stretches outwards, divides into two branches, and extends into the body cavity. The branches are called respiratory trees because they look like trees. The “trees” absorb oxygen. The dorsal blood vessel network is distributed to the outside of the left respiratory tree. The absorbed oxygen enters into circulatory system through the respiratory trees and is carried into other organs by the blood. Carbon dioxide (CO₂) is removed out of the body along the same channel.

Apart from the respiratory trees, the skin also has a respiratory function. Because the walls of the tube feet in ventral side are very thin, the feet could absorb oxygen from the water and remove CO₂ from the body.

d. Circulatory System

There is a hemal ring around the esophagus, from which five radial blood vessels are divided and spread along five canals and under muscle layer, until the posterior end. The dorsal blood vessel and the ventral intestinal blood vessel form the blood network, which covers the loops of the intestine. The left respiratory tree closely connects with the blood network. The blood of S. japonicus is transparent and brown in colour.

e. Water-vascular System

The water-vascular system is located above the haemal ring and surrounds the oesophagus. Five radial canals branch off from the ring canal and send branches forward to tentacles and backward to the tube feet along five ambulacral zones. The ring canal also connects with the Polian vesicle and the stone canal. There is a small pore at the end of the stone canal.

f. Nervous System

Oral nerves: There is a nerve ring in the calcareous ring from, which five radial nerves extend. They send branches forward to the tentacles and backward to the tube feet along the ambulacral zone.

Inner nerves: Nerve ring is absent. There are only five radial nerves, which give off branches to the transverse, longitudinal and radial muscles.

Figure 1. Internal structure of a sea cucumber. (1. pharynx wall; 2. radial water vessel; 3. tentacle vessel; 4. tentacle around mouth; 5. calcareous ring; 6. inner maduciorite; 7. stone canal; 8. water-vascular ring; 9. haemal ring; 10. oesophagus; 11. dorsal blood vessel; 12. gonad; 13. descendant intestine; 14. ventral blood vessel; 15. blood vessel joint; 16. respiratory tree; 17. general cloacal cavity; 18. anus; 19. blood vessel network; 20. longitudinal muscle; 21. vesicle; 22. ascendant intestine; 23. rectum; 24. Polian vesicle; 25. mouth; 26. mesentery; 27. water-vascular canal; 28. tube feet; 29. body wall; 30. radial muscle; 31. radial nerves).

1.4 Ecology

1.4.1 Distribution

S. japonicus is widely distributed. Its vertical distribution is from intertidal zone to 20–30 m depth zone. It is mainly distributed in the West Pacific Ocean. The Northern limits of its geographic distribution are the coasts of Sakhalin Island, U.S.S.R. and Alaska, U.S.A. The Southern limit is Tanega-shima in Japan. In China, it is commonly distributed on the coast of Liaoning, Hebei and Shandong Province, Yantai and Qingdao of Shandong Province. Its Southern limit in China is Dalian Island in Lian Yungang, Jiangsu Province.

1.4.2 Habitat

The environmental factors which have a close bearing on the habitat of S. japonicus are water temperature, salinity, tidal current, substratum, food, attachment and living spot of juveniles. Among them, water temperature, salinity and substratum are the main limiting factors.

a. Water Temperature

S. japonicus lives in the temperate and frigid zones. It cannot adapt to higher or lower water temperature. When the seawater temperature is below 3 °C, it moves slowly, feeds less and lives in semi-dormancy. Its feeding rate is also greatly reduced as water temperature rises to 17–19 °C, and the animal goes estivating as water temperature goes over 20 °C. If water temperature in its living environment remains high or low for a long period, it cannot grow normally.

b. Substratum

S. japonicus commonly lives on reefs and rocks. It is also found on muddy or sandy bottom with eel-grass clumps. However, it is hardly found in pure sandy or muddy bottom.

The distribution of S. japonicus depends on the particle composition of the substratum. Tables 1 and 2 show the distribution density of S. japonicus and the particle composition of the substratum in the coast of Ishigawa County in Japan and Gangdong in Qingdao, China, respectively. Investigations of the above regions show similar features. If the substratum contains more rough sand and rock the larger the distributive density of S. japonicus; less if the stratum is made up of small sand and mud.

* Results in March 6th, 1983.

c. Salinity

Generally, S. japonicus lives in seawater of normal salinity. Its suitable chlorinity range is 13.4–19.2 ppt. It can also adapt to certain habitats where it has lived for a long period. Living around reef and rock, the body colour of S. japonicus is red-brown or brown (hence called red sea cucumber). It adapts slightly to higher salinity and is mostly distributed in areas mainly influenced by oceanic water. Living on sandy and muddy bottom with bush seaweeds, its body colour is greenish yellow or greenish brown (thus also called green sea cucumber). It adapts slightly to lower salinity and is distributed in areas mainly influenced by fresh water.

Its tolerance of low salinity relates to its body colour. When chlorinity of seawater is below 5.1 ppt, both the red and green sea cucumbers show the tendency to die. The half-lethal time is 8–17 hours and the lethal time is 9–24 hours. In seawater with chlorinity level of 5.8–9.9 ppt, some of the green and red cucumbers die in a short period. The higher the temperature, the lower the intolerance, especially the red sea cucumber. At chlorinity level of 11.4 ppt, the green sea cucumber can be adapted for about 10–12 days at normally crawling stage. Over this period it can feed and grow normally. In the case of red cucumber, its tube feet initially lose their ability of adherence after 8–10 days and some dead sea cucumbers with decayed dorsum can be found. Half-lethal time (LT50) is 14 days and all die after 22 days. When chlorinity is 12.6 ppt, the green sea cucumber lives normally, but at 50 ppt the red sea cucumber dies after 30 days. It can only live normally in seawater of 14.1 ppt.

d. Depth

S. japonicus is distributed from the intertidal zone to a depth of 20 m. At different depths, its body weight varies. Juveniles above 3–4 cm in length mostly stay on rocks in the intertidal zone or attach on large subtidal seaweeds. S. japonicus less than 50 g are distributed in shallow water along the coast: those of 50–100 g are found in 5 m depth; 150–200 g in 10–15 m; and above 200 g in the region more than 15 m deep. It is rare to find S. japonicus over 200 g living in intertidal zone or shallow water. On the other hand, juveniles are rarely found in water deeper than 10 m.

