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Diseases of cultured sea cucumber, Apostichopus japonicus, in China

Wang Yin-Geng1, Zhang Chun-Yun2, Rong Xiao-Jun2, Chen Jie-Jun2 and Shi Cheng-Yin1

1 Yellow Sea Fisheries Research Institute, Qingdao, China; 2Ocean University of China, Qingdao, China

Abstract

Ever since the artificial breeding techniques of Apostichopus japonicus were first introduced in the 1980s, the Chinese have been making efforts to develop and improve the rearing protocols. In recent years, sea cucumber aquaculture has developed rapidly along the northern coast of China, where 1-2 billion seeds can be produced and 90 000 tonnes of sea cucumber (live weight) can be harvested every year.

The rapid expansion and intensification of sea cucumber farming has led to the occurrence of various diseases, causing serious economic losses and becoming one of the limiting factors in the sustainable development of this industry. A study has revealed that several new or non-reported diseases have been discovered. The epidemiological study showed that the syndromes of rotting edges, ulceration of the stomach in auricularia stages and autolysis of young juveniles were caused by bacterial agents, whereas skin ulceration, erosion of epidermis and body oedema were triggered by various pathogens including bacteria, fungi and parasites during outdoor cultivation. These pathogens induced high mortality rates, occasionally reaching up to 80 %. Upon the isolation of these etiological agents, morphological, physiological, biochemical and pathological studies have been performed, and a preliminary identification of the isolated agents was conducted in the present study.

Keywords: Aquaculture, sea cucumber, disease, bacteria, fungus, parasite

Introduction

Sea cucumbers belong to the phylum Echinodermata, class Holothuroidea. There are about 1 200 holothurians in the world (McElroy, 1990) and 134 species identified in China, among which about 20 species have commercial value for human consumption (Chen, 2003). The temperate species, Apostichopus japonicus, naturally distributed in Bohai Bay and the Yellow Sea, is the most valuable due to its nutritional and supposed medicinal properties. It has been shown, in particular, that acid mucopolysaccharides from this species have anticoagulant, antifungal and antitumour activities (Li and Lian, 1988; Nagase et al., 1997; Suzuki et al., 1991; Fan, 2001). Additionally, S. chloronotus is also known to promote growth, facilitate internal healing and enhance the immune system in humans (Fredalina and Ridzwan, 1999).

Since 1954, Chinese researchers have conducted studies on artificial breeding of A. japonicus, and the technique was successfully established in the middle of the 1980s. The first sea ranching trials were successfully carried out by Zhang Fengying, Sui Xilin and others in the provinces of Hebei, Shandong and Liaoning (Zhang and Liu, 1998). The recent economic growth in China and, consequently, the improved standards of living have increased the demand for sea cucumber products for human consumption in the national market. Sea cucumber farming has developed rapidly and has become a vigorous industry along the northern coast of China, where every year 1-2 billion juveniles and 90 000 tonnes of sea cucumber (live weight) can be produced and harvested, respectively.

The rapid expansion and intensification of sea cucumber farming has led to the occurrence of various diseases, causing serious economic losses and becoming one of the limiting factors in the sustainable development of this industry. However, the research on diseases of cultured sea cucumber in China has only recently started, so there is little information available on this aspect. This paper presents the results of an epidemiological study, during which several non-reported diseases have been discovered.

Diseases of Apostichopus japonicus larvae and their aetiology

Rotting edges symptom

Epidemiology and clinical signs: This is the first report on this disease. Usually, it occurs during the auricularia stages from June to July, causing a high mortality of up to 90 % in certain cases. This symptom was widely detected in commercial sea cucumber hatcheries in Shandong Province. Compared with the normal larvae (Figure 1), the clinical signs of infected animals are recognized by the darkening of the body edges (Figure 2). Diseased specimens undergo autolysis (Figures 3 and 4) and the body completely disintegrates within 2 days. If metamorphosis is achieved, the pentactulae are weak and the survival rate is rather low.

Figure 1. Normal auricularia of A. japonicus.

Figure 2. Abnormal auricularia with bacterial infection and the presence of darkening edges (arrows).

Figure 3. An affected larva undergoing autolysis.

Figure 4. An autolyzed larva with incomplete edges.

