Name : Nadhirah Binti Baharin

Group : 11 SC 1
SID Number : 1311171150
Title : The effect of temperature on the hatching success of brine
shrimps
Date of Experiment : 3
rd
April 2014
Date of Submission : 11
th
April 2014
Lecturer : Madam Lili Syahani Binti Rusli





Title
The effect of temperature on the hatching success of brine shrimps.
Objective
1. To investigate the effect of temperature on the hatching success of brine shrimps.
2. To determine the temperature that the brine shrimps have the greatest hatching success.
3. To develop certain experimental skills, considering ethical issues arising from the use of
brine shrimps, presenting results, producing reliable results, identifying trends in data and
drawing valid conclusions.

Problem statement
How does temperature affect the hatching success of brine shrimps eggs?
Temperature, PH and salinity of water are among the factors that affect brine shrimps hatching
time. The higher the temperature, the faster the time for brine shrimp eggs to hatch until an
optimum temperature is obtained.
In this experiment, the brine shrimps are tested under different temperature to draw out
conclusion at which temperature do the eggs will hatch the most.

Hypothesis
Alternate Hypothesis
The greater the temperature, the greater the hatching success for the brine shrimps eggs until an
optimum temperature is reached

Null hypothesis
Temperature do not affect the hatching success of brine shrimps.
Introduction

Figure 1 : Brine shrimp

The brine shrimp, Artemia, is a small crustacean living in highly saline environments and
is also used as a general food for fish and shellfish culture. Artemia develops in two different
pathways. During ovoviviparous development, embryos develop directly into swimming naupliar
larvae. In oviparous development, embryonic development is arrested, enters the diapause state,
and forms encysted gastrulae (cysts) that contain a complex shell to defend against extreme
environments. When the cysts are re-hydrated at an appropriate temperature, the embryos
continue to develop into swimming larvae within 24 hours. Thus, Artemiais a good model
organism to study diapause-associated molecular processes. (1)
Brine shrimps are also a nutritious and easy to raise feed for aquaculture, however, are
considered a main vector of pathogenic microbes because they carry opportunistic bacteria that
proliferate during their production. Considering that brine shrimp is also susceptible to some of
those bacteria, their yield during nauplii production could be reduced by those opportunistic
pathogens. (2)
Artemia sinica is widely used in commercial aquaculture, is a valuable biological
resource due to its high unsaturated fatty acid and protein content. A. sinica is easy to acquire,
and its diapause cysts are easy to transport and preserve. These attributes, along with the
relatively short life cycle and simple culturing conditions of this crustacean, make A. sinica an
excellent model system for studies of genetics, development and evolution (3)
A key feature is the production of encysted gastrula embryos (cysts) capable of remarkable
resistance to various stresses, including severe desiccation, anoxia and exposure to UV radiation Under
favorable environmental conditions females generally produce free swimming nauplii, whilst production of
cysts is often the method used in stressful environments. However, there are significant exceptions to this
generality, and control of the mode of reproduction is complex (4)
Studies shows that the effects of temperature and salinity on A. franciscana from Lake
Grassmere, New Zealand reported that more than 90% of the nauplii survived to maturity within
the range 2028C/100170 ppt, and that adults were more tolerant than juveniles. However,
since no other species except A. franciscana from Lake Grassmere were tested, no direct
intraspecific or interspecific comparison can be made.



Apparatus and materials:

Brine shrimp egg cysts, 2 g of sea salt for each treatment, 100 cm
3
of distilled water for each
treatment, 40 cm
3
beaker of salt water, 100 cm
3
beakers (one for each temperature to be tested),
water baths, stirring rod, magnifying glass, pair of forceps, fine glass pipette and measuring
cylinder
Variables
Manipulated variable : Temperature of salt water containing the brine shrimps egg cysts
The temperature of the water is varied by putting the test tube inside water baths with
temperature of 30
o
C and 34
o
C and another temperature which is room temperature of 25
o
C.

Responding variable : The number of brine shrimps larvae successfully hatched
The successfully hatched brine shrimps larvae are put under light microscope and then counted.

Fixed variable : The time allocated for the brine shrimps to hatch and the initial
egg cysts number
All of the brine shrimps egg cysts are allocated with 24 hours to hatched. The initial brine
shrimps egg larvae are also kept constant






Methodology

1. A range of temperature 25
o
C, 30
o
C and 34
o
C are decided to be tested.
2. 2% of salt water is prepared by placing 2g of sea salt into 100 cm
3
beaker.
3. 100 cm
3
of distilled water is added and stirred until the salt completely dissolved.
4. Then, a tiny pinch of egg cysts is placed onto a large sheet of white paper.
5. Magnifying glass is used to count approximately 20 eggs to put into the test tube.
6. Steps 2 until 5 are repeated for all temperature that would be investigated such as 27
o
C
and 30
o
C.
7. All treatments are replicated to obtain average reading number of brine shrimps larvae.
8. On the next day, the number of hatched larvae in each test tube is counted. A bright light
is then placed next to the test tube. Any larvae would swim towards the light. Brine
shrimp is very delicate and care had to be taken when handling them.
9. The larvae that had successfully hatched at each temperature are recorded in the table
below.
10. The brine shrimp is then disposed by boiling the test tube so that the brine shrimp died.