1.4.3 Feeding Habit and Growth

The food of S. japonicus includes unicellular algae (mainly benthic diatoms), protozoans, fish eggs, larvae of sea animals, organic detritus and other small organisms. There is also a lot of sand and pieces of molluscan shells in its digestive tract. The composition of ingested sand corresponds to that of its habitat substratum. It feeds without selectivity; whatever is clinging on its tentacles is brought to its mouth. S. japonicus has been found to feed on 1.5 × 0.7 cm size shell. The feeding habits of the juvenile and young of S. japonicus are closely related to their ecological conditions. Generally, the juveniles and young live under rocks or attached to seaweed in intertidal zone. Apart from some mud and sand, the digestive tract, has mainly benthic algae and detritus. Up to a body wall weight of 2.0–2.5 g, it remains attached. It changes over from attached to benthic conditions when the weight exceeds 2.5 g.

The feeding activity of S. japonicus is characterized by crawling with its tentacles, which at the same time convey food to its mouth. Generally, it feeds on materials found at the surface of the sea bottom, but it also dig deeper when hungry. Even the young (2–3 cm in length) can dig 3–4 mm into the mud or sand to feed.

S. japonicus feeds without intervals, but the amount of food intake during day and night are different. At daytime, the intake is low because of its inactivity, the intake is high during night. The day-night feeding ratio is about 3–4:7-6.

The food intake by S. japonicus and its tract length and weight vary according to seawater temperature. It goes with aestivation at water temperatures over 20 °C. At this stage, it stays under rock or rock crevices and stops moving and feeding. There is no food in its digestive tract, which is reduced to the shortest and lightest stage. After aestivation, it crawls from its shelter and feeds again at 19–20 °C. Its digestive tract recovers gradually, but the length and weight of the tract do not increase. When water temperature is below 3 °C in winter, its normal activity also slows down; its feed intake decreases. When water temperature rises above 3 °C in the following spring, it begins to move frequently and its feed intake increases. At water temperature of 8–10 °C, it moves at the maximum frequency. Its digestive tract can reach as much as 5.7– 6.4 times longer than its body length. At temperature of 17–19 °C, it goes into its reproductive phase. During this period, it feeds less, moves more slowly and its digestive tract degenerates.

Monthly variations in food intake in relation to body wall weight of S. japonicus are delineated in Figure 2. It can be calculated from this figure that the annual food intakes by individuals of S. japonicus with body wall weights of 10 g, 30 g, 50 g and 100 g are 0.8 kg, 2.1 kg, 3.5 kg and 6.8 kg respectively.

Figure 2. Monthly variations in food intake in relation to different body wall weights of Stichopus japonicus.

The retention time of food in the digestive tract of sea cucumber is 21 hours, during which only 52–54 % of organic carbon in the food is absorbed. Carbon digestibility in sea cucumber is poor when compared to that in fishes. There enzymes present in the digestive tract of S. japonicus are amylase, cellulase, pectase, proteinase, dipeptidase, fatty glycerol and high fatty acid.

The movements, feeding and growth of S. japonicus are limited by water temperature and the seasons. Its normal activities and feeding are confined to about half the year only. Hence, it grows slowly. The body length of an S. japonicus that hatches in June is about 5.9 cm after 12 months, while its body weight reaches 15.5 g. After two, three and four years, its body length and weight are 13.3 cm and 122.4 g, 17.6 cm and 307.1 g and 20.8 cm and 472.5 g respectively. It has been reported that S. japonicus could live at least five years.

1.4.4 Respiration

The respiratory trees in the body of S. japonicus are the main respiratory organs, the skin also possessing respiratory functions. Its oxygen consumption varies with temperature level. At its normal temperature range, the adult consumes about 0.4–0.8 ml of oxygen per hour.

For respiration the sea cucumber draws in water several times before expelling the respired water. At water temperatures of 11–14 °C, it draws in water 9–10 times before expelling the water once. At 19–22 °C, it expels water once after drawing in 9–15 times. At 8 °C, it only opens and closes its anus slightly. If the respiratory trees are cut-off, the sea cucumber cannot perform its respiratory activities at the anus. The extent of respiration performed by the skin depends on the temperature. It increases with increasing temperature and can constitute as much as 60–90% of total respiration.

1.4.5 Movement

S. japonicus crawls slowly on the sea bottom through constriction of its longitudinal and transverse muscles and tube feet, it can cover 1 m in 10 minutes. The maximum linear distance that can be covered during one day is 170–180 m (average: 140 m). With abundant food and good environmental conditions, it only moves about 5 m per day. However, if food is scarce and the environmental conditions are poor, it can move for quite a distance and even loosen its body and float with the waves. Under artificial cultivation also, both the juveniles or adults can float. Mainly, this occurs during the night or at dawn.

1.4.6 Predators

The natural predators of S. japonicus are few. Sea gulls prey upon it at the intertidal zone. S. japonicus has been found in the stomach of salmon and trout. Young sea cucumbers (about 3 g) have also been found in the stomach of some other fishes such as bullheads. Starfish may prey on juveniles less than 3 cm long.

1.5 Ecological Characteristics

1.5.1 Eversion of Viscera and Regeneration

Under poor environmental conditions, S. japonicus can evert its viscera, which includes stomach, intestine, respiratory trees, dorsal blood vessel network and gonads. Eversion of viscera usually follows intensive constriction of the body wall. The viscera everted through the cloaca and the anus. The environmental stimuli that induce eversion include sudden increase or decrease of temperature, polluted water and physical or chemical stimulation.

Regeneration capacity is very highly developed in S. japonicus. When its caruncle is cut off, a small convex growth will appear at the same place in about 5–7 days and it grows to 1–2 mm in thirty days. When the tentacle is cut off, the wound heals and a convex growth appears in the same place in about 7–10 days, while the tentacle regenerates fully in 30–35 days and functions normally. Any wound up to 2–4 cm long on the ventral or dorsal side gets completely cured in 5–7 days. The regenerative capacity is stronger in the posterior region.

The regeneration in S. japonicus, which has everted its digestive tract and respiratory trees, proceeds as follows. The ventral central longitudinal muscle and two dorsal longitudinal muscles get connected by a suspended membrane after 5–7 days, the digestive tract is formed after 9 days, one respiratory tree is formed and the linear digestive tract is very obvious from the mouth to the total cloaca after 14 days and in about 25 days the respiratory trees develop noticeably and the digestive tract bulges. At this time, it begins to feed a little and about 33 days later, the respiratory trees resume functioning gradually.

