The purpose of this research is to see if hypoxia (water containing low oxygen) exposure causes changes in growth and survival in the species of fish commonly known as the Atlantic croaker. This research is important because it is one way to draw a direct length between human land use and the consequence for an entire marine ecosystem. The research question being addressed is: Do Atlantic Croaker exposed to hypoxia grow more slowly than those that are not exposed to hypoxia? Hypoxia is an oxygen deficiency in a biotic environment. The scientists involved in this research are looking at the croaker’s otoliths to see if the hypoxic environment is affecting the croaker’s normal diet. Otoliths are three small oval calcareous bodies in the inner ear of vertebrates, involved in sensing gravity and movement. This would be an important topic for a middle school science student to understand and remember so that, they can see that the human impact on a marine ecosystem effects specific organisms found in their local environment.
Methods & Procedures
What we did was to measure the total fish length, spine length, total fish mass, mass of the whole stomach and the mass of the stomach without contents. We also collected otoliths, liver and muscle tissues and when available stomach contents such as crabs and fish. The length of the fish were done using millimeters on a standard metric ruler. The mass of the fish and its’ contents were done using digital scales that measured to the 1,000ths of a gram. The collection of otoliths required a small kitchen knife and forceps. Collection of the liver, stomach, and muscle tissue required the use of small surgical scissors, a scalpel, and forceps. All tissues were stored in small vials with lids and stored on ice.
One of the most important procedures in carrying out this dissection is to prepare all of the materials before one starts. There must be an abundance of paper towels already pulled apart and stacked at arms length. There needs to be DI water (deionized water) in a squirt bottle used for rinsing the tissue samples and otoliths. One also needs a squirt bottle of ethanol for cleaning the cutting board. Two full cups of ethanol to cleans the dissection tools, The dissection tools include large tweezers, forceps, fine point tweezers, kitchen knife, scalpel, and small surgical scissors. One also needs three trays of numbered vials for the future storage of otoliths, liver and muscle tissues. The vials are to be number with a direct correlation to the Fish ID number. For example Fish ID: HMU 789 has three vials labeled HMU 789. These are the vials that will store the previously mentioned otoliths, liver and muscle tissue. One will need a small petri dish for rinsing off the otoliths. There must also be an empty tray to store the newly extracted otolith vials and a bucket of ice to store the liver and muscle tissue samples. Finally, one needs a supply of latex gloves to change out as sometimes they tear while in use.
Once all of the fore mention prep work is done then the data collection process begins. The total length of the fish is measured in millimeters from the point of the nose to the very tip of the tail. The spine length is measured because it is less variable than total length which can be affected by the quality of the tail. The length will later be used to categorize the fish. Then one uses a fine point tweezer to find the end of the spinal cord which is near the tail. Once the end of the spinal cord is found the length is then measured in millimeters. Using a paper towel dry off any moisture on the outside of the fish and place it on the digital scale. Record the mass in grams. Next, one is to place the fish on the cutting board and using the small kitchen knife remove the head. One should cut from between the operculum (also known as the gill cover) and pectoral fin (fins located on the left and right side of the fish just behind the operculum). Once the head is removed used the forceps to open and probe into the fish’s mouth. Sometimes before dying the Atlantic croaker will regurgitate its stomach. So if there is something in it’s mouth the majority of the time it is the stomach. If it is in the mouth remove the stomach and place it on the cutting board. Using either the forceps or one’s hand pull on the lower lip until the entire bottom half of the fish is removed. The gills will be removed in this pull along with the jaws, tongue and throat.
By removing the bottom half of the head one clearly exposes the part of the cranium where the otoliths are stored. Using the forceps remove the cranial cover. Then with the forceps carefully remove the left and right otolith. Place the otoliths in a petri dish. Squirt with DI water and then using the forceps place the otoliths in the vial corresponding to the Fish ID number. Leave the lid open and place in the tray dedicated to holding otoliths. Note that sometimes one will need to use the kitchen knife on the cranial cover because when the head was removed a small part of the spine is still attached to the cranial cover. The small piece of spine makes it impossible to remove the cranial cover with forceps.
