For
the past months we’ve discussed culturing micro-foods to
satisfy the three steps in the food chain required to feed our
potential fish fry. To recap the past columns, we have grown
greenwater (phytoplanktons) to feed to our rotifer cultures.
We’ve covered the use of rotifers as an initial food source
for fry, and then described the culture of ciliates as helper
food items for smaller fish larvae. In this month’s column
we’ll discuss the use of Artemia (brine Shrimp) nauplii as a food source for larger fry.
Introduction
Among
the live diets used in the aquaculture of fish, brine shrimp (Artemia)
nauplii are the most widely used food item, mainly due to its
convenience and availability. [Fig
1] Unquestionably, the ability of this small branchiopod
crustacean to form dormant embryos, called ‘cysts’ (brine
shrimp eggs), helps make this food source convenient, suitable,
and an excellent larval food source for aquaculture. (Dhont,
1993) These cysts are available in large quantities year round,
along the shorelines of hypersaline lakes. After harvesting and
processing, Artemia
cysts have remarkable shelf life and can be stored in containers
for years, to be opened and utilized as a “ready-made live
food source.” Reawakening the cysts with overnight hydration
in saltwater releases wiggling nauplii that are usable directly
as a live food to the larvae of a wide variety of marine
ornamentals. The ease and simplicity of hatching brine shrimp
nauplii makes them one of the most convenient, least
labor-intensive live foods available for aquaculture. Artemia
are nonspecific feeders and will ingest a wide variety of foods,
which makes them even more useful to aquaculturists.
Additionally nutritional enrichment of Artemia
can enhance levels of important marine-based highly unsaturated
fatty acids (HUFA) to equal many copepod species. Furthermore,
the ability of Artemia
to feed on floating particles allows the bioencapsulation of
specific agents tailored to suit the predators’ requirements.
[Fig 2] Use of species-specific bio-encapsulation has had a major
impact on improved larval survival, growth, success of
metamorphosis and quality of many species of fish and
crustaceans. Bio-encapsulation methods are now being developed
for oral delivery of vitamins, chemotherapeutics and vaccines
that will further boost the importance of Artemia in the aquaculture world.
Fig 2diagram of the use of Artemia as a delivery vector
for transfer of
specificcomponents
into the cultured larvae diet.
Fig 3 (from top to
bottom):Artemia
cyst as (a)dispensed from a can of store bought brine
shrimp. (b) photomicrograph of cysts, (c) cysts bursting
“hatching”
Artemia
Life cycle
The
Artemia life cycle
begins by the hatching of dormant cysts. These cysts are
metabolically inactive embryos that can remain dormant for many
years, as long as they are kept dry and oxygen free. When the
cysts are placed back into salt water they re-hydrate and resume
their development. [Fig 3]
After hydration for 15 to 20 hours at 25 oC (77 oF)
the cyst bursts and the embryo leaves the shell. While the
embryo hangs underneath the empty shell (umbrella stage) the
development of the nauplius is completed.Within a short period of time the hatching membrane is
ruptured (hatching) and the free-swimming nauplius is born. In
the first larval stage, the nauplii (first larval stage -instar
I; 400 to 500 µm in length) are a brownish-orange color because
their yolk reserves.Newly
hatched Artemia do not
feed because their mouth and anus are not fully developed.
Approximately 12 hours after hatching, the animals molt into the
2nd larval stage (instar II). Small food particles ranging in
size from 1 to 50 µm are filtered into the digestive tract.
During the next eight days, the nauplii grow and progress
through 15 molts before reaching adulthood. Adult Artemia
average about 8mm long, but can reach lengths up to 20mm in
optimal environments. An adult brine shrimp is approximately 20
times longer and 500 fold larger in biomass than a nauplius.
Male brine shrimp possess a paired penis in the posterior part
of their trunk, and adult female Artemia
can easily be recognized by the brood pouch [Fig
5].
In
low salinity and optimal food levels, fertilized females usually
produce free-swimming nauplii at a rate of up to 75 nauplii per
day (ovoviviparous reproduction). Each female shrimp can produce
10-11 broods over an average life span of 50 days.But under ideal conditions, an adult Artemia
can live as long as three months and produce up to 300 nauplii
or cysts every 4 days. Under deteriorating environmental
conditions, such as high salinity, chronic food shortages and/or
cyclic oxygen stress, cyst production is induced. The embryos
develop up to the gastrula stage, and are then surrounded by a
thick shell. [Fig 6]
Formation of this shell initiates a state of metabolic dormancy
(diapause), and the cysts are released by the female (oviparous
reproduction) where they float to the shoreline and dehydrate.