The speed by which the digestive tract and respiratory trees of S. japonicus regenerate varies at different life stages. At resumption stage after aestivation, regeneration is very rapid. The digestive tract and respiratory trees regenerate and become completely functional in 25–33 days at this stage. However, such regeneration needs 8 weeks during other life stages.

1.5.2 Estivation

Generally, the period of estivation is usually from June to late September. When water temperature is at 3 °C, normal activity is hindered. When the temperature rises to 8–10 °C, the sea cucumber gets into its most active living stage. At 17–19 °C, it moves slowly and feeds less and its digestive tract begins to degenerate. At 20–25 °C, it stops feeding, discharges food in the digestive tract, stays under rock or in the crevice of a rock and begins to estivate. When temperature drops to 17–20 °C, it resuscitates, crawls out of its shelter and begins to feed.

Temperature is generally the main cause of estivation. Estivation is a means of ecological adaptation to high temperature. According to some, aestivation is resorted to recover from exhaustion caused by the discharge of genital products.

1.6 Embryology and Development

1.6.1 Reproduction

a. Gonad Development

S. japonicus is a dioecious animal, but it is difficult to distinguish sexes from outward appearance of the gonads. The colour of gonads after a closer examination is a reliable indicator. The gonad is branched. A tube, called the genital tube, extends forward from the gonad.

According to the developmental stages of the gonadal gland, five reproductive phases are distinguishable: namely spent, proliferative, active, ripe and spawning.

b. Reproduction

The reproduction of S. japonicus is mainly dependant on water temperature. It spawns at 15–23 °C, usually at 18–20 °C. Eggs are discharged at 2100–2400 hrs. Only rarely, it spawns after midnight or in the afternoon. The male releases sperms first and the female releases eggs later. Sperms appear milk-white in the water and eggs are orange-yellow. The female can spawn 1–3 times at an interval of 5–15 minutes or longer. Fecundity is 300,000–500,000.

1.6.2 Development

a. Embryo Development

• Sperm and egg. The mature sperm of S. japonicus does not move inside the testis. It moves actively only after it gets into seawater. Eggs are demersal and only have primary egg membrane, which is formed by the primary cytoplasm of egg.
• Fertilization. Eggs are fertilized in seawater.
• Cleavage. Because it is isolecithal, it cleaves completely.
• Blastula.
• Gastrula.

b. Larval Development

• Auricularia. Figure 3a.     Because the larva of S. japonicus is ear-like in this stage, it is called auricularia and has the following structures:

  1. Longitudinal ciliated band
  2. Larval arm

  3. Left anterior coelom sac, hydrocoel and posterior coelom. From hydrocoel, primary buccal tentacle and radial water-vessel are formed.

• Doliolaria.     Figure 3b.     It has the following characteristics:

  1. Ciliated ring
  2. Water canal ring and Polian vesicle
  3. Developed posterior coelom

  4. Vestibule

• Pentactula.     Figure 3c.     It has the following characteristics:

  1. Oral widened, the anus lost for a period and formed again. Five tentacles protrude from the vestibule.
  2. Ciliated ring degenerates until it disappears.
  3. Digestive tract is prolonged, winded; respiratory trees are going to be developed.
  4. Spicula is formed by primary mesenchyme.

  5. There is a group of cells near the posterior coelom, which will develop into posterior.

• Juvenile. The first tube foot is formed under the anus on the ventral side. Its body colour varies from colourless, half transparent, light brown to dark brown.

Table 3 shows the developmental stages of S. japonicus after fertilization at 20–21 °C.

Figure 3. Three major embryological stages of sea cucumber: a) auricularia, b) doliolaria, and c) pentactula.

CHAPTER II ARTIFICIAL BREEDING OF Stichopus japonicus

2.1 Breeding Status of Stichopus japonicus

As early as 1930, Japanese scholars began to study the artificial breeding techniques of sea cucumber, S. japonicus. For example, in 1937, a Japanese, Inaka Densapuro, succeeded in rearing fertilized eggs to auricularia in 15 days. Until the 1950s, much useful information covering the ecological habit, life history and gonad development was increasingly gathered by Japanese biologists. These informations provided the basis for artificial breeding of sea cucumber. In 1950, Imai Taiju et al. were able to obtain 569 juveniles of S. japonicus in a 1.9 m³ tank using non-colour flagellates as larval diet. In 1977, another Japanese, Ishida Masatoshi, reported that 2.975 auricularia were obtained from induced spawning using thermal shock in the experimental farm of Fukuoka county.

In the USSR, Mokpelsba (1973) carried out artificial breeding of sea cucumber in Big Peter Bay by means of thermal shock and using Phaeodactylum tricornutum and Platymonas spp. as diet of postlarvae. However, no mention was made of the survival rate of juveniles and the report only briefly mentioned the stocking density as 4000 individuals per m².

In China, the study of sea cucumber has focussed on artificial breeding since the late 1970s, dealing with the relation of gonad development, spawning habit, embryonic development and postlarvae development to the physical, chemical and biological factors in the environment. Studies on the feeding habit at every stage of development of postlarvae, selection of diet species and other topics have provided a useful scientific basis for artificial breeding on a commercial scale.

From 1973 to 1985, the Yellow Sea Fisheries Research Institute and its cooperative units worked jointly to improve the techniques of artificial breeding of S. japonicus, including collection of broodstock, spawning, fertilization, rearing of postlarvae and juveniles, diet species and their cultivation method, prevention and control of natural enemies and other aspects. These studies have led to the development of suitable technologies for artificial breeding and rearing of S. japonicus.

2.2 Basic Facilities for Artificial Breeding

2.2.1 Nursery Room and Feeding Room

A new nursery room should be selected near the seashore where the water is calm, free of pollution, and there is no in-flow of freshwater. The nursery room should preferably face south-east, with good ventilation and uniform illumination. The optimum light intensity required in the room is 1000–2000 lux.

The ratio of area of rearing tanks of larvae and unicellular algae should be 1:4. However, in the case of high density cultivation of unicellular algae, their rearing can be decreased.

2.2.2 Broodstock and Postlarvae Rearing Tanks

Rearing tanks are the main components of the nursery room. At present, the general construction of the tanks is of brick and concrete or reinforced concrete. They should be rectangular with rounded corners and should be suitable for rearing with flowing water. The size of the rearing tank should be preferably between 5 m³ and 10 m³. The height of the tank should be less than 1 m. The required volume of the tank for S. japonicus broodstock is relatively small, which is about 1.5–2 m³. The intake and drain pipes should be well integrated for efficient operations.