Aetiology: Two dominant bacterial isolates (LB-1 and LB-2) are obtained from infected sea cucumber specimens. Based on the characterization of the isolated microbes and histopathological analysis, the bacteria LB-1 and LB-2 have been associated with the disease.

The colony of LB-1 was round, yellowish brown, and the surface was smooth, moist and opaque. The cells were Gram-negative, short rods with round ends or curved rods. The edge of the colony LB-2 was smooth with a yellowish dot in the centre, transparent and moist, and a blue glistening could be seen under the light. The particles were Gram-negative, thick and short rods. The morphological, physiological and biochemical characteristics of the above isolates are listed below (Table 1). According to the results, these two bacterial strains possibly belong to the Vibrio genus.

Table 1. Morphological, physiological and biochemical characteristics of the bacteria nominated LB-1 and LB-2 isolated from infected sea cucumber larvae.

Characteristic

LB-1

LB-2

Characteristic

LB-1

LB-2

Gram Stain

-

-

Indole production

+

+

Movement

+

+

H2S production

-

-

O/129 Sensitivity (10µg)

-

+

Lysine decarboxylase

-

-

O/129 Sensitivity(150µg)

+

+

Arginine dihydrolase

+

+

Growth on TCBS

+

+

Ornithine decarboxylase

+

-

0% NaCl, growth (w/v)

-

-

Gas from glucose

-

-

3% NaCl, growth (w/v)

+

+

Arabinose

+

+

6% NaCl, growth(w/v)

+

+

Mannitol

+

-

8% NaCl, growth (w/v)

+

-

Inositol

-

-

10% NaCl, growth (w/v)

-

-

Raffinose

-

-

4°C, growth

+

+

Rhamnose

-

-

25°C, growth

+

+

Sucrose

+

+

28°C, growth

+

+

Glucose

-

+

35°C, growth

+

+

Salicin

+

+

40°C, growth

-

-

Sorbitol

-

-

45°C, growth

-

-

Gelatinase

-

-

Oxidase

+

+

Urease

-

-

Oxidation-Fermentation

F

F

ONPG

+

+

Citrate utilization

-

-

Spore stain

+

-

Vogus-Proskauer reaction

+

+

Methyl red test

-

-

Symbols: +: positive reaction; -: negative reaction; F: Fermentative; x% NaCl: (10x) g NaCl was added to the liquid media consisting of 15 g tryptone, 5 g phytone and 1 litre distilled water.

Histopathology: Infected larvae stained with Haematoxylin & Eosin (H&E) appeared with the edges in dark purple. The epithelium cells showed multiple layers, with enlarged and deeply stained nuclei (Figure 5). Affected cells became necrotic and shed off from the tissues.

Figure 5. H&E staining of sea cucumber larvae with the rotting edges symptom. The thick stomach wall results from the proliferation of the epithelial cells. Insert: Close-up of the edge tissues (arrow).

Stomach ulceration symptom

Epidemiology: The disease typically occurs in summer at high temperatures. It is reported to be associated with pathogenic bacteria and is triggered by unsuitable feeds and high stocking densities (Zhang and Liu, 1998; Liu, 2000; Liu et al., 2002). It appears that the auricularia is susceptible to the infection. The mortality of affected larvae may rise up to 90 % in certain cases.

Clinical signs: Under a microscope, the stomach walls of juveniles are thick, rough, and visibly atrophic in the latter stages (Liu et al., 2002). The ulceration of the stomach usually results in reduced growth and a low metamorphosis rate. The disease often leads to mortality during the metamorphosis from the auricularia to the doliolaria stage.

Aetiology: According to the literature, the aetiology of the disease may be associated with two causes: (1) lack of appropriate food, e.g. feeding juveniles only with chrysophyte or Platymonas sp.; (2) certain bacteria induce the ulceration of the stomach (Zhang and Liu, 1998).

Treatment: Using appropriate feeds that meet the nutritive requirement of juveniles, such as marine yeast. When a microbial flora becomes dominant in the breeding tank, antibiotics such as penicillin or streptomycin, in the range of 3-5 ppm, will be effective (Zhang and Liu, 1998).

Gas bubble disease

Epidemiology: This disease has only been detected in the auricularia stage and causes relatively low mortalities (Zhang and Liu, 1998).

