DNA Analysis of Cytochrome Oxidase 1 to Confirm The identity of Locally Purchased fish Introduction Matthew Chrostek and Alex Doan; Carleton Summer Science Institute 2013, Carleton College, One North College St., Northfield, MN 55057 Discussion Results In 2011, Americans spent 80.2 billion dollars[1] on seafood. A comprehensive study by Oceana found that within the United States, an estimated 33% of commercially bought fish and shellfish were mislabeled[2]. Cheaper fish such as the Escolar ($13/lb) are frequently mislabeled, either intentionally or accidentally, as more expensive varieties, such as Albacore Tuna ($18/lb). Beyond a monetary loss, cheaper fish can also cause a variety of adverse health effects, for example, in the case of Escolar, distressing gastrointestinal effects[3]. We sought to verify the identity of 6 Northfield area fish samples using analysis of the mitochondrial DNA subset Cytochrome Oxidase 1. Of the six samples of fish we analyzed, two were found that did not corresponded to the species of fish they were labeled as. Mislabeling frequently occurs for one of two reasons, either for monetary gains or simply by accident. In the case of the Oreochromis niloticus (Nile Tilapia), this fish species was mislabeled as Lutjanus campechanus (Red Snapper). These two species of fish are not found in the same location, do not resemble each other in appearance, and are not from the same family of fish. Moreover the large price difference between an Oreochromis niloticus and Lutjanus campechanus may make the mislabeling a lucrative mistake for the company selling these mislabeled fish. The other fish that was found mislabeled was the Sander Canadensis (Sauger) that was incorrectly labeled as a Sander vitreus (Walleye). In this case the two species of fish shared similar appearances, could be found in the same location, and came from the same family of fish. In this case of mislabeling it seems probable that a simple mistake was made. By mislabeling fish the health of the customer can be put in jeopardy. Mislabeling may also give the seller an unfair advantage over competitors by allowing them to sell their fish for far cheaper than others. While our conclusions would implicate restaurants or supermarkets, these may not be the guilty parties at all. The mislabeling could occur anywhere in the path of the fish while it moves from ocean to plate. Sequences of Fish Mislabeled Fish Example of DNA Sequence Alignment of Fish 1 [Labeled Red Snapper] Forward and reverse sequences were compared using ClustalW2 to compare sequence similarity and to ensure accuracy of sequencing reaction. Comparative sequences between two mislabeled fish Red Snapper vs. Nile Tilapia [Fish 1] Walleye vs. Sauger [Fish 5] *N indicates unknown base pair [sequencing error] ? Albacore Tuna Escolar Methodology 1.DNA was extracted from six fish samples collected from pet stores and restaurants in Northfield and Proteinase K was used to break down cells, and a DNeasy spin column was used to isolate DNA. 2.DNA was amplified through Polymerase Chain Reaction to isolate the mitochondrial DNA segment of Cytochrome Oxidase 1, about 600 base pairs long. 3.Purified the PCR product to remove unincorporated nucleotides. 4.DNA was sequenced by Elim Biolabs. 5.We compared sequences using the Basic Local Alignment Search Tool, a program which compared our sequences to known sequences to determine fish species. Walleye ($17/lb) Sauger (Unknown Price) Nile Tilapia ($4.5/lb) Red Snapper ($15/lb) Conclusion Actual Fish Species Labeled as Matched Base Pair % Base Pair Similarity Identified as Red Snapper Lutjanus campechanus 91% 440/480 Nile Tilapia Oreochromis niloticus 99% 594/595 Halibut Hippoglossus Hippoglossus 647/649 Goldfish Carassius auratus auratus 100% 618/618 Cod Genus: Gadus 615/615 Greenland Cod Gadus Ogac Walleye Sander vitreus 92% 582/627 Sauger Sander Canadensis 628/631 Tilapia 626/626 In conclusion, we found that our results, like those of the Oceana study, indicated incidents of mislabeling occur. Two of the six fish analyzed did not match the labels. Analyzing the Cytochrome Oxidase 1 Gene was a very efficient way to determine fish species, because even within the same genus, in the case of the Walleye and Sauger, there was a 7% difference in their COX 1 DNA sequences. Despite our small sample size, we found it notable that we discovered one instance of both what seemed to be intentional mislabeling and accidental mislabeling. Going forward, we would suggest further studies with more samples across a larger area. Acknowledgements: We would like to thank the Carleton Summer Science Institute, and Carleton College, Northfield, Minnesota. We would especially like to thank Professor/Men’s Tennis Coach/Biology Chair Stephan Zweifel Ph.D., and Research Assistants/Bagel Deliverers Drew Holmen and Madeline Arnold. References [1]: Consumer Reports Magazine. “Mystery Fish”. December, 2011. http://www.consumerreports.org/cro/magazine-archive/2011/december/food/fake-fish/overview/index.htm [2]: Warner, Kimberly; Timme, Walker; Lowell, Beth; Hirshfield, Michael. “Oceana Study Reveals Seafood Fraud Nationwide”. Oceana. February 2013. http://oceana.org/sites/default/files/National_Seafood_Fraud_Testing_Results_FINAL.pdf [3]: Daily Mail Reporter. “Fish mislabeling widespread in the U.S. and 84% of ‘white tuna’ is a species which causes distressing gastrointestinal side effects”. Daily Mail Online. 24 February 2013, http://www.dailymail.co.uk/news/article-2283880/Fish-mislabeling-widespread-U-S-84-white-tuna-species-causes-distressing-gastrointestinal-effects.html