2.2.3 Settling tank

Suspended solids in the seawater supply, such as silt, bits of organic matter and plankton should be allowed to settle, a process which generally takes more than 24 hours. The settling tank should be covered to darken the tank and hasten the settling of the plankton. The tank should also have a layer of heat insulation to prevent strong sunshine from raising water temperature. Settling tanks must be regularly cleaned to prevent the build up of harmful materials such as H²S, NH³ and so on. The bottom of the tanks should be swept once every 4–5 days. The total capacity of the settling tank may be 2–3 times as much as the volume of water used every day. The tank can be partitioned into several compartments, so that settling and cleaning can be rotated among the compartments.

2.2.4 Filtration tank

The water from the settling tank, after being precipitated, may be used directly in the nursery room, or filtered with a silk-bolting cloth and cotton or sand before it is piped into the nursery room.

1) Sand filtered tank: Water passes through the layer of filtering materials by gravity. The filtering materials consist of layers of different size sand and stone. Cobble, gravel, grit and fine sand are placed in layers and thickness of each layer is about 10–15 cm; the thickness of the fine sand layer should be increased appropriately. The total thickness of the filter is 40–60 cm.

2) High-pressure sand filter tank: This is a closed system, in to which water is forced into the filter tank by pump or from a highly elevated settling tank. Its construction is simple. It is built with reinforced concrete or steel plate, with a sieve plate in the centre of the tank, on which cobble stones and sand are placed (the same way as in a sand filter tank). Sometimes, fish net and window screen are spread on the sieve plate, over which are spread silk bolting cloth and then fine sand.

2.3 Gonad Developmental Stages

2.3.1 Resting Stage

Male ♂ : In this stage, which lasts from the last ten-day period of June up to November, the gonad is small and its epithelium along the wall of the tube is without bumps and holes. The epithelium consists of three layers of spermatogonia or spermatocytes. In general, weight of the gonad is below 0.2 g.

Female ♀ : Gonad is small and its epithelium along the wall of the tube is without bumps and holes; it consists of two layers of oocytes, with diameter of about 10 μm.

2.3.2 Recovering Stage

This stage generally lasts from December to March. The weight of the gonad generally varies from 0.2–2 g, with the gonadal index below 1 %. Gonads develop slowly, colourless or light yellow, some can now be distinguished as female or male. The diameter of oocytes is 30–50 μm; the nucleoli in nucleus are noticeable. The sperms have not been formed.

2.3.3 Developing Stage

This stage can be divided into developing stage I and developing stage II. In general, stage I is from March to the first ten-day period of May. In this stage, gonads gradually grow in thickness and branch more. Weights are generally 2–5 g and the colour apricot or light tangerine. Female and male can already be distinguished. The gonadal index is generally 1–3 %.

The stage II occurs during the second and third ten-day periods of May. In this stage, gonads develop very quickly and gain weight rapidly (generally 3–13 g). Those heavier than 7 g comprise 70 % of the total. The colour becomes darker and the index rises to about 70 %. The diameters of oocytes range from 60–90 μm, sperms have formed.

2.3.4 Ripening Stage

This stage is generally from the last ten day period of May to the first ten-day period of June. Gonads become thicker and the colour turns dark. Those heavier than 10 g comprise fifty percent of the total. The index of about one-half of the total reaches ten percent. The testes are filled with sperms; the diameter of oocytes is 100–130 μm.

2.3.5 Spawning Stage

Releasing of eggs and sperms begins during the first ten-day period of June. Usually, gonads of bigger broodstock develop earlier, faster and mature more rapidly. Initial spawning occurs when the individuals are about 110 g in total weight and 60 g in body wall weight. The number of eggs per g of ovary varies from 220,000 to 290,000. The broodstock whose body weights range from 200 to 350 g generally have a fecundity of 2.5 m to 3.6 m eggs.

2.4 Collection of Broodstock

2.4.1 Collection Timing and Water Temperature

The key to a successful collection of well-developed broodstock is the timing and proper water temperature during collection. 15–16 °C is appropriate temperature to collect the broodstock since the rate of fluctuation of water temperature varies in different areas, there is need for measures to suit local conditions. Before the broodstock are collected, samples should be taken and dissected for observation. Early collection must be avoided. However, if they are raised too long, their gonads will atrophy and if they are collected too late they will lay eggs in the sea.

2.4.2 Broodstock Collection Standards

Measurements have shown that the following information is needed to guide one in selecting broodstock: when the weight of body wall of S. japonicus reaches 255 g (the weight of body wall is about one-half of that of the body), its gonad is 98 g; when the weight of body wall is 130 to 255 g their gonads are 34.7 g on an average and the mean gonadal index is 16.6 %. When the weight of body wall is from 80–110 g, the gonads are 5.6 g.

The above data indicates that the size of S. japonicus to be collected should be over 20 cm and should weigh over 250 g(their body wall would be about 130 g). In general, wall weight is directly related to gonad weight and gonad index to fecundity.

2.4.3 Broodstock Collection Methods

The quality of broodstock has a great bearing on breeding. When broodstock are collected, the following must be strictly observed:

1. Collect large individuals with thick body walls and body length of over 20 cm must only be collected. The body surfaces should be free of injury.

2. In the course of collecting broodstock, they should not be allowed to get in touch with greasy dirt.

3. The broodstock collected from the sea should be put in good quality water and care should be taken to change water often and to avoid direct light.

2.4.4 Broodstock Transportation

After collection, the breeders can be transported by road or boat. Transportation by boat is preferable since it will be free from bumps, which are characteristic of land journey and which may cause injury to the breeders. If the breeders have to be transported by road, they should be put in a plastic bag containing seawater and the bag placed in a canvas bucket filled with seawater. The bucket should be covered with some algae to keep seawater from spilling out and to shut out light. The best time for transportation is early morning or evening.

2.5 Broodstock Rearing

2.5.1 Rearing Density

If density of broodstock is too high, it will result in rapid depletion of dissolved oxygen. An environment of low DO over a long period causes abnormal behavior in the broodstock in that they tend to crimp their bodies and creep incessantly on the surface of the tank wall. It also adversely affects gonadal development, resulting in failure to release the sexual products normally. When the density of breeders is more than 40 individuals per m³, 70 percent of them have been observed to fall to the tank bottom and become stiff, an indication of paralysis, because of dissolved oxygen level dropping to 0.6 mg/1. When the density of breeders is 30 individuals per m³, the DO level is generally below 5–6 mg/1. Therefore, it is desirable to maintain the broodstock at 20–30 individuals per m³.

