1. Lab. No. 7 Production of the brine shrimp Artemia
Marine biotechnology
Lab. No Production of the brine shrimp Artemia

2. The brine shrimp, a small crustacean living in salt ponds, represents an excellent prey for old or large fry due to:
Its nutritional value.
Mobility in water.
Moreover its easy and short production cycle
Commercial availability.

3. As the mouth size of marine fish larvae increases with age, rotifers are gradually replaced by the larger freshly hatched nauplii of the brine shrimp Artemia salina.
Due to the larger size of its mouth at first feeding, some fish fry can accept Artemia as a first prey, thus making rotifer supply not compulsory.
Later on in the rearing process, and before weaning, larger Artemia metanauplii stages replace nauplii for larger fry.

4. Morphology and natural history
Brine shrimp most surprising characteristic is its ability to live in extremely hostile environments, such as salt lakes and man-made brine ponds throughout the world.
They show a remarkable capacity to stand severe environmental conditions such as a water salinity values over 200 ppt.
Its body permeability to salts is very low when compared with other micro-crustacea.

5. Artemia absorbs water from the medium and eliminates salt by defecation.
In this way it can keep the correct osmotic blood values in spite of the hyperhaline environment in which it lives.

6. Description : The adults are characterized by : elongated body
two stalked complex eyes
a linear digestive tract
sensorial antennules
11 pairs of functional thoracopods

9. Males are easily recognizable for their pair of large muscular claspers (the 2nd pair of antennae) in the head region.
Females bear the brood pouch or uterus behind the 11th pair of thoracopods.

19. From an aquacultural point of view, the most important biological characteristic of Artemia is that it produces resistant cysts, containing embryos in diapause (“dormant stage”).
Cysts are formed when environmental conditions become intolerable; their function is to protect the embryos against dehydration and against excessively high temperature and salinity values.

21. After hatching, the Artemia larva goes through about 15 molts with an initial size ranging from 400 to 500 µm.

22. The first two larval stages Metanauplii (instar II) :
Nauplii (instar I): small, rich in egg yolk.
Metanauplii (instar II) :
Large, has a digestive tract .
Has to be enriched with special integrators.
Are the most commonly used in fish fry feeding.

24. Under optimal conditions brine shrimp reach the
10-mm long adult stage in
eight days and can live for
several months, reproducing at rate of up to
300 nauplii or cysts every four days.
Under optimal conditions brine shrimp reach the

25. The majorityof brine shrimp are females.
This is important because the females are able to fertilize their own eggs without the assistance of  the male brine shrimp. This method of reproduction is called Parthenogenesis. 
However, males are required to produce cysts. 
The production of cysts requires sexual reproduction which means that males need to contribute sperm to the egg. 

27. Reproduction :
Parthenogenetic and bisexual Artemia strains exist, where ovoviviparous and oviparous reproduction alternates.

28. Oviparous : eggs hatching out side the body and free swimming nauplii is released
Ovoviviparous :
eggs are retained inside the uterus until embryonic development is fully completed (4- 5 days) and free swimming nauplii are then released.

29. Ovoviviparous
Eggs
uterus
nauplii

30. Oviparous
Eggs
nauplii

33. Fertilized eggs normally develop into free-swimming nauplii (= ovoviviparous reproduction) which are released by the mother.
In extreme conditions (e.g. high salinity, low oxygen levels) the embryos only develop up to the gastrula stage. At this moment they get surrounded by a thick shell (secreted by the brown shell glands located in the uterus), enter a state of metabolic standstill or dormancy (diapause) and are then released by the female (= oviparous reproduction).

34. Change from ovoviviparous to oviparous reproduction seems to be induced by under nourishment or even by an inadequate food quality, rather than by other abiotic factors.

35. Artemia use in aquaculture
The use of Artemia nauplii as live food for the rearing of fish and crustacean larval stages, has been one of the most important steps in the development of marine aquaculture.
The advantages of Artemia are multiple:
Off the shelf and regularly available dormant cysts.
They are easy to produce.
Are visible as prey.
And are highly palatable to the larvae

36. Stages of feeding the fish
Yolk sac (embryo)
Rotifer ( first food )
Artemia( second and last live food )
Artificial feed (weaning)

37. Having a larger size than rotifers, the larval stages of a small crustacean, the brine shrimp Artemia salina are used as the second (after rotifers), and last live food fed to fish larvae before their weaning on artificial feed.
The first Artemia larval forms, the nauplii, which are also the smallest and richest in yolk, are followed by a larger metanauplius, whose nutritional value has to be boosted by feeding them special enrichment diets 12 to 24 hours before being offered to fish to upgrade their nutritional value for fish larvae.

38. Nutritional value of Artemia
A major constraint, in the use of Artemia as a food organism for marine fish larvae is its variable nutritional quality.
This is especially true for the second and larger metanauplius.

39. As Artemia is a non-selective filter feeder, it filters all particles of suitable size - bacteria and algae- from its surrounding environment and special feeds can be given to improve its nutritional value.
Enriched nauplii produce better performances in fish larvae in terms of growth and survival.

40. Essential fatty acids (HUFA’s)
This problem can be circumvented with the use of enrichment techniques adding
Essential fatty acids (HUFA’s)
Vitamins.
Prophylactic
Therapeutic drugs, can also be passed to the fish larvae via the Artemia nauplii acting as carriers.

