The Effect of Temperature on the Hatching Success of Brine Shrimps

7/15/2019 The Effect of Temperature on the Hatching Success of Brine Shrimps

http://slidepdf.com/reader/full/the-effect-of-temperature-on-the-hatching-success-of-brine-shrimps 1/15

BIOLOGY LAB REPORT

TITLE : THE EFFECT OF TEMPERATURE ON THE HATCHING SUCCESS

OF BRINE SHRIMPS

PREPARED BY :

I/C NUMBER

STUDENT ID :

GROUP :

LAB PARTNER :

LECTURER’S NAME :

PRACTICAL DATE :

SUBMISSION DATE :



Abstract

Our earth is experiencing global warming which already reach an alert stage and this will, for sure,

have a profound adverse effects on various form of live, either land or marine life. To develop anunderstanding on how temperature effect the rate of growth and survival of marine life, an experiment

was designed to determine the optimum temperature needed by brine shrimp to hatch and survive.This experiment accomplished by using salt water with 0.2% salinity in three respective test tubes

which was then placed in three different temperature range (22˚C, 29˚C and 32˚C) with about 20 eggcysts of brine shrimp. The number of hatched larvae in each test tube was counted on the next day and

the following day using light and was recorded.

Introduction

1. Global Warming(Greenhouse Effect)

Greenhouse effect is a phenomenon in which earth’s atmosphere traps the heat from the sun and

prevents it from escaping. Atmosphere acts like a greenhouse glass in which sunlight enters through

transparent glass but then, the heat released by plants (infrared radiation form) is not allowed to

escape. Thus the temperature increases inside the greenhouse. This is an analogy for what is happening

to our earth but in this case, certain gases known as greenhouse gases act as atmosphere and this

contributes to global warming.

Figure 1 : How Greenhouse effect happen and how they work (1)



Table 1 : Some of the effect of Global Warming

2. Brine Shrimp

Brine shrimp (Artemia) nauplii of aquatic crustaceans under the Artemiidae family. These Artemia

populations are found around word in saltwater lakes (up to 250% salinity level) and this enable them

to avoid living together with most types of predators. The lack of competitors (or effective predators)

in this extreme environment allows them to build large populations during seasons when the

conditions for reproduction are suitable.

Anatomical features of the Nauplii stage Anatomical features of the Adult stage

The nauplii swim through the water columntowards light (phototaxis) using the

secondary antenna. Mandibles are used to filter water and

capture green phytoplankton.

The nauplii have only one simple eye(photoreceptor) .

The adults swim using swim/filter feedingappendages.

The median eye is accompanied by 2 lateralcompound eyes.

A simple brain forms a ring like structure

around the oral cavity (typical of mostinvertebrates).

Females develop eggs in a ventral egg sac at

a rapid rate during favorable conditions.

Males have longer tufted antennae.

Table 1 : Anatomical features of Ar temia Saline in Naupilii stage and Adult stage(3)



Figure 1: Life cycle of Artemia (4)(5)

Artemia produces cysts which have led to extensive use of Artemia in aquaculture. Artemia cysts have

amazing shelf life and can be stored in containers for years, to be opened and utilized as a “ready-

made live food source. Furthermore, the ability of Artemia to feed on floating particles allows the

bioencapsulation of specific agents tailored to suit the predators’ requirements.(2)



Oviparous reproduction Ovoviviparous reproduction

After copulation, fertilized eggs are surroundedin the brood pouch of the female with a tough brown shell.

The cysts are released into water and will nothatch until they have been completely dehydrated

(in nature by floating ashore and sun-drying).

The embryo inside each cyst is in a state of metabolic dormancy and will not further develop

until hydrated.

Once sufficiently hydrated the embryo further develops into the instar I larva (nauplius) which

will hatch out of the cyst shell.

After fertilization, the eggs are not surrounded by a shell but instead immediately developfurther into naupliae in the broodpouch of the

female.

These naupliae are then released in the water as

free-swimming naupliae

Table 2: Two types of reproduction in Artemia(4)

Oviparous reproduction (Red Artemia ) Ovoviviparous reproduction(Pale whitish

Artemia )

Low O2 content, high salinity High O2 content, low salinity

Strong O2 fluctuation Minor O2 fluctuation

Iron rich food (green algae) Iron low food (organic debris)

Table 3: Environmental factors for the mode of reproduction (4)



Special abilities of Artemia :(6)

1. Artemia can keep its blood hyposmotic to environments more saline than about 10 parts per

thousand.

This is something most marine invertebrates cannot do, at least not to the same extent. Artemia

drinks brine and actively secretes salts from the maxillary glands, epipods, and gut. The

maxillary glands can produce urine four times as salty as the blood. Maintenance of a

hyposmotic blood is facilitated by the impermeability of most of the integument. The

exoskeleton(except epipods) is impermeable to salts. The epipods are major sites of active salt

secretion. Artemia belongs to a predominantly freshwater taxon and presumably evolved from

freshwater.