Clinical signs: Presence of gas bubbles inside the body of the larva, which results in anorexia. The most severe cases lead to death.

Aetiology: According to Zhang and Liu (1998), the aetiology consists of excess aeration in the rearing tanks. Under this condition, larvae can easily swallow bubbles and become affected. However, the mechanism that leads to death is still not well understood.

Treatment: Avoiding excess aeration by adjusting the air flux is effective in treating this disease. Continuous aeration should be avoided. Thirty minutes intervals every 2 hours without aeration are recommended.

Off-plate syndrome

Epidemiology: This disease was first observed about ten years ago, however, little information was recorded. It occurs in juveniles that have settled (normally on PVC plates) after the completion of metamorphosis from the doliolaria to the pentactula stage. Often mortality can reach 100 %.

Clinical signs: The affected juveniles shrink and gradually lose the ability to remain attach onto the available substrate. Meanwhile, the epidermis of infected individuals disappears; the whole body can even dissolve with the autolysing process. In such cases, the spicules can be found on the bottom of the infected tanks as they drop from the dissolved carcasses.

Aetiology and morphology: Three dominant bacterial strains, nominated HB-1, HB-2 and HB-3, have been isolated from specimens collected from different hatcheries. Based on histopathological analysis and bacterial count from infected tissues, these bacteria have been associated with the syndrome. The colony of HB-1 is small and round, with a white aureole around its edge. The cells are short or curved rods and Gram-negative (Figure 6). The colony of HB-2 is thick, moist, opaque and white. The cells are thin, straight short rods and Gram-negative. The colony of HB-3 is flat and medium in size, moist and transparent, and shows a blue lustre under the light. The cells are Gram-negative, thick and short rods about 2mm in length (Figure 7). Preliminary studies on the morphological, physiological, biochemical and molecular characteristics of the three isolates have been done (Table 2). Based on the above results, HB-1 and HB-3 appear to be Vibrio like, while HB-2 has not yet been defined.

Figure 6. Gram-negative HB-1 bacteria where the short and curved rods are visible.

Figure 7. Negative staining of the HB-3 bacteria show thick and short rods with a single polar flagellum under electron microscope.

Table 2. Morphological, physiological and biochemical characteristics of the bacteria nominated HB-1, HB-2 and HB-3isolated from sea cucumber larvae.

Characteristic

HB-1

HB-2

HB-3

Characteristic

HB-1

HB-2

HB-3

Gram Stain

-

-

-

Indole production

-

-

+

Movement

+

+

+

H2S production

-

+

-

O/129 Sensitivity (10µg)

+

-

+

Lysine decarboxylase

-

-

-

O/129 Sensitivity (150µg)

+

-

+

Arginine dihydrolase

-

-

+

Growth on TCBS

-

+

-

Ornithine decarboxylase

-

-

+

0%NaCl, growth

-

-

-

Gas from glucose

-

-

-

3%NaCl, growth

+

+

+

Arabinose

-

-

-

6%NaCl, growth

-

-

-

Mannitol

+

-

-

8%NaCl, growth

-

-

-

Inositol

-

-

-

10%NaCl, growth

-

-

-

Raffinose

-

-

-

4°C, growth

+

+

+

Rhamnose

-

-

NT

25°C, growth

+

+

+

Sucrose

+

-

-

28°C, growth

+

+

+

Glucose

-

+

+

35°C, growth

-

+

+

Salicin

+

+

+

40°C, growth

-

-

+

Sorbitol

-

-

-

45°C, growth

-

-

-

Gelatinase

-

-

-

Oxidase

+

+

+

Urease

-

-

-

Oxidation-Fermentation

F

F

F

ONPG

+

+

+

Citrate utilization

-

-

-

Spore stain

+

-

-

Laetrile

+

-

NT

Methyl red test

-

-

-

Lactose

-

-

-

Tryptophan deaminase

-

-

-

Vogus-Proskauer reaction

+

-

-

Pigmentation

-

-

-

Symbols: +: positive reaction; -: negative reaction; NT: no test conducted; F: Fermentation; x% NaCl: (10x) g NaCl was added to the liquid media consisting of 15 g tryptone, 5 g phytone and 1 litre distilled water.