Female Artemia can
switch from one mode of reproduction to the other.
Adult Artemia
can tolerate brief exposures to temperatures as extreme as -18
to 40oC 104oF.There are differences in optimal temperature for cyst
hatching and adult grow-out between strains.The optimal temperature for the San Francisco bay strain
is 22oC as compared to 30oC for Great Salt
lake Artemia (Bossuyt,
1980).Brine shrimp prefer a salinity of 30-35 (1.022-1.026
specific gravity) and can live in fresh water for about 5 hours
before they die.
Feeding
Artemia
is a non-selective filter feeder of organic detritus, microscopic
algae, and bacteria. Food is not directly consumed, but rather
transferred to the mouth in a packaged form. The gap between Artemia
legs widens as the legs sweep forward. Water is drawn into this
space from below, and small filtering hairs collect food particles
from the incoming water stream. Once the appendages sweep
backward, water is forced out of this space and the food remains
in a groove at the base of the legs.This groove has secretory glands that create adhesive mucus
which clumps food particles into balls.Once the food balls are formed, micro hairs move the food
packages toward the mouth. The optimal size for food is
<50microns for nauplii and < 60 microns for adult brine
shrimp.
Fig
4Artemia embryos 8hrs old. Photo courtesy of
Jason Chaulk
Fig
5Pair of Artemia in riding position. Photo courtesy of
Jason Chaulk
Fig 6(top) Close-up of female Artemia uterusshowing oviparous formation of cysts.
(middle
and bottom) two images of male Artemia demonstrating head and
body structures. Photos courtesy of Jason Chaulk
Cyst hatching
requirements:
Although
hatching small quantities of Artemia
cysts is basically very simple, several parameters need to be
taken into consideration for a successful hatching:
·
hatching container
·
aeration
·
temperature
·
salinity
·
pH
·
cyst density
·
illumination
The
best hatching results are achieved in conical bottom containers,
aerated from the center bottom. (Lavens, 1990)Cylindrical or square-bottomed tanks will have dead spots
in which Artemia cysts
and nauplii accumulate. Transparent hatching containers (like 2L
soda bottles) permit visual inspection of the hatching mix.This is especially useful when one is harvesting the brine
shrimp.
The
intensity of aeration should be sufficient to maintain high oxygen
levels, because increased hatching has been reported with
increasing oxygen level. (Tackaert,
991) Excessive foaming must be prevented, as hydrating
cysts will be carried out of the container on the bubbles. As for
optimal temperature, optimal hatching occurs in the range of 25-28°C;
below 25°C cysts hatch more slowly and above 33°C the cyst
metabolism is stopped. Reports from commercial hatcheries suggest
that strong illumination (approximately 2000 Lux at the water
surface) is essential for maximal hatching, and that this lighting
is essential during the first hours after complete hydration, in
order to initiate embryonic development (Vanhaecke, 1981). Good
circulation is essential to keep the cysts in suspension. The
optimal conditions for hatching Artemia are: 250C, salinity - (1.030 density), pH range
8-8.5 with heavy continuous aeration.
Enrichment
of Brine shrimp
As
mentioned in the introduction, a critical factor affecting the
nutritional value of Artemia
as a food source for marine larvae is the content of essential
fatty acids. Most marine fish fry are incapable of synthesizing
these HUFAs, and therefore must be provided with these fatty acids
in their diets. Therefore to ensure adequate HUFAs, we can provide
fatty acids as food supplements to Artemia
nauplii. Once the nauplii have molted past the second larval stage
(about 8 hours after hatching), one can simply include lipid
products into the culture media of the brine shrimp nauplii. This
technique of bioencapsulation, also called Artemia enrichment (or boosting) and is widely used in fish
hatcheries. Other enrichment products have been created to boost
specific aspects of the predator nutritional diet, and these
products include feeding phytoplanktons, yeast, micro-particulate
diets and self-emulsifying concentrates to the nauplii. According
to (Lavens, 1987) the highest
enrichment levels can be obtained by adding emulsified
concentrates, such as Selco to the Artemia’s
diet. Selco (also known as Selcon or super Selco) is a
self-dispersing complex of selected marine oil sources, vitamins,
and carotenoids and upon dilution in seawater disperses into
microparticles that are readily ingested by Artemia.