2. Artemia have short life span and have ability to remain dormant for long periods.

Artemia can live in water having much more or much less salt content than normal seawater.

They tolerate salt amounts as high as 50%, which is nearly a saturated solution, and can live for

several days in solutions very different from the sea water, such as potassium permanganate or

silver nitrate, while iodine – a frequent addition to edible salt – is harmful to them. The

animal's color depends on the salt concentration, with high concentrations giving them a

slightly red appearance. In fresh water, Ar temia sali na dies after about an hour.



Suitable conditions for the growth of Artemia :(7)

1. Dehydrated cysts of most strains measure between 200 and 270 μm, and weigh 3.5 μg on

average. Dry cysts are very resistant to extreme conditions. Up to 80°C, hatching efficiency is

not affected. Hydrated cysts are killed by temperatures lower than 0°C and higher than 40°C.

Cysts are very hygroscopic and absorb even water from the atmosphere. When stored, water

content should be lower than 0.09 g H2O/g cyst or 10% (no metabolism). At 0.3 g H2O/g cyst

or 25% the metabolism of the dormant cyst starts.

2. At salinities higher than 70 ppt cysts cannot hatch because of the too high osmotic gradient. In

salinities lower than 5 ppt cysts will hatch but resulting nauplii will die quickly. Light

triggering is needed at the beginning of the hatching to start metabolism. In freshwater and

seawater, dehydrated and hydrated cysts sink. In brine (saturated salt solution) they float.

3. In nauplii, growth is optimal at 28°C and 35 ppt and drops below pH 7. Lethal temperaturelimits are 0°C and 37 – 38°C. Salinity changes can be administered very abruptly without harm.

At 0°C, activity will stop but can be reactivated by increasing the temperature. For adults,

mostly salinity tolerance is up to 200 – 250 ppt. Limitation is more caused by oxygen depletion

than by salinity itself. Below pH 7 general appearance of Artemia deteriorates. pH 8 – 8.5 is

optimal.

Various effects of water temperature on the hatching metabolism of Artemia cysts



Objective

To investigate the effects of temperature on the hatching success of brine shrimps.

Problem Statement

Which is the effect of temperature on the hatching success of brine shrimps?

Hypothesis

As temperature increases, the rate of hatching success of brine shrimps also increases until optimum

temperature is achieved.

Apparatus

Fine glass pipette, pair of forceps, magnifying glass, stirring rod, bright light source, water bath or

incubators, refrigerator, 100cmᵌ beaker, 40cmᵌ beaker, 2g of sea salt for each treatment, 100cmᵌ de-

chlorinated water for each treatment, sheets of white A4 paper and graph paper.

Materials

Brine shrimp egg cysts.



Variable :

Types of Variables Ways to control the variables

Manipulated Variable:

Temperature (˚C)

Use different temperatures (22˚C, 29 ˚C and 32˚C) to

incubate the brine shrimps egg cysts.

Responding Variables:

Number of shrimps hatchedCalculated by catching using fine glass pipette and

counting the number of larvae that swim towards light

source for 2 days .

Fixed Variables:

Salinity

pH of water

Use same weight of salt (2g) for each fixed volume of

water (40cmᵌ) per beaker.

pH of water was kept between 8 and 8.5 (close to natural

pH of salt pan habitat)



Procedure

1. Three test tubes were labeled A, B, C and D.

2. 200cmᵌ of salt water was placed into test tube A.

3. A tiny pinch of brine shrimps egg cysts were placed onto a large sheet of white paper. A piece

of graph paper was then dampened using a few drops of salt water and then dabbed lightly on

the egg cysts to pick up approximately 30 eggs.

4. The eggs were counted using magnifying glass and the graph paper was cut so that there were

exactly 20 eggs.

5. The graph paper was then placed into a beaker to wash off all 20 eggs into the water. After

about 3 minutes (calculated using stopwatch), the graph paper was gently removed using a pair

of forceps.

6. Steps 2 to 5 was repeated with beaker B, C and D and all 4 beakers were placed in their

respective incubators at temperatures 22˚C, 29˚C and 32˚C.

Beaker Temperature (˚C) Place

A 22 Inside a.c. room

B 29 At laboratory room

C 32 Water bath

7. On the next day, the number of hatched larvae in each beaker was counted and recorded. A

bright light was placed next to the beakers and the larvae, which are phototaxic will swim

towards the light source. The brine shrimps larvae were counted by each of the group members

and the average number was noted. Step 7 was done for two days for all the three test tubes. On

each day, the test tube content was stirred using glass rod to enable aeration. The number of

larvae that was successfully hatched at each temperature was recorded.