Diseases during aestivating stages of Apostichopus japonicus and their aetiology

Skin ulcer disease

Epidemiology: This infection tends to occur in juveniles during aestivation as a result of high temperatures and stocking densities. The infection rapidly transfers from the diseased individuals to healthy ones making it difficult to control. Occasionally, the whole population can be wiped out in a short time once the infection sets in.

Clinical signs: Infected individuals are weak and anorexic; their body shrinks and eventually takes a rounded shape and becomes white. The skin ulceration begins with the appearance of small white patches, which enlarge and eventually expose the underlying muscle and spicules. Dead juveniles fall from the substrates leaving clearly visible white marks.

Aetiology: A previous study associated this disease with bacteria (Zhang and Liu, 1998) that proliferate on the PVC plates appearing as red, pink or purple-red patches. The skin ulcer disease occasionally breaks out as the bacterial colonies spread over the plates. The characteristics of these bacteria have not been reported.

Treatment and prevention: Preventive measures include: (1) good hatchery management operation; (2) disinfection of tanks, plates and tools before use; (3) removal of excess food, faeces and other organic matter; and (4) provision of high quality water. The disease can be treated with antibiotics (3-5 ppm) such as terramycin, acheomycin and sulphanilamides.

Predatory copepods

Epidemiology: Summer marks the peak of copepod reproduction as the larvae of sea cucumbers develop into juvenile stages. In the presence of high numbers of copepods, the abundance of juveniles decreases acutely within 1 or 2 days. Usually, these predatory copepods will attack juveniles smaller than 5 cm, and often cause high mortalities.

Clinical signs: The juveniles have lesions on their body and become weak. Eventually, the juveniles die off, the body walls dissolve and the spicules disseminate on the bottom of the rearing tanks.

Aetiology: According to the literature, the copepod known as Microsetella sp. is the causative agent. Normally, the rearing conditions for juveniles are favourable for the growth and reproduction of this species. At temperatures between 15-25 °C one adult copepod can produce 90 individuals in 20 days. Generally, the mature female can produce new oocysts within a few minutes following the release of an initial batch. The copepods compete for food and space, as well as bite the young sea cucumber juveniles.

Treatment: Chlorophos is the best option to control the problem at present. A dosage from 2 to 3 ppm is effective; at this concentration all copepods can be killed in two hours without harming the sea cucumber juveniles (Zhang and Liu, 1998; Liu, 2000).

Diseases in outdoor cultivation of Apostichopus japonicus and their aetiology

Bacterial Ulceration Syndrome (BUS)

Epidemiology: Sea cucumber adults are susceptible to this disease during the warm season. It usually results in chronic mortalities, with cumulative rates of 30 %. Generally, the infected sea cucumbers die 15 days after the clinical signs appear.

Clinical signs: The infected sea cucumber shrinks and small lesions usually appear around the mouth. The lesions gradually expand with increased mucus synthesis (Figure 8) over large areas of the body wall. The infected skin becomes eroded with deep ulceration (Figure 9) and assumes a bluish white colouration. Many of the infected sea cucumbers will eviscerate in severe infections, while the lightly infected ones will stop feeding.

Figure 8. White patches around the papillae of an infected sea cucumber adult (arrows).

Figure 9. Deep ulceration visible on an infected sea cucumber adult (arrows).

Aetiology and morphology: Based on the isolation, there are two dominant bacteria (KL-1 & KL-2) (Figures 10 & 11). According to observation of the smear preparations, the infected tissues contain a large amount of bacterial cells, and parasitic nematoda are also found in some cases (Figure 12). The dominant bacteria are considered as the primary infectious agent while the nematode is a secondary invader. The smooth-edged colony of KL-1 is large, flat, moist and yellowish. The cells are Gram-negative with curved and short rods (approx. 2 m.m) with a single polar flagellum. The colony of KL-2 is thick, moist, white and convex. The cells are Gram-negative with thick and short rods (approx. 1.5 mm). Preliminary studies on the morphological, physiological and biochemical characteristics of the two isolates have been carried out (Table 3).

Table 3. Morphological, physiological and biochemical characteristics of the bacteria nominated KL-1 & KL-2 isolated from sea cucumber juveniles.