2.5.2 Broodstock Management

1. For the water in the raising tank to remain fresh and have normal DO, one half or one-third of the volume should be changed once in the morning and again at night.

2. Excreta and dirt in the tank should be removed immediately.

3. The behavior of the individual breeders should be constantly watched.

The discharge of sperms by some of the males and the frequent trips of the females to the tank wall where they hold their head high and keep swaying, are indications that the breeders are ready to spawn. It is necessary to make all the preparations for spawning and further care well in time.

2.6 Spawning and Handling of Eggs of Embryos

2.6.1 Spawning

The main aim of artificial breeding is to successfully obtain high quality zygotes. Natural spawning and the various methods for inducing spawning are detailed below.

1. Natural spawning

   When their gonads are fully mature, the male and female breeders release their gametes naturally, without any inducements. At first, the male releases the sperms, which induces the female to release eggs after about half an hour. The eggs are generally released around 2000–2100 hrs. Some females are able to release eggs continuously for more than 45 mins. One female can release more than 1 million eggs at one time and a total of 4–5 million eggs during one spawning phase.

2. Stripping

   This method was used by a Japanese (Inaka Densapuro) during the 1930s and a Chinese (Fengying Zhang) in the 1950s. The rate of fertilization is as low as 20% and the number of deformed individuals is large. In this method, to start with the back of the breeder is cut open with a scissor from anus upwards. The ovary or testis is taken out and dried in the shade. The ovary is then placed in a container filled with seawater and torn lightly with tweezers or scissors to release the eggs into the seawater. The eggs are filtered off from the water with a gauze and set aside. The testis is placed in another container with seawater and cut to pieces, when the sperms swim out into the seawater. The seawater with eggs in then poured into the one with sperms for the eggs to be fertilized. It is difficult to get a high rate of fertilization with this method and is, therefore, limited only to small-scale experiments.

3. Thermal shock

   This method is often used to induce spawning in marine invertebrates, such as molluscs and echinoderms. The water temperature of filtered seawater can be raised by exposure to intense sunshine, or with an electric heating rod, or by adding hot water with a temperature higher by 3–5 °C than that of the filtered seawater. This thermal shock stimulates the breeders to discharge sperms or eggs. This method is widely used.

4. Stimulation through desiccation and flowing water

   This method can be used after the breeders have been conditioned over 7–10 days. Stimulation for inducing spawning is generally carried out at dusk. First, the tank is emptied of all the water and the broodstock left to dry in the shade for a period of time. They are then subjected to high pressure seawater for several minutes. While applying water pressure, the tank should be scrubbed clean and later filled with filtered seawater. After the breeders have been stimulated for 1.5–2 hours, they begin to move up the tank wall and move about frequently. First, the male will release sperms in about half an hour's time after which, the female is induced to release eggs. This method can generally result in 95–100 % fertilization. With this method, one can plan in advance and work out a programme for use of facilities, food propagation, and other breeding operations.

2.6.2 Fertilization

It is important to ensure a high survival rate in artificial breeding by obtaining high quality eggs. Therefore, it is necessary to handle the eggs carefully as soon as they are released. Two procedures are followed in this regard at present.

1. After they have released their eggs or sperms, the breeders are removed from the tank. The eggs are washed several times, usually in the early morning hours of the day after spawning. However, this method is not very satisfactory, since the large quantity of sperms released into the same tank might pollute the water, resulting in reduced fertilization and a large number of deformed embryos.

2. The second procedure involves the use of an egg-box for keeping the eggs separately. When the high peak of spawning begins, constant observation of the broodstock is needed. A person must be assigned to conduct inspection tours in the evening. As soon as the male is observed to release sperms, the inspection tours to the tanks should be done more frequently. When the female breeders begin to release eggs in large numbers, they should be gently moved to a specially prepared “eggs-box” holding filtered seawater to continue spawning. Because the eggs of S. japonicus tend to sink the water must be stirred with a glass rod as the eggs are being released in order to keep the eggs suspended so as to increase fertilization rate. After the releasing of eggs is completed, the breeders must be moved out at once and the water stirred thoroughly. A sample is taken to estimate the number of eggs and the status of fertilization. In general, matured high quality eggs are spherical and evenly formed. Their diameter is generally about 140–170 μm, while the length of normal sperm head is 6 μm. After fertilization, the zygotes should be transferred with a rubber pipette into a rearing tank, which has been washed clean and contains filtered seawater. The density of eggs put in postlarval rearing tank is 10 million per m³. While the eggs are being piped into the rearing tank, filtered seawater is gradually added so that the eggs are distributed uniformly. This helps to improve the rate of fertilization, which should be aimed at 95– 100%.

   The density of eggs in the eggs-box should be kept below 100 per ml.

CHAPTER III REARING OF POSTLARVAE

3.1 Preparation of Rearing Tanks

Rearing tanks and other tanks used in breeding, especially the new tanks, must be scrubbed clean and then kept filled with water for 25 to 30 days, during which period the water is changed repeatedly in order to lower the pH to less than 8.5. Before the tanks are used, they are scrubbed and filled with water containing 40 ppm bleaching powder and then washed clean with filtered seawater before the larvae are introduced.

3.2 Rearing Density

Strict control of rearing density of larvae, i.e. the number of larvae per ml of water, is required. At present, there are two methods to rear the larvae: still water rearing and flowing water rearing. Auricularia, during their early and middle stages, concentrate at the surface of the water. If the density of larvae is too high, they will cohere into a ball and sink, resulting in death. Therefore, controlling rearing density would ensure a better survival rate. The Yellow Sea Fisheries Research Institute, the Marine Fisheries Research Institute of Liaoning Province, and the Marine Fisheries Research Institute of Shandong Province have separately carried out several experiments on rearing density. The results of the experiments indicate that the desirable density is 300–700 postlarvae per litre.

