Artemia Investigation Report

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Artemia in aquaculture

General Characteristics • Small branchiopod crustacean of the order Anostraca • Inhabit natural and artificial salt lakes worldwide • ~7 strains, Artemia salina most important for aquaculture • Non-Selective filter feeders - Consume micro-algae, bacteria,

protozoa and detritus in size range of 1-50um • Physiologically well adapted to environments free from aquatic

predators & where competition with other filter feeders is low - most efficient osmoregulatory system in the animal kingdom (up

to ~300ppt) (regulators) - respiratory pigments tolerant of low oxygen levels (to ~2ppm) - dormant egg (cyst) production in extreme conditions

• Dispersed by wind & waterfowl (can adhere to body or pass through digestive tracts unharmed)

• Ability to produce cysts has led to extensive use for culturing aquatic animals

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Why is Artemia used so extensively?

• Easily sourced, availability worldwide • Excellent storage capacity (cysts, live & frozen) • High nutritional value, easily enriched • High fecundity, planktonic reproduction • Ease of propagation • Cost effective • Appropriate predator vs prey size • Natural attractant to predators – motility &

colour

Reproductive Biology of Artemia

• Bisexual & parthenogenetic strains (no males)

• Females of most Artemia strains can reproduce sexually via either:

Ovoviviparous - live young (favourable conditions)

Oviparous - cyst formation (unfavourable conditions)

• Favourable conditions: females produce 2n eggs (either parthenogenically or sexually) that hatch into 2n nauplii

• Longevity: 30-60 days under optimal conditions

• Sexual maturity (15 moults) in ~8 days

• Each brood 50-110 nauplii, up to 75 / d (~1,500-4,000 /life) - rapid population growth

• In extreme conditions (eg. salinity>150ppt, low DO) cysts are produced - sometimes in vast quantities

• Embryo is ~300 cells and at gastrula stage when encysted. Embryo now in diapause

• Cysts float, harvested at 10-100kg / ha / yr

• When environment becomes suitable diapause deactivation occurs and cysts hatch

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Cyst Characteristics • Chorion

– hard, brown, protective outer layer

– lipoproteins impregnated with haematine & chitin (secreted by the uterus)

– interconnecting channels filled with air create buoyancy

– removed via decapsulation procedure

• Outer cuticular membrane

– under the chorion

– thin multi-layered membrane that acts as a molecular sieve, only allowing movement of small molecules (O2, H2O and CO2)

• Embryonic cuticle

– fibrous, transparent & elastic membrane surrounding embryo (ametabolic in diapause)

Hatching Process • Dehydrated cyst – concave & dimpled appearance.

Embryo inert at moisture content of <5% • Hydrated cyst – ~2hrs after immersion in water &

exposed to light, cyst becomes spherical & aerobic metabolism commences (osmotic pressure inside cyst increases)

• Breaking time – cyst shell ruptures due to increasing pressure

• Tear drop - Embryo leaves cyst shell enveloped in hatching membrane

• Hatching (Instar 1 nauplii) – nauplius swims with head appendages & has large yolk reserves. Is lecithtrophic. No functional openings & cannot feed. Approx 8-10h @25°C

• First moult (Instar 2 nauplii) – functional digestive tract, can feed on particles from 1 - 40µm. 27% less energy than Instar 1, swims faster, lower fatty acid & amino acid content

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Development to Adult • Larval development to adult (8-10mm long),

occurs over 15 moults. Paired appendages in the trunk region differentiate into Thoracopods; 3 parts; telopodites & endopodites (locomotion & filter-feeding) & the exopodites (membranous gills - gas transfer, osmoregulation)

• Lateral stalked eyes develop

• From instar 10 the antennae lose their locomotion function: develop into hooked graspers in males, the female antennae change to sensory appendages

• Males have a paired penis in the posterior trunk, females have a brood pouch or uterus behind the 11th thoracopod.

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P. Jones

Watch Artemia development sequence video. Author; Paul Jones

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Read

Introduction, biology and ecology of Artemia

http://www.fao.org/docrep/003/w3732e/w3732e0m.htm

Wikipedia: Brine shrimp

https://en.wikipedia.org/wiki/Brine_shrimp

Cyst Quality & Selection Criteria • Numerous Strains – eg, GSL, SFBB, Thailand, Canada, Australia • Factors vary between strains & seasons • Price can range from $50 to $300/kg depending on grade & supply

HATCHING QUALITY • Cysts per gram (typically 100,000-300,000 cysts /g) • Hatching Percentage (%H) - how many of the cysts will hatch. Takes into

account the hatching method but not the level of impurities • Hatching Efficiency (HE) (nauplii / g product) - reflects %H, impurities such

as sand, salt, broken shell, individual cyst weight, hatching method • Hatching Times - time until nauplii release (T0- incubation to first nauplii,

T10- incubation to 10% of total hatch (instar 1= 18-24h; instar 2 = 36-48h) • Hatching Synchrony; Ts = T90 - T10 (uniformity of hatch - nauplii size range

& nutritional implications) • Hatching Output (dry wt nauplii / g product) - best ~600mg / g. (determine

total food / predator -FCR value) • Nutritional properties typically low in HUFA (marine larvae) - require

boosting

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Cyst Quality & Selection Criteria Cyst Packaging • Dehydrated before storage (moisture <5%) • Humid conditions reduce hatchability • Storage under nitrogen or vacuum • Oxygen reduces hatchability over time • Kept at low temperature • When frozen, kept at room T for a week prior to incubation Naupliar Size • Hydrated cysts = 225 - 285um, Instar 1 = 428 - 517um • Predator preference • different size strains can improve larval rearing protocol Naupliar Food Value • EFA content varies between strains and batches • Amino Acid content reasonably constant