Characteristic

KL-1

KL-2

Characteristic

KL-1

KL-2

Gram Stain

-

-

Indole production

+

-

Flagella

m

-

Esculine

-

+

Movement

+

+

Glucose

-

-

O/129 Sensitivity (10µg)

+

+

Nitrate reduction

+

+

O/129 Sensitivity (150µg)

+

+

Arginine dihydrolase

-

-

Growth on TCBS

-

-

Raffinose

+

-

0%NaCl, growth

-

-

Gas from glucose

-

-

3%NaCl, growth

+

+

Arabinose

-

-

6%NaCl, growth

+

-

D-Mannose

-

-

8%NaCl, growth

-

-

Mannitol

-

-

10%NaCl, growth

-

-

N-Acetylglucosamine

-

-

4°C, growth

+

+

Capric acid

+

-

25°C, growth

+

+

Sucrose

+

+

28°C, growth

+

+

Maltose

-

-

35°C, growth

+

+

Lactose

-

-

40°C, growth

-

-

Salicin

-

-

45°C, growth

-

-

Glucoheptonate

-

-

Oxidase

+

-

Cellobiose

-

-

Xylose

-

-

Gelatinase

-

-

Oxidation-Fermentation

O

-

Urease

-

-

Methyl red test

-

-

Adipate

-

-

Pigmentation

-

-

Malate

-

-

Citric acid

-

-

Phenylacetic acid

-

-

Symbols: +: positive reaction; -: negative reaction; m: monotrichous; O: Oxidation; x% NaCl: (1 0x) g NaCl was added to the liquid media consisting of 15 g tryptone, 5 g phytone and 1 litre distilled water.

Figure 10. Negative staining of the KL-1 bacteria showing curved cells and short rods with a single polar flagellum.

Figure 11. Staining of the Gram-negative KL-2 bacteria. Insert: Negative staining of a cell under EM, showing thick and short rods without flagella.

Figure 12. Parasitic nematoda found in the lesion associated with the Bacterial Ulceration Syndrome (BUS) (400 ×).

Figure 13. Eroded papillae (white arrows) and a large area of body surface appearing bluish white (black arrows) of an infected sea cucumber.

Fungal disease

Epidemiology: Fungal diseases frequently occur in pond cultured sea cucumbers from April to August. Both juveniles and adults can be infected by the fungi, but no case has been found in the larval stages. Although this disease does not cause widespread death, it will result in an unhealthy appearance and poor quality of the final product.

Clinical signs: The papillae of the sea cucumbers become white during the early stage of the infection (Figure 13). With the development of the infection, large areas of body wall appear bluish white as the skin is eroded by the fungi. Unlike bacterial infections, there is no obvious mucus around the lesions. In some cases, the whole body surface becomes discoloured and transparent; the body wall becomes thinner and the affected individuals develop oedema (Figure 14).

Figure 14. Oedema condition associated with fungal infection in sea cucumber.

Aetiology and morphology: Two fungal species have been found to be associated with the disease. Microscopic observations of smear preparations show that one of the fungi is quite large with branched hyphae and macroconidia that contain more than 8 spores (Figure 15), while the second species is thin, with straight hyphae and small sporangium.

Histopathology: Histopathological observations show that the fungal hyphae and spores can be detected in the muscular tissues (Figure 16). This is an indication that the fungus can invade the body wall and grow deep into the body tissues. Connective fibre tissue turns necrotic and disintegrates in heavy infections.

Figure 15. Fungi with branched hyphae (black arrow) and distinct macroconidia (white arrow) growing on sea cucumber.

Figure 16. Fungal spores detected in the muscular tissue (arrow) of a sea cucumber.

Parasitic diseases - protozoan infection

Epidemiology: The infection generally occurs in young animals and adults, but generally does not cause a serious mortality problem.

Clinical signs: Compared with normal behaviour, the infected animals tend to be weak and sluggish. The body usually shows no conspicuous lesions, however the intestine, respiratory tree, etc. would be eviscerated in severe infections.

Aetiology: The disease is caused by a relatively large protozoan (about 70 mm), with a distinct nucleus and numerous ciliae covering the whole body surface. A large number of the parasites have often been observed in the inner wall of the respiratory tree (Figure 17).

Histopathology: Histological sections reveal large numbers of infusorians attached only to the inner wall of the respiratory branches. The head of the infusorian penetrates through the epithelial tissues of the inner wall from which they ingest nutrients (Figure 18). The epithelial tissue is usually damaged.