Proud sponsor of this column
Decapsulated
cysts
The hard egg shell
that covers a Artemia cyst can be completely removed by short exposure to a bleach
solution. This procedure is called decapsulation and decapsulated cysts
offer a number of advantages to the home hobbyist compared to non-decapsulated
ones:
a) Brine shrimp
egg shells are not introduced into the culture tanks. When hatching
normal cysts, the separation of 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 fry, often times the
egg casing can not be digested and may obstruct the gut.
b) Nauplii that
are hatched out of decapsulated cysts have a higher energy content and
individual weight (30-55% depending on strain) than regular nauplii.
c) Decapsulation
results in a disinfection of the cysts. A culture infection due to
contamination by “dirty” brine shrimp eggs is common in aquaculture
facilities, fortunately not so much in home culture.
d) Decapsulated
cysts can be used immediately as an energy-rich food source for fish and
shrimp larvae.
e) The
illumination requirements for hatching decapsulated cysts are lower.
The procedure to
decapsulate Artemia cysts
requires an initial hydration of the cysts (as complete removal of the
envelope can only be performed when the cysts are spherical), removal of
the brown shell in a bleach solution, followed by washing and
neutralization of the remaining bleach. These decapsulated cysts can be
hatched into nauplii immediately, or dehydrated in concentrated brine
solution then stored for later hatching. Lastly, the decapsulated eggs
can be used for direct feeding. Decapsulated Artemia
cysts can be stored for a few days in the refrigerator without any noted
decrease in hatching. Okay enough of the scientific background; let’s
get into the important part.
Fig
7The2 L soda bottle brine shrimp hatchery.Shown here is a basic layout of what is needed to
construct this simple Artemia hatchery. Image
courtesy ofMatt Lindenfelser (http://www.killi.net/)
Baby
Brine Shrimp
Baby
brine shrimp are an excellent food source for larger fish fry.
Usually after initial feedings on rotifers or copepod larvae, fish
fry require a large food type. Additionally, baby brine shrimp
make a good substitute macro-plankton for some filter-feeding
invertebrates.
Home culture of
Brine Shrimp
Hatching brine shrimp is quite simple. A brine
shrimp hatchery can be built out of almost anything cone shaped.
Household items such as 2L soda bottles make excellent hatcheries.
If you prefer to go the high-tech route, transparent 1 gal
inverted cone hatcheries are available online. Many local pet
stores sell Sally’s’ shrimp hatcheries and these hatcheries
utilize a sturdy rubber base in which an inverted 2L soda bottle
is attached. Again almost any container will work, however ensure
that the container has a conical bottom as this ensures water flow
and prevents dead spots. Airline tubing pressed to the very bottom
of the container is connected to an air pump, and your hatchery is
complete.[Fig
7]
Hatching of brine
shrimp consists of adding brine shrimp cysts to seawater.
Wilkerson recommends a low-salinity, cost-effective method,
using the cheapest aquarium salt you can find. (Wilkerson, 1998)
Add 2L of dechlorinated tap water and 2 teaspoons of aquarium
salt. Once the salt is dissolved, aerate for 1 hour and then mix
1/4 teaspoon (or less) of brine shrimp cysts. Sufficient
aeration is critical for a good hatch rate and to ensure the
nauplii remain healthy. Airstones are not recommended;
therefore, a rigid airline tube can be used and pressed to the
bottom of the hatchery. Once the air pump is started, do not be
timid in providing air to the culture. The amount of bubbling
can reach a rolling boil. While the cysts are hydrating, that
many of the cysts may get stuck on the bubbles that are formed
at the container surface and be carried out of the water, so a
lower initial aeration rate may be helpful. Ensure that your
shrimp hatchery is near a light source for the first day and
within 16-20 hrs later you should observe baby brine shrimp.
These freshly hatched nauplii are the most nutritious and can be
feed directly to your fish fry. The addition of the yolk sac
causes the nauplii to appear orange. As an aside, if you are
feeding clownfish fry, you must use freshly hatched or no more
than 30 hr old nauplii -- waiting any later nauplii will be too
large for the fry to ingest and unless they have been enriched
are now depleted of nutrients. So, if you do wait for 24 hrs,
the next day the egg yolk will be depleted and the nauplii are
feeding and can be enriched by the addition of a particulate
food to their media.