41. Critical Parameters for Optimum Hatching
Hatching of Artemia
Critical Parameters for Optimum Hatching
hatching container
aeration
temperature
salinity pH
cyst density
illumination

42. Hatching of Artemia
• Hold the temperature constant during hatching at 25° to 30 °C. Below 25 °C, the cysts will hatch slowly, and above 33 °C, cyst metabolism is greatly affected. • Maintain the salinity at 5 ppt. { Research has shown that nauplii have a higher energy content when hatched at low salinity.}

43. Oxygen levels should be above 2 mg/L (2 ppm)
Oxygen levels should be above 2 mg/L (2 ppm). Constant aeration is necessary during hydration and hatching; it helps disperse the cysts.
• The cyst density should be 5 g of cysts per liter of water.
• A bright, continuous light above the cysts is necessary during hatching.
• Cyst disinfecting is highly recommended to assist with improving hatching yields and killing bacteria that are often found on the cysts and could be harmful to the larvae being fed the Artemia nauplii.

44. Disinfection of brine shrimp cysts
Artemia cyst shells are usually contaminated with bacteria, spores of fungi and other microorganisms.
Fish larvae can be infected when untreated empty shells, unhatched cysts or cyst hatching medium residues are introduced into the larval rearing tank.
Before hatching, cysts should therefore be disinfected.
This process also improves hatching by reducing the bacterial load of the hatching medium.
Disinfection is done by keeping the cysts for a few minutes in a hypochlorite solution.

45. Hatching
1. Transfer the rinsed cysts to the hatching container. As soon as the cysts are placed in water, the embryos inside the cyst start their metabolic activity. 2. Start aeration, and leave the cysts in hatching medium (5 ppt seawater). The free swimming nauplius, called instar I, hatches after about 20 to 24 hours if incubated under optimal conditions as follows:

46. • Hatching container design: round with conical bottom, a drain with a valve is installed in the cone tip. Cylindrical or square-bottomed tanks will have dead spots in which Artemia cysts and nauplii accumulate and suffer from oxygen depletion. • Filtered and sterilized seawater, Optimal hatching can be obtained in the range 5-35 ppt.

47. Water temperature 28-30°C.
Strong aeration to provide a vigorous water agitation, to keep cysts in suspension: air is provided through the open end of PVC pipe placed close to the container bottom;
Dissolved oxygen level above 4 ppm;
pH over 8; if needed, add sodium bicarbonate (NaHCO3) at the rate of about one gram (previously dissolved) per litre;
A strong illumination of 2000 lux at the water surface during the first incubation hours; light can be provided by two neon tubes (2 x 58 W) placed few centimeters near the hatching container;
Cyst density for incubation: 5 g per litre

48. For the first few hours, the embryo hangs beneath the cyst shell and at this stage it is called as the umbrella stage.

50. Harvesting of nauplii
Artemia should be harvested when at the energy-rich instar I larval stage, just after hatching.
This occurs in about 22 h at 28°C. To assess the proper time, sample the incubation medium with a 5-ml glass pipette and check for nauplii and umbrella stages (embrios still attached to their cyst) under the stereomicroscope.
1. After 24 hours, stop and remove the aeration.
2. Let the cysts settle for 5 to 10 minutes.

51. A distinct separation should occur:
Empty cyst shells will float to the surface.
The nauplii will tend to concentrate near the bottom.
The unhatched cysts and debris accumulate at the very bottom underneath the nauplii.

52. 3. Open the valve on the hatching container to drain the unhatched eggs and other debris.
Close the valve when the newly hatched nauplii begin coming out. This procedure should be repeated later to ensure that all of the newly hatched nauplii are harvested.
Nauplii can also be concentrated with light, since they are positively phototactic at this stage.

54. Decapsulation of Brine Shrimp Cysts
The hard shell that encysts the dormant Artemia embryo, the chorion, can be completely removed by short-term exposure to a hypochlorite solution. This procedure is called decapsulation.

55. Decapsulated cysts offer a number of advantages compared to the non-decapsulated ones:
Cyst shells are not introduced into the culture tanks. When hatching normal cysts, the complete separation of Artemia nauplii from their shells is not always possible. Unhatched cysts and empty shells can cause deleterious effects in the larval tanks when they are ingested by the predator: they can not be digested and may obstruct the gut.

56. 2. Nauplii that are hatched out of decapsulated cysts have a higher energy content and individual weight than regular instar I nauplii, because they do not spend energy necessary to break out of the shell, thus, the hatching nauplii have better nutritional value. In some cases where cysts have a relatively low energy content, the hatchability might be improved by decapsulation, because of the lower energy requirement to break out of a decapsulated cyst.

57. 3. Decapsulation results in a disinfection of the cyst material. 4
3. Decapsulation results in a disinfection of the cyst material. 4. Decapsulated cysts can be used as a direct energy-rich food source for fish and shrimp. 5. For decapsulated cysts, illumination requirements for hatching would be lower.

58. One disadvantage, however, is that decapsulated cysts are not buoyant and will settle out if extra circulation or aeration is not provided. Most hatchery managers prefer to aerate gently and not circulate or exchange water at all during the first week, since the larvae are quite fragile.

59. Treatment in a decapsulation solution (chlorine solution).
The decapsulation process consists in four steps:
Hydration of the cysts.
Treatment in a decapsulation solution (chlorine solution).
Washing and deactivation of the residual chlorine used in the decapsulation.
Feeding the eggs directly to larvae, or waiting until the nauplii hatch and then feeding them to larvae.

60. The decapsulated cysts can be either stored or, unless not commonly, be fed directly to fish.
For short-term storage, up to one week, keep in the refrigerator at a temperature of 0 - 4°C.
Decapsulated cysts that have to be stored for a longer period have to be dehydrated in a saturated brine solution.