Playing with Plutei

Hailey Conrad, Rutgers University

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Me! Photo Credit: Kady Palmer

Ocean acidification is known as climate change’s evil twin. When the pH of ocean water drops, carbonate ions in the water form carbonic acid instead of calcium carbonate. Calcium carbonate is the form of calcium that marine animals that have calcium-based skeletons (like us!) and shells use to build their bones and shells. Having smaller and weaker skeletons or shells impacts their ability to survive. However, some individuals within certain species or populations of species have genes that make them more resistance to ocean acidification. If these individuals are able to pass on these genes to their offspring, then the species has the ability to evolve in response to ocean acidification instead of going extinct. This summer I’m working with Dr. Bob Podolsky in College of Charleston’s Grice Marine Field Station to study the extent to which ocean acidification affects Atlantic purple sea urchins, Arbacia punctulata. We are specifically trying to see if any individuals within a population from Woods Hole, Massachusetts, have any heritable genetic resistance to the negative impacts of ocean acidification. We hypothesize that there will be genetic resistance given that the northern Atlantic coast naturally has lower levels of saturated calcium carbonate, so a population that has evolved to live in that type of environment should have some resistance to lower calcium carbonate levels already (Wang et al 2013). We’re using a basic cross breeding technique to rear Arbacia punctulata larvae to their plutei stage, when they have four main body rods. At this stage they look less like sea urchins than they do like Sputnik!

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A sea urchin larvae during the 4-spined pluteus phase

Secondarily, we are looking to see if any males produce sperm that is more resistant to ocean acidification by looking at the percentage of eggs they are able to fertilize when bred with different females in water with different levels of carbon dioxide. Then, we will look to see if any of the male parents consistently produce male offspring that are more resistant to ocean acidification that others or consistently produce sperm that results in higher rates of egg fertilization than others.  If males like these exist within this population, then the species has the capacity to evolve in response to ocean acidification, instead of going extinct! This is a very big deal, and could potentially be very hopeful. Even if we don’t get the results that we are hoping for, the results of this research could inform policy and management decisions.

Literature Cited:

Wang, Z. A., Wanninkhof, R., Cai, W., Byrne, R. H., Hu, X., Peng, T., & Huang, W. (2013). The marine inorganic carbon system along the Gulf of Mexico and Atlantic coasts of the United States: Insights from a transregional coastal carbon study. Limnology and Oceanography, 58(1), 325-342. doi:10.4319/lo.2013.58.1.0325

Thank you to the National Science Foundation and College of Charleston’s Grice Marine Laboratory for funding my project. And, special thanks to Dr. Bob Podolsky for being a wonderful and supportive mentor!

 

Expect the Unexpected in Science

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Alessandra Jimenez, Whitworth University

As this internship has recently come to an end, I now begin to reflect on the wonderful yet challenging experience I had conducting observational research on Atlantic brown shrimp (Farfantepenaeus aztecus). In the last few weeks of this 10-week summer program, there was a fascinating yet unexpected turn of events. In particular, results of the experiment pointed to conclusions that I initially found myself unprepared for!

In summary, the focus of this experiment was to test effects of immune response on the ability to escape predators in shrimp. The escape mechanism, called tail-flipping (see video below) is actually powered anaerobically. However, recovery from this energetic behavior absolutely requires oxygen (is aerobic). As further explained in previous blog posts (click here and here), a recently discovered consequence of mounting an immune response against bacterial infection involves depression of aerobic metabolism. So, my mentor and I decided to focus on the recovery aspect (aerobic) of the escape response and predicted that this aerobic process would be impaired in shrimp injected with bacteria. At the same time, we predicted that the anaerobic part of this mechanism would be significantly impacted.

A slow-motion video of an Atlantic brown shrimp juvenile tail-flipping in an experimental tank (c) Alessandra Jimenez

The last few weeks of the internship mainly consisted of analysis, arriving at conclusions, and publicly reporting results. After testing tail-flipping ability (click here for an explanation of how this was tested) in a total of 42 shrimp juveniles, 30 of these were chosen for final analysis. Using a statistics software called Sigmaplot (version 12.5), I conducted tests that basically compared experimental groups based on the two variables I investigated: treatment type (bacteria or saline) and time given after injection (4 or 24 hours). Afterwards, results were deemed important based on significance values assigned by these Sigmaplot tests.

Significant results were very surprising!  Overall, results suggested that metabolic depression (indirectly caused by the immune response) did not have an impact on recovery (aerobic). At the same time, the most unexpected finding of all suggested that bacterial exposure actually increased anaerobic tail-flipping activity in Atlantic brown shrimp juveniles! Thus, this result called for a complete change in focus from the aerobic part to the anaerobic part of this particular escape response.

So, how could I possibly explain the increase in anaerobic processes found through this experiment? After much pondering and going through scientific literature, I formulated a new hypothesis. An important enzyme in crustaceans called arginine kinase is involved in the storage and creation of anaerobic energy that can be used for tail-flipping. Recent studies involved injecting bacteria into live crustacean tissue and comparing arginine kinase expression levels with controls. Results indicated a significant increase in expression in bacteria-injected tissue, especially in abdominal muscle (important for tail-flipping!). Based on these investigations, I now think that there may be a link between immune response and levels of anaerobic metabolism. Further research is required to explore this.