Fig
82L soda bottle hatcheries. Shown are 3 brine
shrimp hatcheries and 4 bottles of phytoplankton. The
airline tubing have been removed from the hatcheries to
collect the nauplii. The phytoplankton is used to enrich
the nauplii. Photos courtesy of Ray Jay (http://www.angelfire.com/ab/rayjay/)
Feeding and enriching Artemia
Artemia are non-selective filter feeders and therefore will ingest a
wide range of foods. The main criteria for food selection are particle
size, digestibility, and nutrient levels. (Dobbeleir, 1980) Possibly the
best foods for Artemia are
live microalgae such as Nannochloropsis,
Tetraselmis,
Isochrysis, and Pavlova
(see home phytoplankton culture). In fact combinations of live
phytoplanktons fed to Artemia
cultures have demonstrated superior enrichment characteristics (i.e.,
increased HUFAs) over feeding single phytoplankton species. (d’Agostino,
1980) [Fig 8]However, not all species of
unicellular algae are appropriate for sustaining Artemia
growth. For example, Chlorella, and Stichococcus have a thick cell wall that cannot be digested by Artemia.
Moreover, Artemia
cultures can be enriched by feeding a wide variety of processed foods,
which is very convenient to the home fish breeder. Processed foods
include yeasts (such as brewers yeast), fish meal, soybean powder, egg
yolk, and micronized rice bran. Preserved microalgae such as
phytoplankton cryopastes (such as Tahitian blend from www.brineshrimpdirect.com)
have also been used with success.One
problem with using processed foods is that you have to be careful not to
overfeed; processed foods will quickly foul the Artemia culture. The simplest way to measure food levels in the Artemia
cultures is by visually assessing the clarity of the water. According to
Schumann (website below) this is easily accomplished with a small wooden
dowel (at least 30cm long) in which measuring marks (marked off in
centimeters) are notched into the dowel; on the end of this dowel attach
a white disk with black fields, like a small Secchi disk (http://sebagolakeassc.org/secchi.htm).
The depth at which the contrast between the white and black fields on
the disc disappears determines the light penetration into the tank.
Increasing amounts of food in the culture decreases optical
transparency. With a density of 5000 nauplii per liter, the transparency
should be 15-20 cm for the first week, and 20-25cm thereafter. For
optimal production and enrichment of nauplii, it is best to maintain an
optimal food level in the culture media, so frequent or continuous
feedings work best. I find feeding of phytoplankton to the Artemia
cultures works extremely well and fry fed phytoplankton-enriched nauplii
grow quickly. To supplement the phytoplankton I also recommend the use
of selcon enrichment to boost HUFA levels. For enrichment, 24-30 hr old
nauplii can be transferred into a new culture container, where
enrichment emulsion (Selcon) is added at 1 ml/liter and aeration is
started. Six hours later, the enriched nauplii are harvested. In my
experience there is no need to rinse the nauplii; use them directly.
Harvesting nauplii
Harvest
the nauplii by simply turning off the air, and allow the culture to
settle for approximately ten minutes. Hatched, empty shells float to the
surface, and unhatched cysts will sink to the bottom. The newly hatched
nauplii will concentrate just above the unhatched cysts on the bottom.
Since the newly hatched nauplii are attracted to light, shining a
flashlight at the center of the bottle will concentrate the nauplii
where it is easy to siphon them off, or in a commercial hatching tank,
one can drain the cysts off the bottom by using the valve, then drain
the nauplii onto another container. An alternative method to collect
nauplii is to utilize a brine shrimp net (which is a small mesh
material) after decanting the floating hatched eggs off the top. An
inexpensive siphon can be constructed out of rigid air (1/8")
tubing with 2-3 ft of flex tubing attached. Then just siphon the
wriggling shrimp off into a brine shrimp net, and add to your fish fry
growout tank.
Sources of brine Shrimp eggs
Eggs
can be bought in most pet shops or by mail order (see online sources
below). Eggs bought in bulk (such as 1 lb cans) will be less expensive
than the tiny tubes sold in stores and will often be fresher and in
better condition. The cans may be held in the freezer, with 2-3 weeks
worth of supply held in a small, sealed jar.