3.3 Selection and Counting of Larvae

After the fertilized eggs are moved to the rearing tank, they develop into the early auricularia stage in about 30-hours. The bottom of rearing tanks should be cleaned completely. Healthy larvae occupy the surface layer of water, while the deformed larvae and dead embryos generally stay in the lower layer of the water column or at the tank bottom. All the dead individuals, deformed larvae and sediment should be siphoned out in order to clean the tanks. After the tanks are cleaned, the rearing water is stirred lightly up and down for the larvae to be evenly distributed. A sample is then taken for counting of larvae. Samples are taken separately from the two ends and the middle of the tank with a 250 ml beaker; in turn three smaller samples are obtained from the first sample with a small cylinder or measuring pipette. These are used for counting. The average of three counts is taken as an indication of the density of larvae. The result of the count would show whether the density is desirable or not. When auricularia are in their early stage, they are reared at a density level of about 500 per litre. The period of auricularia development can be divided to three stages: early, middle and late stages. As they develop from one stage to the next, the bottom of tanks must be cleaned completely once, or the larvae moved to another tank. The sediment must be removed to keep the water fresh. An up-to-date information on the survival rate at each developing stage is necessary.

3.4 Water Management

In the course of rearing, the larvae eject faeces and consume dissolved oxygen constantly. Some of the larvae die in course of time. These and the leftover food produce harmful substances, such as H₂S, and NH₃. In addition, bacteria reproduce rapidly with rise in temperature. Poor water quality directly affects the normal development of larvae. Therefore, proper water management and sanitation is essential. Cleaning of tanks and changing water are essential. There are several methods of changing water. In the still-water rearing method, which is used in some places, the tank is gradually filled with water during the earlier stage of rearing. The dirt and deformed larvae on the tank bottom are siphoned out every day. Net cages are used for changing water. Attention must be paid to the mesh size of the silk-bolting cloth of net cages; the mesh size must be smaller than the body width of larvae, otherwise they will be washed away. While the water is being changed with net cage, someone should constantly stir the water lightly all around the tank. This will prevent the loss of larvae during water change, since siphoning would normally force the larvae to stick to the net cages, which might cause mechanical injury to the larvae. The sediments on the bottom of tanks should be siphoned out completely every three or four days.

Since 1982, the Yellow Sea Fisheries Research Institute has used the method of rearing in flowing water, which maintains a better water quality and avoids injury to larvae. The water flow in a 7m³ tank should be maintained at 6,000 ml/min. With the flow at this volume for 8–10 hrs every day, the quantity of water that is changed is more than half of the total volume. The water flow should be stopped for one hour while food is being put in.

3.5 Food Species and Feeding Rates

Suitable and high quality species of diet and appropriate feeding schedule are the key to successful rearing.

As the larva of S. japonicus develops into early auricularia stage, its alimentary canal is linked up and the larva must be given its diet at once. The feeding mechanism of the larvae consists of conveying the suspended bits of organisms and unicellular algae into the alimentary canal through the mouthparts by the swaying of the peristomial cilia. The effectiveness of several unicellular algae as larval diet has been studied. The results show that Platymonas, even though it can be easily bred on a large scale, cannot be ingested by larvae because of its big size and powerful moving ability. In addition, the larvae cannot easily digest the thick cell wall of Platymonas. Thus, the larvae fed on this species develop slowly, have low survival rate, and more deformed individuals. Dunaliella sp. is effective, since it has no cell wall and can be easily digested. Hence, the larvae develop quickly and show high survival rate. Phaedocatylum tricornutum is small in size, has weak moving ability and can be easily propagated on a large scale. Its effectiveness as a diet is also very good. A notable point in using this diet is that the optimum temperature for its reproduction and growth is 14–18 °C, which is identical with the temperature requirement of the early part of larval development. Dicrateteria sp., whose temperature requirement for reproduction and growth is 18–28 °C, can meet the diet requirement of the later part of larval. Some also use Chaetoceros sp. and Isochrysis sp.

The larvae require different quantities of diet during different development stages. Unicellular algae are fed twice a day, but the quantity given each time depends on the particular stage of the larvae. In general, it is from 19,000–30,000 cells per ml of rearing tank water. The quantity of diet given should be increased or decreased depending on the quantity of food in the stomach of sea cucumber, which has to be checked every day. Unicellular algae during the peak period of their reproduction are the most preferred diet for the larvae. Combinations of a few algae are more effective than a single species diet.

3.6 Environmental Factors

3.6.1 Temperature

The optimum temperature for rearing larvae of S. japonicus is 18–22 °C. The water temperature should be measured twice a day, in the morning and afternoon.

3.6.2 Dissolved Oxygen

Dissolved oxygen level varies with water temperature. The higher the temperature, the lower the DO level. Two units are used for DO level, viz. ml per l, and mg per l and their conversion relation is as follows:

1 mg/l = 0.7 ml/l, or 1 ml/l = 1.43 mg/l

3.6.3 pH

Under normal conditions, the rearing seawater is generally alkaline with a pH of 7.5–8.6. Tests have shown that S. japonicus larvae and juveniles adapt to a fairly wide range of pH. However, when pH rises over 9.0 or drops below 6.0, the moving ability of the larvae weakens and growth stops. Therefore, pH value of the water must be kept between 6.0 and 9.0.

3.6.4 Salinity

The salinity of normal seawater is 32–34 ppt. If the temperature of the rearing water is 18–22 °C and salinity 1.5–12.9 ppt all the larvae will die in 1–2 days. The larvae reared in 19 ppt salinity seawater for 4 days stop developing further. In 26.2–32.7 ppt salinity, they develop normally, but in 39.3 ppt salinity they develop slowly and their size remains small. The lethal critical salinity is 12.9 ppt. The optimum salinity for larval development ranges from 26.2–32.7 ppt. In this range, higher the salinity, quicker is their development. Both too high and too low salinity values adversely affect the normal development of embryo and larvae, resulting in a large number of deformed individuals or even causing death. Salinity measurement is, therefore an important routine work throughout the entire rearing period. A salinity refractometer is now commonly used for salinity measurement. If a specific gravity meter is used, the measured value can be converted into salinity value using the following formula or by consulting the salinity-specific gravity index chart (Table 4):

1. When the water temperature is over 17.5 °C,

   S(%o)= 1305 (specific gravity - 1) + (t - 17.5) × 0.3.

2. When the water temperature is below 17.5 °C,

   S(%o)= 1305 (specific gravity - 1) - (17.5-t) × 0.2.

3.6.5 Ammoniacal Nitrogen

The ammoniacal nitrogen content of seawater is very low. Its sources in breeding tanks are mainly the metabolites of the larvae, the unconsumed diet and decomposing organisms. Too much accumulation of NH₃ can be harmful to the larvae. The larvae can develop normally with an ammoniacal nitrogen content of 70–430 mg per m³ water. When its content is over 500 mg per m³, it will have a harmful effect on the development and growth of larvae.

