A Bioassay Using Sea Urchin Larva

Lytechinus variegatus Sea Urchins

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A Brief Summary of the Project

Studying the larval growth of the Lytechinus variegatus sea urchin can provide valuable information that applies to many organisms from simple sea creatures to humans because of the similarity of the gametes. Urchins are valuable as a food source in many parts of Asia and are needed to control algae and plant growth in the wild. In this experiment, the urchin larva were subjected to different levels of pH and salinity and their growth was monitored. This helps to predict what could happen if acid rain or point source runoff change the water quality around coastlines. In the salinity experiments, the urchin larva survived in the normal ranges and in slightly higher salinities, but died in lower salinities and extremely high salt contents, showing that the urchins will not tolerate any drop in salt content due to runoff. In the pH experiments, the larva survived in the normal ranges and in slightly lower ones, showing that the urchins may be able to withstand a small rise in pH due to acid rain.

Use the following links to learn more about this project:

Pictures

Further Introduction

Procedure for Experiment

Conclusions

Results --Including Charts

Pictures of Developing Larva

Links to other sites


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Pictures

This is the tank where the Adult Urchins are Held

This is the Microscope and Camera Equipment Used to Monitor the Larva

Here are the tools used to measure the water samples. The refractometer is on the left, and the pH probe is on the right.


Further Introduction

This experiment is a continuation of a project that was started in 1998. During that time, the procedures for obtaining the gametes of the sea urchins, fertilizing them, and bringing the larva to the pluteus stage were studied, and the goals were reached. This year the project will involve doing a bioassay on the sea urchins by testing the resistance of the larva to changes in salinity and pH. The reason for choosing these parameters as the independent variables is that these are two parameters of the marine environment that could change with the increased population along coastlines. Runoff from paved areas goes directly into the coastal water, when it would normally soak into the ground. This could cause the salinity of the coastal waters to drop after rains. Also, acid rain is a large concern with power plants that push tons of sulfur rich smoke into the air, which raises another concern: The heavy runoff could significantly increase the pH of coastal waters (Dahm et al, 1999). Sea urchins are valued as food in Asia, especially in Japan, and the United States is the largest Exporter of sea urchins (Penfold et al, 1999). Urchins also play a large part in the environment by providing food to many animals and by controlling the growth of aquatic algae and plants (Steele, 1999). Studying sea urchins will hopefully provide information about how the changing environment could affect the wild urchin populations. The sea urchin is an echinoderm and is a member of the phylum echinodermata, which includes over 6,000 species (Friese, 1996). The name means "Spiny-skinned animal of the sea," and refers to the spines on the skin of all echinoderms (Fenner, 1998). Other members of this phylum include sea cucumbers, starfish, and sand dollars. All members have pentamerous (five-part) symmetry and a water vascular system that makes it possible for movement with the use of tube feet (Wells and Haywood, 1989). Sea urchins belong to the class echinoidea, which is Greek for, "Like a Hedgehog," and refers to the many spines covering the body of these animals. Sea urchins belong to the order exocycloida. More than 800 species of sea urchins have been observed and recorded. The species used in these experiments is Lytechinus variegatus, the Variegated Sea Urchin. Urchins have a rigid endoskeleton called a test. It consists of five close-fitting rigid calcareous plates. The globular body is covered with moveable spines. Between these spines are tiny, highly flexible arms called pedicellaria. These arms are used to move about, hold food, manipulate it to the mouth, and as a defense. The mouth of the sea urchin is beaklike and consists of five teeth and supporting muscles and articulating structures. The whole mouth structure is called the Aristotle's lantern (Wells and Haywood, 1989). There are gonads in each of the five sections of the urchin's body. The endoskeleton of the sea urchin allows the animal to resorb calcium from the test if insufficient limestone (calcium carbonate) is provided. This causes the animal to shrink if conditions are not appropriate (Fenner, 1998). Sea urchins are herbivorous grazers and scavengers. They seem to prefer shallow-growing turtle grass and algae, especially the macro algae of the genus Calurpa (Rudloe, 1999). Urchins can be found in huge groups grazing the rocks. Urchins are nocturnal, that is, they are most active during the nighttime hours. Urchins are extremely sensitive to impurities in the water, especially metals. They show signs of stress at even the most subtle changes, and for this reason, they are good indicator animals for the environment. Public aquariums often use them as indicator animals for water quality. TABLE 1. Urchin Larva Development 1 Time Stage Reached 2 Minutes Fertilization Membrane Forms 10 Minutes Union of Pronuclei 50 Minutes First Cell Division Occurs 80 Minutes Second Cell Division 100 Minutes Third Cleavage 7 to 8 Hours Blastula Formation 2 Days Gastrula Stage is Reached 5 Days Pluteus Stage is Reached 1-3 Months Metamorphosis into Juvenile Urchin When a sperm cell enters and fertilizes an egg, it creates a zygote. The zygote then goes through a series of cleavages, or divisions, to form a solid ball of cells called a morula (Hoegler, 1999). After this, the cells in the morula begin to migrate, and the embryo changes from a solid ball of cells to a hollow sphere of cells one layer thick known as a blastula. During this stage, the embryo breaks the fertilization membrane and becomes free floating. The blastopore is a point on the embryo at which the cells begin to migrate toward to form a primitive gut. This is called gastrulation, and an embryo in this stage is called a gastrula (Poppen and Piercy, 1999). The embryo further develops into the pluteus stage and looks somewhat like an upturned easel. During this entire period, the larva is free floating and feeds on single celled organisms and algae, but after a few months in the pluteus stage, the larva undergoes a metamorphosis and becomes a juvenile urchin. At this point the urchin looks like the adult urchin and begins life on the sea floor (Carolina Pamphlet, 1998).