To open up the abdomen use the surgical scissors. Insert the scissors into the rectum and start to cut open the abdomen by cutting a straight line all the way up to the where the head used to be. When done correctly one will cut between the pelvic fins (Pair of fins located on the underside of the fish nearest the head). Once the abdomen is dissected then the abdomen opens up like french doors. On the side where the head was removed and inside the abdomen one might find the liver. Sometimes one will find the liver or the left side, sometimes it is on the right and sometimes it is on both sides. The liver can be a variety of colors from dark red to light brown. Using the forceps and place the liver on the cutting board and rinse with DI water. Using the forceps place the liver sample in a vial that corresponds to the Fish ID number, close the lid and place it in the ice bucket.
Note that sometimes the liver will liquify and thus one will not be able to collect a sample. In that case toss the liver vial corresponding to the Fish ID and write in the lab book, on the right hand margin for that Fish ID number “no liver.” The removal of the stomach is challenging. Although there is not a complex digestive system, it is messy and tangled. Using the forceps one has to pull apart the gooey and entangles substances and structures. One should be looking for an oval shape structure that Vs off. At one end there will be a dangly structure that looks a bit like tentacles this is called the pyloric caeca and the other end will connect to the intestinal tract. Using any of the cutting tools remove the pyloric caeca and the intestinal tract. Using forceps place the stomach on the balance and find its mass and record under the category of whole mass. The balance will measure grams. Most of one’s results will be less than one gram. Our balanced measured the mass in thousandths of a gram. Remove the stomach from the balance, place it on the cutting board. Using the forceps push out the stomach contents and then measure the mass of the stomach without contents and record the data. The vast majority of fish have nothing in their stomach except goop like fluid. The research scientist in our study want to collect an identifiable organisms or parts of an organism such as crab claws or crab legs.
When one does find organisms or organism parts then they are placed in a sealable container, filled with ethanol, labeled with the appropriate Fish ID number and put aside with any other stomach samples. The final sample is the muscle tissue. Lay the fish on the cutting board with the end where the head used to be facing the left side. Using the scalpel remove the muscle sample located between the vertebrae (backbone) and the lateral line (visible line on the outer surface of the fish which runs horizontally). When removing the muscle sample be careful not to include any scales, skin or bones. Any of these three structures will compromise the chemical signature that the research scientist are looking for. Once the muscle tissue has been removed, place the tissue on the cutting board and rinse with DI water. The DI water will also help to remove any scales that may have attached to the sample. Place the muscle tissue in a vial that corresponds to the Fish ID number, close the lid and place the sample in the ice bucket. Throw away the fish carcass and any fish parts that are remaining on the cutting board. Using a paper towel wipe down all dissection tools and place them in the cups of ethanol. Using another paper towel wipe clean and dry the cutting board. More than one towel may be necessary. Finally, squirt ethanol on the cutting board and wipe down with a clean paper towel. The ethanol disinfects the work surface.
The ETEAMS participants were not able to see the results of the project due to its longevity. After all extracted liver is made into a powder, scientists will begin to analyze the chemical components of each fish’s diet. Scientists will also look at each fish’s otoliths to see how the hypoxic water affected them in their particular environments.
The results of the data collected will not be known until after September. The tissue samples collected in the summer of 2016 will be freeze dried in September. The research scientists will grind the dried tissue to a fine powder. The powder will then be analyzed for a chemical signature. The chemical signature will tell the scientists what the Atlantic croaker has eaten.
If the stomach of the Atlantic croaker has mush in it then it either has not eaten anything or it regurgitated its contents. If there is shrimp or crab in its stomach then it is feeding on the expected water column (feeding zone). If there are anchovies in its stomach then that indicates that it came up from the bottom to feed. Anchovies are a schooling fish and are found in higher areas of the water column. Coming up is potentially a cause of hypoxia. The purpose of collecting tissue samples is because that will tell the scientist what they have been eating. The liver tissue will indicate short term consumption. Indicating what has been eaten within the past month. The muscle tissue is a long term indicator of what the fish has been eating. Analyzing the muscle tissue scientist can determine what has been eaten within the past three months. When examining the otoliths under a microscope; if the bands are small then this indicated slow growth and large bands indicate rapid growth. The next step for these scientist is a more detailed look at the potential for hypoxia displacing these fish in the water column thus collecting more dietary samples. This research is relevant to middle school science students especially those in the coastal regions because the Atlantic croaker is a local species that can greatly impact local marine environment. Students who are familiar with the Atlantic croaker will be excited to dissect this organism and explore its contents developing a deep understanding and appreciation of the species.