Figure 17. Protozoans distributed along the inner wall of the respiratory tree of the sea cucumber. Insert shows the morphology of the protozoan.

Figure 18. Histological sections of the respiratory tree of a sea cucumber showing large numbers of infusorians. Insert shows a protozoan that has penetrated into the wall of respiratory tree.

Parasitic diseases - Platyhelminthiasis

Epidemiology: The worms can infect both aestivated juveniles (larger than 1 cm) and adults. In all cases, the disease causes severe infection resulting in high mortality (over 90 %) within one month.

Clinical signs: Infected juvenile sea cucumbers are weak, anorexic and easily fall from the substrate to the bottom of the tanks (Figure 19). The body of the infected individuals is stiff and is covered in excessive mucus. The entire viscera or part of it (intestine, respiratory tree, etc.) is usually expelled as the infection progresses. Early ulceration usually occurs around the mouth or anus, and then spreads over the dorsal and ventral surfaces of the body and eventually the infected specimen dies (Figure 20).

Aetiology: One unidentified platyhelminth has been observed that caused heavy damage to the skin. Based on a series of biopsies numerous worms have been seen under the microscope. The presence of round projections budding from the segmented body of the parasites is an indication that asexual reproduction may take place (Figure 21). The size of the worm is variable, normally ranging from 50 to 130 mm.

Histopathology: Histological observations demonstrate that there is an abundance of worms in the tissues of the lesion area. The worms occupy a large space within the tissues and cause topical necrosis and scattering of the musculature (Figure 22).

Figure 19. Infected sea cucumber juveniles are weak, anorexia and easily drop from the settlement substrate to tank bottom (arrow).

Figure 20. Infected sea cucumbers with severe ulceration resulting in the exposure of the inner body tissues.

Figure 21. Platyhelminth isolated from sea cucumber skin lesions, showing round projections budding from the segmented body. Insert shows two pieces of worms stained with iodine.

Figure 22. Skin histological sections showing infestation with Platyhelminth worms (arrows) within the muscular tissue (H&E staining, 1000×).

Summary

Study into the diseases of sea cucumbers is a relatively new area of research. So far, parasites e.g. sporozoans, turbellarians and gastropods have been reported in wild sea cucumbers (Smith, 1984; Jangoux, 1987a, b, 1990). Nevertheless, the rapid expansion of sea cucumber farming in China has led to the occurrence of various diseases causing serious economic losses and becoming one of the limiting factors in the sustainable development of this industry. The causative agents become increasingly complex to pinpoint as several kinds of pathogens such as bacteria, fungi, parasites and copepods have been detected. The diseases can occur during breeding, aestivation and outdoor cultivation stages. Several non reported diseases have been discovered.

Preliminary studies have revealed that several types of sea cucumber diseases could cause high mortalities. Amongst the pathogens, bacteria are the most significant aetiological agent, often through vibriosis, while parasites and fungi are regarded as secondary agents. As to the classification of these pathogens further studies on characterization and identification of the pathogens, reinfection processes, pathogenesis, rapid diagnosis as well as treatment methodology need to be undertaken.

Regarding prevention and treatment of these diseases, very little information is available since the fundamental studies have not been conducted yet. Thus, the basic solutions to the current problems rest in proper management. The following issues should be considered: (1) broodstock for reproduction should be healthy without any signs of pathogens to avoid vertical infections; (2) the stocking density should be adapted to the environmental conditions, culture method and experience - overcrowded conditions would lower resistance to diseases; (3) sea cucumbers should be fed with high quality diets that are supplemented with necessary elements including vitamins and minerals; (4) water quality should be maintained by keeping an optimum stocking density, avoiding over feeding and increasing water exchange rates while monitoring the water quality on a daily basis; (5) total bacterial counts should be conducted routinely to monitor and prevent occurrence of diseases; (6) all equipment and tools should be disinfected before use or before transfer from one tank to another and excess food, faeces and other organic wastes should be siphoned and removed as quickly as possible; (7) as soon as a disease is detected, actions and effective measures should be taken and moribund individuals should be removed from the tanks and be properly disposed; and (8) antibiotics should only be used when the causative pathogen(s) has been confirmed and sensitive tests for antibiotics have been carried out.

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