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 LECTURERS 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 an understanding 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 (22C, 29C and 32C) with about 20 egg cysts 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 earths 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 column towards 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 feeding appendages. The median eye is accompanied by 2 lateral compound eyes. A simple brain forms a ring like structure around the oral cavity (typical of most invertebrates). 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 Artemia 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 readymade 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 After copulation, fertilized eggs are surrounded in the brood pouch of the female with a tough brown shell. The cysts are released into water and will not hatch 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.

Ovoviviparous reproduction After fertilization, the eggs are not surrounded by a shell but instead immediately develop further 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 Strong O2 fluctuation Iron rich food (green algae)

High O2 content, low salinity Minor O2 fluctuation 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, Artemia salina dies after about an hour.

Suitable conditions for the growth of Artemia: (7) 1. Dehydrated cysts of most strains measure between 200 and 270 m, and weigh 3.5 g on average. Dry cysts are very resistant to extreme conditions. Up to 80C, hatching efficiency is not affected. Hydrated cysts are killed by temperatures lower than 0C and higher than 40C. 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 28C and 35 ppt and drops below pH 7. Lethal temperature limits are 0C and 3738C. Salinity changes can be administered very abruptly without harm. At 0C, activity will stop but can be reactivated by increasing the temperature. For adults, mostly salinity tolerance is up to 200250 ppt. Limitation is more caused by oxygen depletion than by salinity itself. Below pH 7 general appearance of Artemia deteriorates. pH 88.5 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, 100cm beaker, 40cm beaker, 2g of sea salt for each treatment, 100cm dechlorinated water for each treatment, sheets of white A4 paper and graph paper.

Materials Brine shrimp egg cysts.

Variable : Types of Variables Manipulated Variable: Temperature (C) Use different temperatures (22C, 29 C and 32C) to incubate the brine shrimps egg cysts. Ways to control the variables

Responding Variables: Number of shrimps hatched Calculated by catching using fine glass pipette and counting the number of larvae that swim towards light source for 2 days .

Fixed Variables: Salinity Use same weight of salt (2g) for each fixed volume of water (40cm) per beaker.

pH of water

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. 200cm 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 22C, 29C and 32C. Temperature (C) 22 29 32

Beaker A B C

Place Inside a.c. room At laboratory room 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 the experiment. 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) 22 29 32

First Day (After 24 hours) 8 9 3

Second Day (After 48 hours) 10 12 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 (22C, 29C and 32C) by day count. This data will be analysed according to temperature at which the brine shrimps larvae incubated and hatched. At 22C 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 29C, 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 29C and the lowest at 32C.

DISCUSSION From the result obtained, it can be seen the highest number of brine shrimp hatched at 29C and the lowest hatched at 32C. Thus, this means among all three temperatures, 29 C is the optimum temperature for the egg cysts of brine shrimp to hatch. At 22C, 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 29C, 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 29C. At 32C, 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 40C whereby the hatching enzyme has already been fully denatured. So, at 32C, 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 22C and 29C, 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 22C and 29C 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 (40cm) 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 1980 Available 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 7th April 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 from http://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