* a_t = (reading - 1) × 1000

CHAPTER IV REARING OF JUVENILES

4.1 Types of Settling Bases

When the larvae develop to the late auricularia stage, their bodies contract to half their original length. When they begin to develop into doliolaria, the shelf of settling base should be placed in time. Different units or institutes use different types of settling plate bases. For example, the Yellow Sea Fisheries Research Institute uses a 60×60×80 cm frame welded with a 0.6 cm diameter hard plastic tube. The other types used elsewhere are i) silk bolting cloth and transparent plastic film tied to the frame at 45° angle to each other, ii) No. 8 zinc plated iron wire covered with a plastic tube, the two ends of which are sealed to form the frame, iii) wooden frame, to the middle of which are inserted plates of polyethylene or some other material, iv) tiles hung in the tank and v) stones of different size placed at the bottom of the tank.

The settling bases should have the following characteristics:

1. Juvenile S. japonicus that settle on them can be observed, handled and managed conveniently.

2. Have no toxicity to juveniles and do not spoil water quality.

3. Allow maximum settling of juveniles per unit area.

4. Easily available and inexpensive.

The size of settling base depends on the specific situation of each unit and cannot be set rigidly. In addition, the settling bases should be covered with a layer of diatoms, so that as soon as the juveniles settle on the bases, they can have ready food.

4.2 Diet of Juveniles

Just after completing their metamorphosis, the juveniles have only weak moving ability and their tentacles are short. If food cannot be provided on time, they would die. Studies have been made on the type and composition of diets appropriate for the juveniles. The seaweeds tried for this purpose include Sargassum thumbergii, S. kjellmanianam, Pelvetia siliquosa, Laminaria japonica, Undaria pinnatifida and Ulva lactuca. The seaweed was ground with seawater and the liquid filtered off through a silk-bolting cloth was mixed with the filtered liquid of ground sea grass and fed to the juveniles. Of all the diets tried, S. thumbergii gave high survival rate and faster growth. Juveniles with body length shorter than 2 mm take mainly benthic diatoms as diet. Unicellular algae and filtered liquid of ground S. thumbergii should also be fed every day. When the body length of the juveniles reaches 2–5 mm, their diet should mainly consist of filtered liquid of S. thumbergii, to be fed twice every day. The quantity of diet is increased daily as the juveniles grow.

4.3 Density of Settled Juvenile

When the larvae develop to the juvenile stage, they begin to crawl. Most of them stay on the settling bases. About 15 days after they have settled on the bases, they can be seen by the naked eye. They should now be counted. A random sample is taken with a 5 cm² or 10 cm² counting frame. The sampling area of each tank must be over 5 % of its total area.

In order to achieve increased survival rate, it is necessary to control appropriately the settling density on settling bases to kept it at the optimum level. Tests have shown that too thick density of settling and insufficient diet will be adverse to growth and survival. Hence, after they are counted, their density should be adjusted to the optimum, which is 200–500 individuals per m².

4.4 Juvenile Rearing in Flowing Water

The flowing water method to rear juvenile is beneficial in two ways: it keeps the water fresh and enhances the growth of benthic diatoms on setting bases. The water flow can be appropriately adjusted to about 8–10 l/min. Still water rearing requires a larger volume of water changes, especially in juvenile rearing. Change of water is critical during this stage, because of the need to maintain good water quality. Adequate diet and optimum temperature are other important requirements. The juvenile rearing period coincides with the high temperature period.

Individual juveniles begin to attain different sizes. Since larger individuals will monopolise feed utilization, the juveniles should be segregated by size for the smaller and weaker juveniles to develop properly. The bigger individuals must be taken out and placed in a separate tank. When the juveniles have grown to a certain size, they are transferred to the sea for further growth.

CHAPTER V
PREDATORS AND THEIR CONTROL

5.1 Predation

The juveniles begin to settle from the last ten days of June to July. This is also the period of high water temperature, when predators such as harpacticoids and other copepods are at the peak of their reproduction. These do much harm to juveniles that are smaller than 0.2–0.5 mm. Harpacticoids harm the juveniles by:

1. Reproducing very rapidly in rearing tank and competing for food with the juvenile. (Unicellular algae and Sargassum thumbergii have been found in their alimentary canals).

2. Wounding the body surface of the juveniles with their mouth parts and tearing the epidermis of the juveniles, exposing the bone plates and making them vulnerable to further predation. The parasites eat away at the juveniles until they die. The infested juveniles assume a ball shape and die gradually.

5.2 Predator Control

Control trials on harpacticoids and other copepoda with different chemicals at different concentrations have been conducted. Harpactocoids are sensitive to organic phosphorus. Thus, dipterex, kogor and other chemicals containing organo-phosphorus have been tested. The results show that all harpaticoida can be killed with 2 ppm dipterex in two hours with no harmful effects on the juveniles. However, it is necessary to give careful attention to the preparation of dipterex solution of appropriate concentration. The solution is evenly sprinkled into the tank and the water in the tank must be changed completely after two hours.

PUBLICATIONS AND DOCUMENTS OF THE REGIONAL SEAFARMING DEVELOPMENT AND DEMONSTRATION PROJECT RAS/90/002 (RAS/86/024)

Working Papers
RAS/86/024

NACA-SF/WP/87/1. Lovatelli, A. Status of scallop farming: A review of techniques. 22 pp.

NACA-SF/WP/88/2. Lovatelli, A. Status of oyster culture in selected Asian countries. 96 pp.

NACA-SF/WP/88/3. Lovatelli, A. and P. B. Bueno, (eds). Seminar report on the status of oyster culture in China, Indonesia, Malaysia, Philippines and Thailand. 55 pp.

NACA-SF/WP/88/4. Lovatelli, A. Status of mollusc culture in selected Asian countries. 75 pp.

NACA-SF/WP/88/5. Lovatelli, A. and P. B. Bueno, (eds). Seminar report on the status of seaweed culture in China, India, Indonesia, ROKorea, Malaysia, Philippines and Thailand. 79 pp.

NACA-SF/WP/88/6. Lovatelli, A. and P. B. Bueno, (eds). Seminar report on the status of finfish culture in China, DPRKorea, Indonesia, ROKorea, Malaysia and Singapore. 53 pp.

NACA-SF/WP/88/7. Lovatelli, A. Seafarming production statistics from China, Indonesia, ROKorea, Philippines, Singapore and Thailand. 37 pp.