Safety precaution and Risk Assessment

In order to avoid any accident or injury during the experiment in laboratory, the precautionary

steps should be taken and applied. Wearing lab coat and a pair of suitable shoes are compulsory

when conducting an experiment in the lab at all times to protect the skin and clothing from spillage

of any chemical substance. Hands need to be thoroughly washed before and after performing theexperiment. This is to avoid ourselves from getting infected from any of the microorganism.

Furthermore, the glassware such as beaker and magnifying glass should be handled with full care

because they are fragile. The apparatus such as forceps is also used very carefully to avoid any

unexpected injury. After using all samples and apparatus at the end of experiment, they should be

discarded properly and returned back to their places to avoid injuries and unnecessary accidents

that may result fatal results.

Ethical Issues Regarding the Use Brine Shrimps in the Experiment:

Since brine shrimps are living organism, it contributes to ethical issue regarding the usage of brine

shrimps egg cysts for the research purpose. The brine shrimps were released back into the sea

water after the experiment done, thus no harm was done to their life. Furthermore, the shrimp are

abundant in nature, so using them for experiments will not affect biodiversity thus; it is

environmentally ethical if considered. Economically, the shrimps were bred for food thus they will

die eventually. Anatomically, shrimps are invertebrates which mean they have low awareness of

pain thus; any pain that was hardly caused in this experiment can be regarded as none. In the

aspect of biology, the shrimps used may be clones, hence there is no resultant loss of genetic

variation and the gene pool is unaffected.



Results

Temperature (˚C) First Day (After 24 hours) Second Day (After 48

hours)

22 8 10

29 9 12

32 3 5

Table 2: Number of Brine Shrimps hatched in respective temperature by day count

Graph 1: Number of Brine Shrimp Hatched in respective temperature by day count



Data Analysis

Table 1 above show the number of brine shrimps in respective temperature (22˚C, 29˚C and

32˚C) by day count. This data will be analysed according to temperature at which the brine shrimps

larvae incubated and hatched. At 22˚C on the first day, about 8 larvae were noted to swim towards the

light source (torchlight). This is about 40% from total amount of brine shrimp egg cysts that was used

in this experiment. On the second day, about 10 larvae were noted and this means about half of the

brine shrimp egg cysts amount is hatched. At 29˚C, for the first day, about 9 (45%) larvae were noted

and this further increased in the number of hatched brine shrimp larvae on the second day which is at

about more than half percentage (60%). This is the highest number of hatched brine shrimp among all

three different temperatures. For the last temperature (32˚C), only small number of hatched brine

shrimp larvae was noted. On the first day, only 15% (3) of larvae were noted and for the second day, it

has further increase of two more larvae which give about 25% of total amount of egg cysts used. Thus

it can be concluded that the highest number of brine shrimp hatched at 29˚C and the lowest at 32˚C.

DISCUSSION

From the result obtained, it can be seen the highest number of brine shrimp hatched at 29˚C

and the lowest hatched at 32˚C. Thus, this means among all three temperatures, 29˚C is the optimum

temperature for the egg cysts of brine shrimp to hatch.

At 22˚C, about 8 larvae (40%) were noted to swim towards the light source on the first day and

about 10 larvae on the second day were noted and this means about half of the brine shrimp egg cysts

amount is hatched. At 29˚C, there is about 9 out of 20 (45%) larvae were noted to hatch and this

further increased on the second day which is about more than half percentage (60%) of hatched brine

shrimp. This shows that as temperature increases, the enzyme reaction for hatching enzyme of brine

shrimp eggs cyst increases until the optimum temperature is achieved. Thus, it can be concluded the

optimum temperature in hatching success of brine shrimps is approximately between 22˚C and 29˚C.

At 32˚C, only 3 larvae were noted to swim towards the light source which is roughly about

15% and on the next day, the amount increase to 5 giving about 25% of total amount of egg cysts

used. Here, the number of hatched brine shrimp is far lower than the other two temperatures. Thus,

beyond optimum temperature, the enzyme reaction decreases and is predicted to completely stop at

about 40˚C whereby the hatching enzyme has already been fully denatured. So, at 32˚C, it can be said

that the hatching enzyme already starts to denature causing the hatching rate to drop tremendously.



Any further increase in temperature will halt the hatching of brine shrimp egg cyst as the

enzymes in the eggs are temperature-dependent. Since the brine shrimp grows best in between 22˚C

and 29˚C, the cysts burst and the embryo able to leave the shell. Further increase in temperature

beyond the optimum temperature range will affect the hatching of brine shrimps.