The final stages of the internship included creating and presenting a Powerpoint presentation of our work, and submitting a manuscript of my summer investigation. Overall, this REU internship experience has been challenging yet exciting, and has confirmed my love for marine biological research. As I mentioned at the end of my presentation, “expect the unexpected in science”.

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Picture of me right before giving my Powerpoint presentation (c) Alessandra Jimenez

References:

Burnett, L. E., Holman, J. D., Jorgensen, D. D., Ikerd, J. L., & Burnett, K. G. (2006). Immune defense reduces respiratory fitness in Callinectes sapidus, the Atlantic blue crab. Biological Bulletin, 211(1), 50-57.

Gruschczyk, B., Kamp, G., 1990. The shift from glycogenolysis to glycogen resynthesis after escape swimming: studies on the abdominal muscle of the shrimp, Crangon crangon. J Comp Physiol B, 753-760.

Scholnick, D. A., Burnett, K. G., & Burnett, L. E. (2006). Impact of exposure to bacteria on metabolism in the penaeid shrimp Litopenaeus vannamei. Biological Bulletin, 211(1), 44-49.

Yao, C., Ji, P., Kong, P., Wang, Z., Xiang, J., 2009. Arginine kinase from Litopenaeus vannemai: Cloning, expression, and catalytic properties. Fish Shellfish Immunol 26, 553-558.

Many thanks to College of Charleston for hosting my project, Dr. Karen Burnett and Hollings Marine Laboratory for guidance and work space, and NSF for funding the REU program.

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A day in the Shrimp Lab

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Alessandra Jimenez, Whitworth University

Have you ever wondered what it’s like to be a lab researcher who works with live animals? Through this internship, I am experiencing this firsthand in Hollings Marine Laboratory, along with all the responsibilities involved!

A normal workday in the life of a “shrimp intern” is like this: A big part of it is animal care and maintenance. It starts in the morning with a daily visit to the wet lab, where approximately 80 brown shrimp juveniles are kept in four large tanks with circulating water. After feeding them a round of commercial shrimp pellets, I test the salinity of the water in each tank using a refractometer to make sure that each tank has a certain salinity value: 30 parts per thousand, to be exact. I use dechlorinated freshwater and seawater to adjust this value if needed. Besides salinity, I also need to watch out for harmful levels of ammonia (it’s a part of shrimp waste!), nitrates, etc. In usual circumstances, I conduct a water change (replacing old water with new) once a week in order to dilute these chemicals. For the past couple of weeks, however, I have been conducting water changes daily in order to keep ammonia levels neutral in three tanks. Ah, the life of a caretaker of tons of baby shrimp!

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Wet lab. @AlessandraJimenez

Besides animal husbandry, I work on my experiment involving the effects of injection of bacteria on tail flipping (Want to learn more about what I’m doing? click here). I have two shrimp at a time in separate, well-aerated tanks, and they are both from the same treatment group. Shrimp are randomly assigned to one of four treatment groups. These treatment groups are designated according to the treatment type (injection of bacteria or saline) and according to the amount of time between the moment of injection and the tail-flipping procedure (4 or 24 hours). I randomly select two shrimps from the wet lab, weigh them, and keep them in the two experimental tanks overnight so they can get used to the new environment, temperature, etc. The next day, I take each shrimp out of the tank momentarily and quickly inject them with bacteria, or a saline buffer if they are part of the control group. Then, I give them 4 or 24 hours (depending on group type) to rest before conducting the actual tail-flipping experiment. Using a stir-rod (basically, a straight stick), I poke the shrimp lightly to induce tail-flipping, and count how many flips they perform before fatigue. The number of flips here is called ‘initial activity’. Then, I give them 20 minutes to recover in the tank before tail-flipping them again. The number of flips this time is called ‘recovery activity’.

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Experimental tanks @AlessandraJimenez

Why tail-flip them twice? Well, we hypothesize that recovery activity will be impaired in bacteria-injected shrimp versus the controls, while initial activity would probably not be. This is based on how recovery from tail-flipping activities involves aerobic (or oxygen-fueled) metabolism. Since bacteria accumulate in the gills of shrimp and block oxygen uptake (want to learn more? click here), it would make sense that recovery activity would be reduced. Stay tuned for results later on!

Works Cited:

Gruschczyk, B., Kamp, G., 1990. The shift from glycogenolysis to glycogen resynthesis after escape swimming: studies on the abdominal muscle of the shrimp, Crangon crangon. J Comp Physiol B, 753-760.

Scholnick, D. A., Burnett, K. G., & Burnett, L. E. (2006). Impact of exposure to bacteria on metabolism in the penaeid shrimp Litopenaeus vannamei. Biological Bulletin, 211(1), 44-49.

Many thanks to College of Charleston for hosting my project, Dr. Karen Burnett and Hollings Marine Laboratory for guidance and work space, and NSF for funding the REU program.

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