Fig 9 16oz can of
brine shrimp cyst and home 2L hatchery. This is a common
setup for the home culturist. This can will provide brine
shrimp nauplii for 6months to 1yr depending on the amount
of nauplii needed. In between uses this can should be
refrigerated to preserve viability. Photo courtesy of
James Wiseman
Maintenance of the brine shrimp
culture
Most
people who hatch brine shrimp do so in a low volume batch. Once
the nauplii are hatched, the culture is fed, and utilized for 2 or
3 days. After 3 days, the culture is often depleted of nauplii and
a new culture, that was started the day before replaces the
previous one. Some people desire a continuous culture, and this
presents a few new problems. In smaller containers, water quality
can deteriorate rapidly, especially as biomass and feeding
increase. Furthermore, excess foods not
eaten by the Artemia
will decompose in the culture medium by bacteria, thereby
deteriorating water quality due to a gradual build up of toxic
substances such as ammonia and nitrite. Try to prevent overfeeding
by observing the amount of food added during each feeding and wait
until the culture begins to clear before you add more food.
Once
a sizable population of nauplii is generated, it is often
difficult to balance the need to feed the culture, and the
resulting decrease in water quality from the pollution that
feeding causes, especially when non-living feeds are used.Performing a 50% water change weekly will help overcome
this problem. Additionally, siphoning and cleaning the bottom of
the culture vessel every few days will remove accumulated detritus
and prevent further water quality issues. If the goal is to have
long-term culture then one should consider adding a small sponge
filter or other nitrifying filter to the culture.
Troubleshooting an Artemia
culture
The
two problems I have repeatedly encountered when culturing Artemia, are slow growth and the culture crashing.Slow growth is usually attributed to environmental issues such as
low temperatures, an improper pH, too low or too high salinity, and
insufficient food or poor food quality. There are several possible
causes for a Artemia culture
crash.The easiest problem
to resolve is low dissolved oxygen.This is remedied by increasing the amount of air and amount of
water motion thru the culture. Secondly, the health status of Artemia
can be visually assessed by observing their swimming motion.Healthy cultures react to photo stimuli, and rapidly gather at
the light source.Slow,
dispersed swimming indicates that the animals are in poor condition.
Health can also be assessed by visually inspecting the Artemia
under a microscope. Well-fed Artemia
have a completely filled gut and release compact fecal pellets.
They should also have clean swimming appendages and a clean mouth region
suggesting that the animal is in good health.
Underfed animals have an empty or barely filled gut and tend to release
loose fecal pellets. If their mouth parts or appendages are
covered with food particles, this suggests the animal is feeding poorly.Another reason for crashing cultures is a bacterial or protozoan
contamination of the culture. This can be avoided by decapsulating the
cysts before hatching.
Conclusion
Brine
shrimp nauplii are an easy and convenient food source for larger
fish fry. The ability to nutritionally enrich Artemia
nauplii provides us with a delivery platform to specifically
target potential predators’ nutritional requirements and met
these needs. Feeding and providing proper nutrition to fish larvae
is the greatest hurdle faced by all potential marine fish
breeders. With a bit of closet space, spare tanks, extra aquarium
supplies, and the information provided in the past Breeder’s
Net columns, virtually any marine hobbyist should be able to
raise enough live food to consider breeding marine fish at home.
Over the next few months we’ll begin to produce columns on the
breeding requirement of specific fish species suitable for home
culture, plus information on additional micro foods.
Proud sponsor of this column
How to
Decapsulate Brine Shrimp eggs
HYDRATION STEP
· Hydrate cysts
by placing them for 1 h in water (< 100 gm/l), with aeration, at 25°C.
DECAPSULATION
STEP
· Collect cysts
on a 125 µm mesh sieve, rinse, and transfer to the hypochlorite
solution.
· The
hypochlorite solution can be made up (in advance) of either liquid
bleach NaOCl (fresh product; activity normally =11-13% w/w) or bleaching
powder Ca(OCl)2 (activity normally ± 70%) in the following
proportions:
* 0.5 g active
hypochlorite product (activity normally labeled on the package,
otherwise to be determined by titration) per g of cysts; for procedure
see further;
* an alkaline
product to keep the pH>10; per g of cysts use:
¨ 0.15 g
technical grade NaOH when using liquid bleach;
¨ either 0.67
NaCO3 or 0.4 g CaO for bleaching powder; dissolve the
bleaching powder before adding the alkaline product; use only the
supernatants of this solution;
¨ seawater to
make up the final solution to 14 ml per g of cysts.