The enzyme involved in the hatching of brine shrimp cysts is known as hatching enzyme (HE),

which is secreted from hatching gland cells in hatching larvae for digesting their protective

extracellular coats, is used as an important tool in the process of enzymatic hatching in many animal

species including brine shrimps. The HE provides a typical model in the studies of certain cell

differentiation, specific protein synthesis, and special gene expression regulation during a certain stage

of early embryos at the morphological and molecular level. It will be of great importance to

understand its biochemical properties and gene structure in terms of embryogenesis and embryo

pharmacology. To affect hatching, diapausing cysts have to be activated, while the quiescent portion

has to be triggered by suitable environmental conditions.(8)

Limitations

There are several limitations that have been identified throughout this experiment.

The bright light source that was used to count number of hatched larvae may have heating effect

which subconsciously may affect the hatching rate of brine shrimps rate and alter the results.

The beaker contents need constant aeration in order to provide sufficient amount of oxygen for

the cysts to hatch and to keep the cysts in suspension.

Counting the number of egg cysts is a tedious work. The eggs might overlap each other and

make it hard to count leading to inaccurate number of eggs being put into the test tubes.

Sources of errors

Several sources of error in this experiment were identified and steps were taken to minimize these

errors to make the result more accurate.

Each and every members of the group count the number of hatched brine shrimp in order

to get reliable result.



Conclusion

The rate of hatching of brine shrimp increases as the temperature increases until an optimum

temperature is attained. The optimum temperature is known to be between 22˚C and 29˚C via this

experiment. Thus, the hypothesis is accepted.

Further Investigation

Another experiment can be carried out using different level of salinity to study the effect of salinity

level on the hatching rate of brine shrimp. Temperature and pH level are kept control while salinity

level can be manipulate using different amount salt (2g, 4g, 6g and 8g) to dissolve in each fixed

volume of water (40cmᵌ) per beaker.

References

1. http://www.fao.org/docrep/u8480e/U8480E9d.jpg. Accessed on 24th July 2012

2. Martin Daintith (1996). Rotifers and Artemia for Marine Aquaculture: a Training

Guide. University of Tasmania.OCLC

3. http://www.michaelsharris.com/12ubio/text/projects/brineshrimplab.htm. Accessed on 24th

July

2012

4. Manual On Artemia Production In Salt Ponds In The Philippines. Quezon City, October 1980Available from http://www.fao.org/docrep/field/003/AC062E/AC062E03.htm. Accessed on

24th

July 2012.

5. http://2.bp.blogspot.com/_mWXp8U9LnvI/R54BudCEh5I/AAAAAAAAAoY/eKL6qUZpEKE/s1600-h/ARTEMIA-male-%26-female-for-.jpg. Accessed on 7

thApril 2012

6. Invertebrate Anatomy Online. Last modified on 19 June 2006. Brine Shrimp. Available from

http://lanwebs.lander.edu/faculty/rsfox/invertebrates/artemia.html. Accessed on 24th

July 2012.

7. Artemia. Last modified on 13 June 1995. Brine Shrimp. Available fromhttp://web.cecs.pdx.edu/~davidr/discus/articles/artemia.html. Accessed on 24th July 2012.

8. Experiment with baby Brine shrimp. Available from

http://www.waynesthisandthat.com/brineshrimp.htm. Accessed on 24th

July 2012

http://www.fao.org/docrep/u8480e/U8480E9d.jpg

http://en.wikipedia.org/wiki/University_of_Tasmania

http://en.wikipedia.org/wiki/Online_Computer_Library_Center

http://www.michaelsharris.com/12ubio/text/projects/brineshrimplab.htm

http://www.fao.org/docrep/field/003/AC062E/AC062E03.htm

http://2.bp.blogspot.com/_mWXp8U9LnvI/R54BudCEh5I/AAAAAAAAAoY/eKL6qUZpEKE/s1600-h/ARTEMIA-male-%26-female-for-.jpg




http://lanwebs.lander.edu/faculty/rsfox/invertebrates/artemia.html

http://web.cecs.pdx.edu/~davidr/discus/articles/artemia.html

http://www.waynesthisandthat.com/brineshrimp.htm

http://www.waynesthisandthat.com/brineshrimp.htm

http://web.cecs.pdx.edu/~davidr/discus/articles/artemia.html

http://lanwebs.lander.edu/faculty/rsfox/invertebrates/artemia.html



http://www.fao.org/docrep/field/003/AC062E/AC062E03.htm

http://www.michaelsharris.com/12ubio/text/projects/brineshrimplab.htm

http://en.wikipedia.org/wiki/Online_Computer_Library_Center

http://en.wikipedia.org/wiki/University_of_Tasmania

http://www.fao.org/docrep/u8480e/U8480E9d.jpg

Documents

The Effect of Temperature on the Hatching Success of Brine Shrimps