Procedure For Experiment

A saltwater aquarium was prepared last year for this experiment, but it needs to be set up at least a month in advance to the arrival of the urchins to allow the tank to "cycle." Ten hours of lighting were supplied per day. The temperature was adjusted to 22°C, and the specific gravity was adjusted to 1.024g/ml. After the water parameters were adjusted, Aquarium rock was added to introduce beneficial microorganisms and bacteria. A power filter was used that contains a way of mechanically filtering the water, as well as a place for activated carbon and other nutrient absorbing resins and pads (Tullock, 1997). The urchins were not put into this aquarium before use, as they might have spawned prematurely, but they were put into this tank as soon as gametes were obtained (Figure 1). As the urchins arrived, an algae culture was started to feed the larva, as they needed food right away. This was done by culturing isochrysis and minochrysis single-celled marine algae. The algae was grown in a petri dish in seawater under fluorescent plant lighting with a photoperiod of ten hours (Figure 2). The algae needed light also, and were given the same photoperiod as the urchin tank. Starting the cultures as soon as the urchins arrived ensured that food was always available for the larva (James, 1978). When the urchins first arrived, the shipping bags were opened, the water was changed, and an airstone was added to each one. If they had been put directly into the tank, they might have spawned prematurely. Although spawning can be induced by several methods, including electricity and various chemical solutions, the following seems to be the most effective: Obtain or prepare a solution of 0.5M KCl (potassium chloride). Injections of 1-2 ml of the solution are sufficient to induce the animals to spawn. A small needle is better, as it reduces the size of the injury to the urchin (a #25-#30 needle works fine). Inject the solution into the soft tissue surrounding the mouth of the urchin. Turn the urchin slowly upside-down and right side-up to mix the solution inside the sea urchin. If the urchin is shaken too hard, the delicate internal tissues and membranes can be damaged (Petti and Terry, 1999). Shortly after the injection, the urchins began to release gametes. When the yellow-orange eggs appeared, the urchin was placed over a beaker of seawater so the top of the urchin just touched the water. If the eggs are not going to be used right away, they should be rinsed several times in seawater prior to use or storage in a refrigerator. If they are going to be used right away, as these were, the eggs should simply be rinsed in seawater. When the white sperm began to appear, the urchin was placed over a dry beaker to collect the sperm. The sperm was placed in a test-tube or vacuum tube and placed in a refrigerator. The sperm can be stored for a few days in this manner as long as seawater is not added. Once the sperm comes into contact with seawater, they are activated, and it is only useful for about 10-20 minutes (Poppen and Piercy, 1999). The first time this year that gamete collecting was attempted, seven urchins were brought from the home of the researcher that had been used in the 1998 experiments. They were kept well fed over the months, and when they were injected, three males released sperm. The sperm was stored in a vacuum tube in the refrigerator. As no females released eggs, another shipment of urchins had to be used. This proves that it is feasible to use the same urchins more than once if a recovery period is provided. It was the intention of the researcher to be able to use the same urchins periodically without purchasing additional animals, in order to save money and reduce the number of urchins being taken from their natural habitat. During the recovery period, a heater failure in the greenhouse caused the temperature in the large tank where the urchins were being kept to drop. Although no urchins were lost, the cold temperature may have caused the females to resorb their eggs, as the failure occurred shortly before the urchins were used (Carberry, 2000). Once the gametes were collected, they were properly diluted before they were mixed for fertilization. To dilute the sperm, 0.2cc (about 2 drops) of sperm were mixed in 10cc of seawater (as the sperm are now "activated," they need to be used within the next 10 minutes). Two drops of this solution contains enough sperm to fertilize a beaker of eggs. After the gametes were mixed, the solution was gently stirred to mix the eggs and sperm. Sperm penetration is very rapid, so if it is to be seen, it is best to perform the mixing on the slide under a microscope. After a beaker of fertilized eggs was obtained, they were put into petri dishes. The petri dish has a large surface area and allows for adequate oxygen exchange. A pipette was used to transfer a small number of eggs to each petri dish of seawater. The larva will need food fairly soon after fertilization. As soon as the larva were placed in the petri dishes, a few drops of the algae culture were added to each one daily to ensure that the larva always had food available. Some water was changed in the petri dishes each day to make sure that the water was fresh and that nutrients were removed. To perform the bioassay on the larva, solutions of water with the correct water parameters were prepared in advance. All solutions were prepared using reverse osmosis (RO) water and Instant OceanÒ salt mix. The pH was adjusted using commercially available solutions for marine aquariums. Water samples ranging from a pH of 6.4 to a pH of 8.8 in .2 increments were prepared. Water samples from a salinity of 1.012g/ml to 1.034g/ml in .002g/ml increments were also prepared. A small amount of each solution was stored in a sealed container for doing the daily water changes on the dishes. The pH solutions were prepared using a pH electrode and amplifier which was used in conjunction with the appropriate computer software. Once the device was calibrated, it yielded very accurate readings. The salinity solutions were prepared using a refractometer (see Figure 4). The device was calibrated using RO water. Eggs should were fertilized at normal temperature (20°C). A few drops of the egg/sperm solution was placed into each dish, and the appearance and growth of the larva was recorded. Since the early development of sea urchin embryos is highly synchronous, when a batch of eggs is fertilized, the deviation from the standard time (the control time) can be interpreted as a difference in conditions, and not simply the different speed for each larva (Epel 1999). A microscope was used with 20X and 40X objective lenses. The 40X is an oil-immersion lens, but the researcher discovered that this lens could be used to get very sharp and detailed images if a drop of the solution to be examined was placed on a deep-well slide and the lens immersed in the seawater. A camera was attached to the microscope, and images were captured using a computer.


