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HORSESHOE CRAB CULTURE

Horseshoe crabs (Tachypleus spp.) are marine arthropods, almost all of which are found in temperate and tropical oceans (Fig. 2.1). During their reproductive season, they actively swim towards intertidal areas from deep sea to dig shallow holes into which the eggs are laid. During this reproductive migration the inhabitants of numerous coastal communities often collect them to feed on the ripe ovary as it is considered a nutritious and delicate product. In addition to the eggs, the blood of these organisms is also collected for the preparation of a pharmaceutical reagent, which is highly priced in the regional market. As a result of indiscriminate harvesting, this natural resource has been severely damaged. To protect and conserve the horseshoe crab, efforts are being made to culture it in captivity.

Figure 2.1.

Figure 2.1. Dorsal (a) and ventral (b) view of Tachypleus tridentatus.

2.1 Biological habitat

Horseshoe crabs live in temperate and tropical waters. During winter, they move into deep waters to over-winter. Only in late spring or early summer, when the seawater temperature rises, do they begin their reproductive migration towards shallower waters. Prior to egg-laying, sexually mature female and male specimens embrace each other with their walking legs for mating. Following copulation, the female will search for a proper spot in the intertidal zone with a sand-muddy substrate to lay the fertilized eggs. It takes about 5–6 weeks before the larvae start to hatch. Prior to sexual maturation, a young female crab will moult 18 times while a male will moult 19 times. Sexual maturation is attained when the specimens are about 15 years old when ecdysis finally ends.

The horseshoe crab has five pairs of walking legs, which are used for locomotion, digging and catching food organisms.

2.2 Culture method

Horseshoe crabs can be cultured indoors or outdoors. In most cases, indoor culture tanks are made of concrete with a depth of 120 cm. If the ponds are built outdoor, the site should be located in the upper to mid-intertidal area in order to facilitate water exchange. During the winter season, when the seawater temperature drops below 10 °C, the crabs tend to bury themselves under the sand throughout the cold months. In artificial farming conditions, heating facilities are often provided to prolong the blood collection period.

Razor clam and oyster meat are favorite food organisms, however they occasionally consume frozen fish. Experimental results have shown that the feeding rate of a horseshoe crab is closely correlated to water temperature, with the highest feeding rate occurring at 20–28 °C (Fig. 2.2). At temperatures above 30 °C or below 16 °C, the feeding rate sharply declines.

Figure 2.2

Figure 2.2. Relationship between feeding rate of a horseshoe crab and water temperature.

2.3 Importance of horseshoe crab rearing

The blue blood taken from the horseshoe crab is prepared into a special pharmaceutical reagent, which is now widely utilized to examine bacteria endotoxin in the pharmaceutical industry, food industry, and for disease diagnosis of patients. Due to the high price of the reagent, the crab's narrow distribution range, and the extremely long time it takes to reach sexual maturity, it is easy to deplete beyond renewal the natural resource of the horseshoe crab. Therefore, cultivation of the horseshoe crab is not only important for blood extraction and production of the special reagent, but also for its conservation.

2.4 Reagent preparation

2.4.1 Blood collection

The animals used for blood collecting are initially disinfected with 1% iodine tincture and 70% alcohol. As a hypodermic needle (18G) is inserted into its artery, the blue blood is made to flow into a clean container filled with an anticoagulant such as caffeine or theophylline. The pH value of the blood is buffered to 7.2 with tris-HCl solution. About 80–100 ml of blood can be collected every time from one mature crab. Blood extraction from one specimen can be done 4–8 times a year.

2.4.2 Haemocytes isolation

There are two methods for isolating haemocytes:

  1. The blood with the anticoagulant is left standing for 48 hrs, during which the amoeba cells will sedimentate while the plasma remains on the top layer. The plasma is slowly decanted and the cells washed three times with a 3% NaCl solution. Under bacteria-free conditions, the cells with 3 parts of 0.9% NaCl are homogenized for 2 hrs and then refrigerated for 24 hrs. The cell content is isolated by centrifugation.

  2. The haemocytes are separated from the plasma by centrifugation and washed with a 3% NaCl solution. The haemocytes are then diluted with distilled free pyrogen water and placed in refrigeration for 48 hrs. Two days later, the cell content is isolated.

2.4.3 Reagent purification

A mixture of one volume of haemocytes and two volumes of chloroform is cooled and shaken for two hours, and then transferred to a separator funnel to isolate the cells. CaCl, MgSO and NaCl are then added into the isolated cell portion until the concentration of calcium and magnesium ions will be 0.03–0.05 M/L. The final step is to desiccate the product by means of freeze-drying.


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