The Biodiversity of Aquatic Invertebrates in Bayside Marina and Fort Totten Park Seohee Lee1, Seoyeong Lee1, Gabriella Ng1 & Jessica Cohen, Ph.D.1 1Francis Lewis High School Location Scientific Name Common Name PBE-01 Bayside Marina Diptera Fly PBE-02 PBE-03 Fort Totten Park Diptera or Telmatogeton japonicus Fly or Marine midge PBE-04 Trichocera sp. Winter crane flies PBE-05 Cereratulus lacteus Milky ribbon worm PBE-19 Leitoscoloplos robustus Marine worm Abstract Water pollution is common in many waterways where people use lakes, rivers, and coastlines for transportation, industrial uses, etc. The purpose of this experiment is to test if water pollution affects the biodiversity in Bayside waterways. Samples were collected from Bayside Marina and Fort Totten. Since Bayside Marina allows docking and fishing, and is located beside a highway, the biodiversity of marine invertebrates near the Bayside Marina may be affected by pollution. The samples of invertebrates from Fort Totten, an area with no docking, were collected as well for comparison. PBE-01, PBE-02 and PBE-05 were from Bayside Marina which were identified as a fly, fly and milky ribbon worm, respectively. PBE-03, PBE-04, and PBE-19 were collected in the Fort Totten area and identified as a marine midge, winter crane fly, and marine worm, respectively. Because there were only 3 samples from each site, a definitive conclusion could not be reached. Introduction Water pollution has become increasingly common in many waterways where people use lakes, rivers, and coastlines for transportation, industrial uses, recreational uses, and more. However, due to these actions, the quality of water has decreased enormously along with the quality of water resources. According to a study, the availability of water is greatly decreasing and will continue to in the future (Postel et al. 1996; Postel et al. 1997). Preventing water pollution in water sources is needed to combat the effects of water shortage and poor water quality (Carpenter et al. 1998). Numerous types of water transportations can pollute rivers, lakes, or oceans. Water transportations not only cause chemical pollution, but also cause noise pollution. Anthropogenic noise increases auditory masking, leading to cochlear damage, changes in individual and social behavior, altered metabolisms, hampered population recruitment, and can subsequently affect the health and service functions of marine ecosystems (Peng et al. 2015). Materials & Methods Marine invertebrate samples were collected from Bayside marina, 28-05 Cross Island Pkwy, Bayside, NY 11360 and Fort Totten Park, Little Bay Rd, Bayside, NY 11359 where permission was granted to collect. Bayside Marina is located beside a highway and allows people to dock and moor boats whereas Fort Totten Park do not have any boat activities. Around 10 samples of marine invertebrates were collected from each place. Most of the samples were collected from the muddy area under the water from each place by using a spade on 12/03/17 and 03/28/18. Some samples were collected by an insect-catching net. The species, using pictures, websites, books, and a taxonomic key, were then tried to be visually identified. When brought to the lab, DNA was isolated, amplified and analyzed by using PCR and a COI primer mix. The results were run through gel electrophoresis to check for successful amplification. PCR projects were then sent to GENEWIZ for sequencing. Bioinformatics was conducted with the blue line of DNA Subway. Results After visiting the Bayside Marina and Fort Totten, marine worms and flies were collected. Out of 19 samples, only 6 samples had DNA that was present. The samples were PBE-001, PBE- 002, PBE-003, PBE-004, PBE-005, and PBE-019. PBE-001 and 002 were identified as Diptera sp., which is fly species. PBE-003 had a similar DNA sequence as Diptera sp. and Telmatogeton japonicus, which is the marine midge. PBE-004 was identified as Trichocera sp., which is winter crane fly. PBE-005 was Cereratulus lacteus, a milky ribbon worm. Finally, PBE-019 was identified as Leitoscoloplos robustus, which is a marine worm. Figure 5. Alignment of sequences. Samples were utilized as well as BLAST results with the least mismatch scores. Fruit Fly was inserted as an outgroup. Figure 3. Sample identification and location. A total of 19 samples were collected from Bayside Marina and Fort Totten Park. Only 6 samples were submitted for sequencing due to errors in the DNA extraction and successfully sequenced. Figure 1. Google Map showing sample locations- Bayside Marina and Fort Totten Park. Diptera https://australianmuseum.net.au/flies-and-mosquitoes-order-diptera https://insectcop.net/mosquito-eaters/ Trichocera sp. Figure 6. Sequence Similarity Chart. This chart assesses the similarity between the samples. None of the samples showed novelty, since they have more than 95% sequence similarity with the known sequences. (Cereratulus lacteus) (Trichocera sp.) (Diptera sp.) (Telpatogeton japoni) (Leitoscoloplos robustus) Telmatogeton japonicus Trichocera sp. http://www.aphotomarine.com/marine_fly_thalassomya_frauenfeldi.html Cerebratulus lacteus http://v3.boldsystems.org/index.php/Taxbrowser_Taxonpage?taxid=599506 Leitoscoloplos robustus https://www.agefotostock.com/age/en/Stock-Images/Rights-Managed/C54-1417088 Figure 2. Number of Samples per each site. A total of 19 samples collected. 9 samples were collected from Bayside Marina. 10 samples were collected from Fort Totten. 3 samples per each site were successfully sequenced. Of the sequenced, there were 3 different genera identified from Fort Totten, and 2 different genera from Bayside Marina. Figure 7. Maximum Likelihood Model Phylogenetic Tree of Samples and Related BLAST results. The Fruit Fly was used as an outlier for this tree. Analysis shows that PBE01 and PBE02 are likely of the genus Diptera, PBE03 of Telmatogeton japonicus, PBE04 of genus Trichocera, PBE05 of Cereratulus lacteus, and PBE19 of Leitoscoloplos robustus. Of the samples, PBE04 is the most distantly related from the rest. Figure 4. Image of collected organisms. Discussion The fly and milky ribbon worm were identified from the Bayside Marina; a marine midge, winter crane fly, and marine worm were identified in the Fort Totten area as well. However, some difficulties arose while attempting to do gel electrophoresis. For instance, out of 19 samples which were collected and went through gel electrophoresis, 13 samples were rejected due to the lack of DNA. The error could have occurred when using micropipettes or grinding tissue DNA since there were minor difficulties. This could have affected the success rate of the gel electrophoresis. Additionally, because there were only 3 samples from each site, there was not enough evidence to test the hypothesis, that biodiversity of Bayside marina would have been affected by pollution caused by boats. For future experiments, more samples will be collected, as only 6 samples were viable for gel electrophoresis. To attempt to lower the number of samples rejected, every step of the experiment will be done carefully to ensure no mistakes are made. References Carpenter, S.R., N.F., Caraco, D.L. Correll, R.W. Howarth, A.N. Sharpley, V.H. Smith. 1998. Nonpoint pollution of surface waters with phosphorus and nitrogen. Ecological Application 8(3): 559-568 Peng, C., X. Zhao, G. Liu. 2015. Noise in the sea and its impacts on marine organisms. International Journal of Environmental Research and Public Health. 12: 12304-12323 Postel, S.L., G.C. Daily, P.R. Ehrlich. 1996. Human appropriation of renewable fresh water. Science 271: 785-788 Postel, S.L., S.R. Carpenter. 1997. Freshwater ecosystem services. Nature’s services. 195-214 Acknowledgements We would like to thank Dr. Cohen, Dr. Wang, Mr. Liang, Ms. Jaipershad, Dr. Marmor FLHS Science department and research program. We would also like to thank the DNA Learning Center West and Barcode Long Island.