Conclusions

The original hypothesis for this experiment is as follows: If sea urchin larva are subjected to varying levels of salinity, the urchins will not fare well in the lower salinities or higher salinities than normal seawater, and if the larva are also subjected to varying levels of pH, they will not survive lower levels, but will tolerate slightly higher ones. The data supports the hypothesis in some ways, and in some ways, proves the hypothesis to be wrong. The urchins seem to be able to withstand higher salinities better than previously thought, but it did slow their growth and cause deformities. The extremely low salinities caused the rupturing of the cells due to the extreme osmotic pressure on the membrane. The urchin larva could not survive a salinity level lower than 1.020g/ml. In the pH tests, some surprising results were recorded. The results contradict the hypothesis in that the urchins do not seem to be able to withstand higher pH's, but do seem to be able to tolerate lower ones, and even thrived under these conditions. This research may conclude that the runoff from areas near the coastline will greatly affect sea urchin populations, but acid rain will not affect the urchins directly if it is not severe. One problem developed during the research that may have affected the outcome. The heating system in the school malfunctioned between the 2nd and 3rd day, and the temperature in the room was around 30°C. This killed some of the larva and one adult urchin which was in the holding tank at the school. The experiment should be repeated to make sure the overheating did not change the results. If the experiment could be done over, the researcher would try to reserve time to check the growth progress of the larva much more frequently. It might also help to have several dishes for each solution, to ensure that the data is consistent. The larva from this experiment are continuing to feed and grow, and the researcher is planning to try to grow them for as long as possible and to continue collecting pictures and other data. The original hypothesis for this experiment is as follows: If sea urchin larva are subjected to varying levels of salinity, the urchins will not fare well in the lower salinities or higher salinities than normal seawater, and if the larva are also subjected to varying levels of pH, they will not survive lower levels, but will tolerate slightly higher ones. The data supports the hypothesis in some ways, and in some ways, proves the hypothesis to be wrong. The urchins seem to be able to withstand higher salinities better than previously thought, but it did slow their growth and cause deformities. The extremely low salinities caused the rupturing of the cells due to the extreme osmotic pressure on the membrane (see Figure 7). The urchin larva could not survive a salinity level lower than 1.020g/ml.


Results

Data was collected over three days, and the stage of growth of the larva in each dish was recorded. The data was categorized by the stage in growth that the larva reached. Some of the larva in the extreme pH and salinity solutions died, while the larva in the normal conditions (control) developed fairly fast. Each day a sample of the water from the dish was put under the microscope for examination, and the cells were looked at carefully. Some of the cells continued to grow, but some were deformed. The tables illustrate the growth of the larva in stages. The stages are categorized as follows: Cell=0, Early Blastula=1, Blastula=2, Early Gastrula=3, Gastrula=4, Early Pluteus=5, Pluteus=6.







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Larva Development

A series of pictures is presented of the control group. These pictures show the standard form of the cell in that stage of development.


Links to other Urchin Sites