NACA-SF/WP/88/8. Lovatelli, A. Site selection for mollusc culture. 25 pp.

NACA-SF/WP/88/9. Lovatelli, A. and P. B. Bueno, (eds). Seminar report on the status of finfish netcage culture in China, DPRKorea, Indonesia, ROKorea, Malaysia, Philippines, Singapore and Thailand. 56 pp.

NACA-SF/WP/88/10. Chong, K. C. Economic and social considerations for aquaculture site selection: an Asian perspective. 17 pp.

NACA-SF/WP/89/11. Chen J. X. and A. Lovatelli. Laminaria culture - Site selection criteria and guidelines. 30 pp.

NACA-SF/WP/89/12. Chen J. X. Gracilaria culture in China. 18 pp.

NACA-SF/WP/89/13. Seafarming Project, RAS/86/024. Site selection criteria for marine finfish netcage culture in Asia. 21 pp.

NACA-SF/WP/89/14. Lovatelli A. Seafarming production statistics from China, India, Indonesia, ROKorea, Philippines, Singapore and Thailand. 47 pp.

NACA-SF/WP/89/15. Chong K. C. and D. B. S. Sehara. Women in aquaculture research and training. 20 pp.

Working Papers
RAS/90/002

SF/WP/90/1. Chen J. X. Brief introduction to mariculture of five selected species in China. 36 pp.

SF/WP/90/2. Lovatelli, A. (ed.). Selected papers on mollusc culture. 74 pp.

SF/WP/90/3. Lovatelli, A. Artificial propagation of bivalves: Techniques and methods. 56 pp.

SF/WP/90/4. Lovatelli, A. Seafarming production statistics from China, Hong Kong, India, Indonesia, Malaysia, Pakistan, Philippines, Korea (Rep.), Singapore and Thailand. 50 pp.

SF/WP/90/5. Tiensongrusmee, B. Site selection for Eucheuma farming. 18 pp.

Bibliography

NACA-SF/BIB/88/1. Selected bibliography on seafarming species and production systems. 20 pp.

NACA-SF/BIB/88/2. Selected bibliography on seafarming species and production systems. 52 pp.

NACA-SF/BIB/89/1. Selected bibliography on seafarming species and production systems. 49 pp.

Training Manuals
RAS/86/024

Manual on seaweed farming: Eucheuma spp. (Training manual No. 1). 25 pp.

Culture of the Pacific oyster (Crassostrea gigas) in the Republic of Korea. (Training manual No. 2). 64 pp.

Culture of the seabass (Lates calcarifer) in Thailand. Training manual No. 3. 90 pp.

Training manual on marine finfish netcage culture in Singapore. (Training manual No. 4). 275 pp.

Culture of Kelp (Laminaria japonica) in China. (Training manual No. 5). 204 pp.

Training Manuals
RAS/90/002

Training manual on Gracilaria culture and seaweed processing in China. Training manual No. 6. 155 pp.

Training manual on artificial breeding of abalone (Haliotis discus hannai) in Korea DPR. Training manual No. 7. 124 pp.

Training manual on pearl oyster farming and pearl culture in India. Training manual No. 8. 103 pp.

Technical Publications

Integrated Fish Farming in China. NACA Technical Manual 7. A World Food Day Publication of the Network of Aquaculture Centre in Asia and the Pacific, Bangkok, Thailand. 278 pp.

Meeting Reports
RAS/86/024

Report of the First National Coordinators' Meeting of the Regional Seafarming Development and Demonstration Project, 27–30 October 1987, Bangkok, Thailand. 71 pp.

Report of the Second National Coordinators' Meeting of the Regional Seafarming Development and Demonstration Project, 20–23 September 1988, Singapore. 102 pp.

Report of the Third National Coordinators' Meeting of the Regional Seafarming Development and Demonstration Project, 24–27 August 1989, Qingdao, China. 103 pp.

Report of the Fourth National Coordinators' Meeting of the Regional Seafarming Development and Demonstration Project, 9–12 January 1991, Bangkok, Thailand. 133 pp.

Workshop Reports
RAS/86/024

Report of the FAO Asian Regional Workshop on Geographical Information Systems: Applications in Aquaculture, 5–23 December 1988, Bangkok, Thailand. FAO Fisheries Report No. 414, FIRI/R414. 13 pp.

Report of the Workshop and Study Tour On Mollusc Sanitation and Marketing, 15–28 September 1989, France. FAO/UNDP Regional Seafarming Development and Demonstration Project RAS/86/024. 212 pp.

Workshop Reports
RAS/90/002

Report on the Regional Workshop on the Culture and Utilization of Seaweeds, 27–31 August 1990, Cebu City, Philippines. Volume I. FAO/UNDP Regional Seafarming Development and Demonstration Project RAS/90/002. 183 pp.

Report on the Regional Workshop on the Culture and Utilization of Seaweeds, 27–31 August 1990, Cebu City, Philippines. Technical Resource Papers. Volume II. FAO/UNDP Regional Seafarming Development and Demonstration Project RAS/90/002. 180 pp.

Report on an ADB/NACA Study and Workshop on Fish Health and Fish Diseases. FAO/UNDP Regional Seafarming Development and Demonstration Project RAS/90/002. In press.

General Reports

Progress report on the 1988 Regional Training/Demonstration Courses organized under the Regional Seafarming Development and Demonstration Project (RAS/86/024). 26 pp.

Report of the Seafarming Resources Atlas Mission. Regional Seafarming Project RAS/86/024, July 1989. 74 pp.

Seafarming Atlas Series
RAS/90/002

(SF/ATLAS/90/1. Lovatelli, A.). Regional Seafarming Resources Atlas: (Volume I). FAO/UNDP Regional Seafarming Development and Demonstration Project (RAS/86/024). 83 pp.

SF/ATLAS/91/2. Lovatelli, A. Regional Seafarming Resources Atlas: Volume II. FAO/UNDP Regional Seafarming Development and Demonstration Project (RAS/90/002). 67 p.

Audio-visual Materials
RAS/86/024

Culture of the Pacific Oyster (Crassostrea gigas) in the Republic of Korea. 71 slides.

Culture of the seabass (Lates calcarifer) in Thailand. 40 slides.

Marine finfish netcage culture in Singapore. 37 slides.

Culture of Kelp (Laminaria japonica) in China. 30 minutes video.

Audio-visual Materials
RAS/90/002

Artificial breeding and culture of abalone in Korea (DPR). 60 minutes video.