Hatching Techniques

• Conical based tank to maximise mixing, vigorous aeration from base of vessel

• DO >2ppm, Temperature 25-30°C, Salinity 10-40ppt • pH ~8 near hatching (can add NaHCO3 @ 1g/L) • Cyst densities for hatching 2-5g/L • Strong illumination (2000lux) essential especially after hydration

to trigger hatching mechanism • Harvest by manipulating nauplii +ve phototactic response • Must be separated from hatched shells & unhatched cysts • Air off for ~5 min, remove empty cysts & cyst shells (float) &

bottom debris (exuviae, unhatched cysts & dead animals) (note: un-decapsulated unhatched cysts float)

• Essential to rinse separated nauplii in freshwater with 150um screen to remove contaminants and hatching metabolite

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Commercial Artemia hatching vessels

Experimental Artemia hatching vessels

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Storage of Artemia Freshly hatched nauplii (Instar 1) • Can be stored @ 4°C under light aeration for up to 48h (to 8 million /L)

Decapsulated cysts • Short term (up to 2 d), place drained cysts in fridge • Long term (up to 3 months), dehydrate cysts in saturated salt solution (NaCl - 350g/L) for 3 h with gentle aeration. Then store in fresh saturated salt solution in a fridge in convenient quantities

Dehydrated cysts • Vacuum packed • Store in cool environment or in freezer • Last for years

Why Decapsulate Artemia? • Improves hatch rate

• Better separation of nauplii & shells

• Disinfection

• Nauplii have a better nutrient profile (don’t waste energy breaking out of shell)

• Unhatched cysts & shells are indigestible to larvae & clog the digestive tract

• Decapsulated cysts may be used as a feed source (bacteria free, smaller than Instar 1, partially digestible), but are non-moving and non-buoyant & tend to settle out.

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Enrichment Why do we enrich Artemia? • DHA:EPA ratio is important for fish – pigmentation, growth,

survival, brain/retinal development, DHA and phospholipids important for elevated stress tolerance & disease resistance

• Artemia are low in HUFA (especially EPA, 20:5n-3 and DHA, 22:6n-3)

• As most marine organisms cannot synthesise these compounds from smaller unsaturated fatty acids (eg. 18:3n-3), Artemia are enriched

• Enrichment procedure may also include vitamins, phospholipids, pigments and therapeutics.

• From instar II, Artemia is a non-selective filter feeder & can be bioencapsulated/boosted/enriched

Enrichment (cont) Methods of enriching Artemia • Microalgae – Feed Artemia with algae rich in HUFA • Direct enrichment - using nutrient emulsions (i.e. Cod

Liver Oil + Yeast) • Commercial products – i.e. Selco, Super Selco,

Frippak and Nutri-Pak (off the shelf reliability & long storage life, but very expensive)

• Freshly-hatched nauplii are transferred to an enrichment tank at ~300 nauplii/ml (<24 h enrichment). An enrichment emulsion is added in consecutive doses of 300ppm every 12 h. Enriched nauplii are harvested after 12, 24, or 48h

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Enrichment (cont)

0

10

20

30

40

50

60

70

Instar 1 - T18 Instar 2 - T24 DC Selco DC Super Selco

H U

F A

( m

g /g

d w

t)

EPA

DHA

HUFA

• Nutritional values of Artemia nauplii

Non Enriched Artemia

12 h Enriched Artemia

24 h Enriched Artemia

Instar I (m)

Instar II (600m)

Instar III (800m) Artemia packed with the HUFA enriched microparticulate diet

Artemia semi-packed with HUFA enriched micro-particulate diet

Nutritional value dependant on composition of nauplii

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essential nutrients pigments

drugs prophylactics

Artemia can be used as a vector for the transfer of specific components into cultured larvae & broodfish

Artemia enrichmnet tanks

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Watch Murray cod larvae feeding on Artemia video. Author; Paul Jones

Response of flounder (Paralichthys olivaceus) (day 50) to either unenriched rotifers & Artemia

(control) or high-DHA enriched live food c o n t r o l D H A

s u r v i v a l ( % ) 1 . 8 2 1 . 5

l e n g t h ( m m ) 1 9 . 1 2 8 . 7

s t r e s s r e s i s t a n c e ( % )

4 0 . 0 9 3 . 0

correctly enriched larval diets result in: increased survival, more uniform growth, better stress resistance, better pigmentation, reduced deformities, better swim bladder inflation, & increased larval vigour

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Commercial Applications of Artemia

• Early feeding stages for larval marine/freshwater fish & crustaceans (1st and 2nd feed, spp. specific)

• On-Grown Artemia (live & frozen) - broodstock/supplementary diets for ornamentals - seahorse culture - weaning diets for fish fingerlings harvested from ponds - stimulate feeding in stressed fish

• Cyst production is big business worth millions

Artemia great salt lake cyst harvesting

Cyst plume

Harvest boat station

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Artemia biomass production ponds

Artemia production ponds; predator netted

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Adult Artemia Biomass

Artemia breeding ponds; San Francisco bay

https://en.wikipedia.org/wiki/Brine_shrimp

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Artemia blister packer