Brine Shrim- Artemia

 Brine shrimp or Artemia

 

Artemia is Brachiopod, forms an important zooplankton that can be stored in the form of cyst. It can be transported from one place to another and can be again rejuvenated whenever required.

8.1. Brine shrimp classification

    Kingdom: Animalia
      Phylum: Arthropoda
        Sub-phylum: Crustacea
          Class: Branchiopoda
            Order: Anostraca
              Family: Artemiidae
                Genus: Artemia

 

8.2. Brine shrimp-Introduction
Brine shrimp is the English name of the genus Artemia of aquatic crustaceans. Artemia, the only genus in the family Artemiidae, have evolved little since the Triassic period. Artemia were recorded from Lake Urmia, Iran, while Schlösser was the first person to give drawings of Artemia in 1756. Artemia are found worldwide in inland saltwater lakes, but not in oceans. Artemia are zooplankton, like copepods and Daphnia, which are used as live food in the aquarium trade and for marine finfish and crustacean larval culture. The cost of good quality cysts fluctuates with supply and demand. Normally 200,000 to 300,000 nauplii might hatch from each gram of high quality cysts.

 

8.3. Brine shrimp-morphology and physiology

Morphology of Artemia cyst: There is a variation in the size, dry weight and energy content of the strains. Hatching quality, percentage hatching rate and efficiency varies although hatching quality mainly depends on collection site. Temperature and salinity significantly affects survival and growth. Total lipid content and amino acid composition also varies depending on the strain.

 

Cyst consists of three layers: The first layer is hard with lipoprotein, chitin and hematin. The hematin imparts dark brown colour to the layer. This layer provides protection against any kind of mechanical and UV radiation. This layer can be removed by oxidation (decapsulation) by hypochlorite. The Second layer is the outer cuticular membrane which is a multilayered membrane with special filter. This protects the embryo from molecule larger than CO² and acts as permeability barrier. The Third layer is the embryonic cuticle which is transparent, highly elastic. It develops into hatching membrane during hatching.

 

8.4.1. Embryo : Undifferentiated gastrula which is a metabolic at <10% H₂O in the absence of oxygen. Presence of oxygen or cosmic radiation results in formation of free radicals which destroy specific enzyme system in the ametabolic artemia cyst.

 

8.4.2. Physiology of the hatching process

The development of an Artemia cyst from incubation in the hatching medium till nauplius release is shown in the figure.

8.4.3. Development of an Artemia cyst: Development of an Artemia cyst from incubation in seawater until nauplius release, when incubated in seawater the biconcave cyst swells up and becomes spherical within 1 to 2 h. After 12 to 20 h hydration, the cyst shell (including the outer cuticular membrane) bursts (= breaking stage) and the embryo surrounded by the hatching membrane becomes visible. The embryo then leaves the shell completely and hangs underneath the empty shell (the hatching membrane may still be attached to the shell). Through the transparent hatching membrane one can follow the differentiation of the pre-nauplius into the instar I nauplius which starts to move its appendages. Shortly thereafter the hatching membrane breaks open (= hatching) and the free-swimming larva (head first) is born. Dry cysts are very hygroscopic and take up water at a fast rate i.e. within the first hours the volume of the hydrated embryo increases to a maximum of 140% water content. However, the active metabolism starts from 60% water content onwards, provided environmental conditions are favourable.

The aerobic metabolism in the cyst embryo assures the conversion of the carbohydrate reserve into glycogen (as an energy source) and glycerol.

 

8.4.4. Ideal conditions for hatching Artemia cysts

 The optimal conditions for hatching Artemia are: temperature above 25^oC with 28^oC being optimum; salinity of 5 ppt; heavy continuous aeration; constant illumination (example: two 40 watt fluorescent bulbs for a series of four 1-liter hatching cones); and pH of about 8. Stocking density is set by adding no more than 5 grams of cysts per liter of water. Good circulation is needed to keep the cysts in suspension. A container that is V-shaped or cone-shaped is best (2-liter bottles work well; glue a valve on the bottle cap and invert it). The best container is a separation column, found in any lab supply, although it is more expensive. Unhatched cysts, empty shells and hatched nauplii can be easily removed separately. The hatching percentage and density are usually a function of water quality, circulation, and the origin of the cysts.

 

 8.4.5. Brine shrimp-decapsulation and hatching of cyst

Process involved in decapsulation and hatching of cysts and its direct usage to produce nauplii, Artemia cysts are either hatched naturally by incubation in seawater for 24–48 hours or hatched after decapsulation. Decapsulation is the removal of the outer membrane of a cyst called the chorion by dissolution in hypochlorite, without affecting the viability of the embryo. The outer shell often causes problems when not removed since it can harbour bacteria and other organisms which may be harmful to the species feeding on Artemia. Also, non-hatched cysts and their shells cannot be digested and may cause blockage of the gut in fish and crustaceans.

 

8.5.1. Hydration: Hydrate the dry cysts in natural seawater. Use a transparent conical tank or funnel-shaped container (e.g., glass or plastic cylinder, thick plastic bags formed into the desired shape) and keep the cysts in continuous suspension by aerating from the bottom of the apparatus for one hour. Upon hydration, the dry cysts which are deflated like bean seeds become round-shaped. Full hydration is necessary to insure that the inner part of the indented dry cyst shell will be completely exposed when the decapsulation solution is added.

 

8.5.2. Reaction with decapsulation solution (Hypochlorite): Prepare the decapsulation solution using 1N NaOH, Sodium hypochlorite (NaOCI) and seawater. Allow the hydrated cysts to react with the decapsulation solution (hypochlorite) for 7–15 minutes. To prevent damage of embryo, keep the temperature below 40°C by adding ice cubes to the suspension or by using a water bath. A change in colour of the cysts from brown to white to orange usually indicates that the reaction is complete. Check under the microscope if possible.

 

8.5.3. Sieving and washing: Drain the suspension of decapsulated cysts into a fine-mesh sieve and rinse immediately with seawater 6–10 times or until the smell of the hypochlorite is removed. Decapsulated cysts may be fed directly to the cultured fish / crustacean or stored in saturated brine solution at low temperature for future use.

 

8.5.4. Incubation: Incubate the decapsulated cysts for 24–48 hours in natural seawater at a density not greater than 5 grams cysts per liter of incubation medium. For optimum hatching, keep the temperature at 30°C and the pH at 8–9. Provide sufficient light at least during the first two hours or preferably continuous illumination of about 1000 lux (attained with 40-watt flourescent light tube, 20 cm away from the hatching container). Maintain the dissolved oxygen at levels close to saturation, with cysts kept in suspension throughout the incubation period. If culture to the adult stage is not intended, it is preferable to feed the Artemia to the fish or shrimp larvae immediately upon hatching to take advantage of the yolk in the nauplii.

 

8.5.4.1. Harvesting processing and packaging of Artemia cyst

After 15-20 hours of incubation, most of the cysts will be hatched and there will be noticeable colour change in the culture from brown to orange. At this time soft aeration and the pinkish orange nauplii will be seen swimming. An empty or undissolved shell tends to float, while the unhatched cyst and debris sink. A siphon also can be used to remove first the debris and then the nauplii from the bottom. The nauplii should then be collected on a 100-120 micrometer screen, washed with clean water, and placed in a small volume of water. Washing removes contaminants and hatching metabolites. Wash harvested nauplii for feeding.