· Cool the
solution to 15-20°C (i.e. by placing the decapsulation container in a
bath filled with ice water). Add the hydrated cysts and keep them in
suspension (i.e. with an aeration tube) for 5-15 min. Check the
temperature regularly, since the reaction is exothermic; never exceed 40°C
(if needed add ice to decapsulation solution). You can assess how well
the process is working by checking for decapsulation under a jewelers
eyepiece or microscope.
WASHING STEP
· When cysts turn
grey (with powder bleach) or orange (with liquid bleach), or when
microscopic examination shows almost complete dissolution of the cyst
shell (= after 3-15 min.), cysts should be removed from the
decapsulation suspension and rinsed with water on a 125 µm screen until
no chlorine smell is detected. It is crucial not to leave the embryos in
the decapsulation solution longer than strictly necessary, since this
will affect their viability.
DEACTIVATION
STEP
· Deactivate all
traces of hypochlorite by dipping the cysts (< 1 min.) either in 0.1
N HCl or in a 0.1% Na2S2O3 solution, then rinse
again with water. Hypochlorite residues can be detected by putting some
decapsulated cysts in a small amount of starch-iodine indicator (=
starch, KI, H2SO4 and water). When the reagent
turns blue, washing and deactivation has to be continued.Alternatively, a DPD chlorine test method may be used.
USE
·
Incubate the cysts for hatching, or store in the refrigerator (0-4°C)
for a few days before hatching incubation. For long term storage cysts
need to be dehydrated in saturated brine solution (1 g of dry cysts
per 10 ml of brine of 300 g NaCl.l-1). The brine has to be renewed
after 24h.
References:
Bossuyt, E.;
Sorgeloos, P. (1980). Technological aspects of the batch culturing of Artemia
in high densities. The Brine Shrimp Artemia,
Ecology, Culturing, Use in Aquaculture 3: 133-152.
D’Agostino, A.S.
1980. The vital requirements of Artemia: physiology and nutrition. In:
The Brine Shrimp Artemia, Vol. 2, Physiology, Biodiversity, Molecular
& Biology. G. Personne, P. Sorgeloos, O. Roels and E. Jaspers (Eds).
Universa Press, Wetteren, Belgium, pp 55-82.
Dobbeleir, J.;
Adam, N.; Bossuyt, E.; Bruggeman, E.; Sorgeloos, P. (1980). New aspects
of the use of inert diets for high density culturing of brine shrimp.
The Brine Shrimp Artemia,
Ecology, Culturing, Use in Aquaculture 3: 165-174
Dhont J., Lavens,
P. and Sorgeloos, P. 1993. Preparation and use of Artemia as food for shrimp and prawn larvae. In: CRC Handbook of
Mariculture. 2nd Edition. Vol 1: Crustacean Culture. J.V. Mc Vey (Ed).
CRC Press, Inc., Boca Raton, Florida, USA, pp 61-93.
Lavens P., and
Sorgeloos, P. 1991. Chapter XIII : Production of Artemia in culture tanks. In: Artemia
Biology. R.A. Browne, P. Sorgeloos and C.N.A. Trotman (Eds). CRC Press,
Inc. Boca Raton, Florida, USA, pp 317-350.
Lavens
P., and Sorgeloos, P. 1987. Design, operation, and potential of a
culture system for the continuous production of Artemia
nauplii. In: Artemia Research
and its Applications. Vol. 3. Ecology, Culturing, Use in Aquaculture P.
Sorgeloos, D. Bengtson, W. Decleir and E. Jaspers (Eds) Universa Press,
Wetteren, Belgium: 339-345.
Tackaert,
W. and Sorgeloos, P. 1991. Semi-intensive culturing in fertilized ponds:
287-315. In: Artemia Biology.
Browne, R.A., Sorgeloos, P. and C.N.A. Trotman (Eds), CRC Press, Inc.,
Boca Raton, Florida, USA, 374 p.
Vanhaecke, P.;
Cooreman, A.; Sorgeloos, P. (1981). International study on Artemia
: 15. Effect of light intensity on hatching rate of Artemia
cysts from different geographical origin. Mar. Ecol. Prog. Ser. 5:
111-114.
Wilkerson,
J., Clownfishes, Microcosm Limited; ISBN: 1890087041; June 1998