Results
Incubation
temperature
(C)
Number of hatched brine shrimp larvae in 1cm
3
salt
solution
Average
of
hatched
larvae in
1 cm
3
salt
solution
Average
of
hatched
larvae in
25 cm
3
salt
solution
Group 1 Group 2 Group 3 Group 4 Group 5
Set
1
Set
2
Set
1
Set
2
Set
1
Set
2
Set
1
Set
2
Set
1
Set
2
25 20 23 3 13 2 3 3 4 16 13 10 250
30 7 8 17 13 14 36 11 10 3 12 13 325
34 10 2 8 13 8 16 10 10 8 13 9 225



Discussion
Results
From the above table, it can be seen that different temperature have different effects to
the hatching success of the brine shrimps. In 25
o
C, the number of larvae successfully hatched are
20, 23, 3, 13, 2, 3, 3, 4, 16, 13 for different replication which result in the mean hatching success
in 1 ml is 10. For 30
o
C , the larvae counts are 7, 8, 17, 13, 14, 36, 11, 10, 3, 12. This makes the
average larvae successfully hatched is 13. Whereas for 34
o
C, the hatching success of brine
shrimps are 10, 2, 8, 13, 8, 16, 10, 10, 8, 13 for the different replication. An average of 13 larvae
successfully hatched is confirmed.
From the result we can know that the temperature in which the hatching success of the
brine shrimp is the greatest is 30
o
C which is 13, followed by 34
o
C which is 13 and the least
successful temperature is 35
o
C.
Brine shrimps shell are temperature sensitive. A change in temperature is correlated to
the success of hatching. During the hatching process, the water diffuse into the shell through
osmosis and will then burst as the wall of shell exert pressure and become tensed. A type of
enzyme help to catalyse the reaction by detecting the temperature and makes the whole reaction
to take place in faster pace. The brine shrimp, Artemia franciscana, produces encysted gastrulae
(cysts) that enter a deep dormancy, termed diapause (5)
There might be some ethical issue regarding using the brine shrimps as an experimental
animal. However, the pros outweigh the cons so it is considered ethical to some scientist. This is
due to the fact that brine shrimps have much less complex nervous system. This means that the
brine shrimp have less or no pain while we are experimenting with them. The brine shrimp must
be disposed appropriately as well.

Sources of error
The sources of error that result in probability of unreliable result are accounted while
carrying out this experiment. First, while transferring the cysts, the exact count of brine shrimps
eggs are not determined. We do not fixed the number of eggs for every experiment. This could
affect the data produced as the greater cysts number might produce greater hatching success.
Next, the eggs might have been damaged or no living embryo present. Since it is in
dormant state, it is quite hard to determine this. The damage to might resulted in from the
mishandling of the eggs. There might be broken egg shells too.
In addition, while transferring the brine shrimps larvae, there are several larvae that are
attached to the wall of the pipette. Thus, they were not counted. The result obtained are less.
The salinity of water inside the test tube might differ or not suitable to the brine shrimp.
The salinity of the water affect the hatching success of the brine shrimp.

Limitation of experiment
Some limitation in the experiment are the pH of the water. Brine shrimp do not facour an
acidic condition. Next, not all of the brine shrimps eggs are in good condition. Some might
already invalid due to damages to the wall or the embryo. We also can not control the light
intensity and fix the intensity. There are experiments which are incubated in dark in the water
bath and one which is room temperature are in inconsistent exposure to light due to switching on
and off the light. The room temperature too, might varied. During the evening the room
temperature might be hotter than late in night. This difference do affect the hatching success of
the brine shrimp.

Precaution
Several precaution include handling the brine shrimps cysts with extra care. This is to
prevent any damage to the cysts. Next, while taking the brine shrimps from the test tube to be
counted, the test tube must be stir gently. This is to distribute the larvae evenly. Shaking the test
tube too hard will cause damage to the cysts.

Conclusion
Different temperature result in different successful hatching of brine shrimps.
Hypothesis is accepted
Reference

1. Expression and characterization of the JAK kinase and STAT protein from brine shrimp, Artemia
franciscana. Chenga, Chia-Hsiung, Chen, Gen-Der and Ye, \Maw-Sheng. 2010, Fish & Shellfish
Immunology, pp. 774782.
2. Proliferation, colonization, and detrimental effects of Vibrio parahaemolyticus and Vibrio harveyi
during brine shrimp hatching. Quiroz-Guzmna, Eduardo, Balczarc, Jos L. and Ricardo. 2013,
Aquaculture, pp. 8590.
3. Cloning and expression of the sorbitol dehydrogenase gene during embryonic development and
temperature stress in Artemia sinica. Wanga, Ting, Hou, Ming and Zhaoa, Na. 2013, Gene, pp. 296302.
4. Two highly diverged New World Artemia species, A. franciscana and A. persimilis, from contrasting
hypersaline habitats express a conserved stress protein complement. Clegga, James S. and Gajardo,
Gonzalo. 4, 2009, Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology,
Vol. 153, pp. 451456.
5. A small heat shock protein from Artemia franciscana is phosphorylated at serine 50. Qiua, Zhijun and
Vinerb, Rosa I. 2004, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, pp. 7583.