Rationale:
Currently there are hundreds of freshwater bioassessment protocol in use globally, however expert error rates as high as 65% have been observed at the genus and species levels. There is no standard freshwater bioassessment method, especially one that leverages the power of DNA Barcoding. The World Economic Forum lists water scarcity as one of the greatest global risks of the coming decade. It is forecast that 66% of our population will experience water scarcity within a decade, leaving us more dependent on surface water for drinking. This requires more filtration infrastructure, and more bioassessment of surface water sources. DNA Barcoding of Chironomidae, the most widespread macroinvertebrate family, may be a move toward a global bioassessment method. Bland Altman statistical analyses will be conducted to further validate DNA Barcoding of Chironomidae Method as a more accurate and precise waterway measurement health data, adding significant value for monitoring scarce water resources. This project exploreskjhg the optimal standard taxonomic level for waterway health assessment globally as well as the statistical power at each taxonomic level. Molecular methods, such as DNA Barcoding from a region of the mitochondrial gene COI (cytochrome c oxidase subunit 1), have begun to enhance biomonitoring programs. DNA Barcoding offers the promise of a more rapid, accurate (less human error), and precise (species level) identification of macroinvertebrate taxa. This project builds on the use of DNA Barcoding to measure waterway health with the larval non-biting midge Chironomidae (order Diptera).

Research Questions: 
1) What is the optimal level of taxonomic identification to apply to a global standard method of using DNA Barcoding of Chironomidae for bioassessment of surface water sources?
2) How does level of taxonomic identification influence statistical power?
3) How can this bioassessment method help visualize the impairment along a stream channel used for municipal water.
4) How can a DNA Barcoding capability and proof of concept enhance citizen science monitoring programs?
5) How can a DNA Barcoding capability enhance monitoring of threatened and endangered species, as well as invasive species?

Procedures:
1) Further validite the method of DNA Barcoding with Chironomidae using Bland Altman statistical analysis.
2) A statistical sampling plan will be designed.
3) Selection of a local waterway that feeds into a municipal drinking water source to test.
4) Water Quality Chemical Analysis: Water quality chemical analysis certifications relevant to this project are up to date. Chemical sampling will be performed with LaMotte water test kit and procedure. Nitrates, orthophosphates, dissolved oxygen, pH,
and turbidity will be monitored.
5) Benthic Macroinvertebrate Sampling: Water quality biological sampling and taxonomic identification certifications relevant to this project are up to date. Sampling will be performed per NJDEP procedure. Freshwater macroinvertebrate samples will be collected with D-frame net. The percentage of net jabs taken in each habitat type will correspond to the percentage of each habitat type’s presence in the stream reach. The sample will be stored in ethanol. Macroinvertebrates from the Chironomidae family (order Diptera) will be identified under a microscope and removed for DNA Barcode
analysis.
In order to not be contaminated with anything local, samples will be collected in the Arctic Circle for the microbiology lab proof of concept study.
6) DNA Isolation Procedure: The membrane-bound organelles such as the nucleus and mitochondria will be dissolved with lysis solution. A sterile plastic pestle will be used to liquify the macroinvertebrate sample in a 1.5ml tube. Silica resin will be used to bind the DNA. The nucleic acids will be eluted from the silica resin with laboratory grade distilled water. Samples will be stored at -20 C prior to PCR amplification.
7) Polymerase Chain Reaction (PCR) Amplification: Primers will be selected based on sample type. Different methods of PCR amplification will be tested:
a) Ready-To-Go PCR Beads will be activated by adding a mix of loading dye and COI primers LCO1490 and HC02198. After bead dissolves, DNA sample will be added with micropipette. The PCR tubes will then be mixed by lightly flicking, and centrifuged for 30 seconds at 13,400 RPM to spin the liquid to the bottom of the tube. Samples will be thermal cycled with the appropriate temperature profile programmed.
b) NEB Taq 2X Master Mix: 10μL of loading dye per sample will be mixed with 12.5μL of NEB Taq 2X Master Mix per sample, combined in a 1.5ml tube, and
shaken gently for mixing. 2μL of sample DNA will then be added with micropipette to the correspondly labeled PCR tubes. 23μL of the LCO1490 and
HC02198 primer mix will be added to each PCR tube. The PCR tubes will then be mixed by lightly flicking, and centrifuged for 30 seconds at 13,400 RPM to spin the liquid to the bottom of the tube. Samples will then be thermal cycled with the appropriate temperature profile programmed.
8) Gel Electrophoresis: Agarose gel will be poured. When it is solid it will be placed into the electrophoresis chamber. Tris/Borate/EDTA (TBE) buffer will be added. PCR samples will be loaded, the gel will be run at 130V and the image will be captured. Images for samples prepared with PCR Beads and with Master Mix will be used to verify DNA amplification.
9) Samples will then be sent for DNA Sequencing. When the sequences are returned they will be trimmed, analyzed, and compared to multiple genetic sequence databases.
10) Perform phylogenetic tree analysis.
11) GIS software will be used for visualization and analysis.

Risk and Safety:
These procedures involve use of ethanol, a Lamotte Water Quality test kit, and microliter amounts of DNA isolation, PCR amplification, and gel electrophoresis reagents. MSDS sheets were reviewed. Personal protective equipment will be used to protect against risk of chemical
exposure. Adult sponsor will supervise use of chemicals. Waste liquid will be collected and given to Clean Harbors, a company specializing in hazardous waste disposal. Training completed and up to date for all equipment and chemicals used in laboratory and field.

BIBLIOGRAPHY
Bolton , Michael J. Ohio EPA Supplemental Keys to the Larval Chironomidae (Diptera) of Ohio and Ohio Chironomidae Checklist. Ohio Environmental Protection Agency Division of Surface Water, Nov. 2012.

“Cape Town: What It's Like to Live Through Water Crisis.” Time, Time, time.com/cape-town-south-africa-water-crisis/.

Cañedo-Argüelles, Miguel, et al. “Are Chironomidae (Diptera) Good Indicators of Water Scarcity? Dryland Streams as a Case Study.” Ecological Indicators, vol. 71, 2016, pp. 155–162., doi:10.1016/j.ecolind.2016.07.002.

Carew, M. E., Pettigrove, V. J., Metzeling, L., & Hoffmann, A. A. (2013). Environmental monitoring using next generation sequencing: rapid identification of macroinvertebrate bioindicator species. Frontiers in Zoology, 10(1), 45. doi:10.1186/1742-9994-10-45

Ekrem, Torbjørn, et al. “Females Do Count: Documenting Chironomidae (Diptera) Species Diversity Using DNA Barcoding.” Organisms Diversity & Evolution, vol. 10, no. 5, 2010, pp. 397–408., doi:10.1007/s13127-010-0034-y.

Ekrem, Torbjørn, et al. “DNA Barcode Data Reveal Biogeographic Trends in Arctic Non-Biting Midges.” Genome, vol. 61, no. 11, 2018, pp. 787–796., doi:10.1139/gen-2018-0100. Folmer, Ole & Black, Michael & Wr, Hoeh & Lutz, R & Vrijenhoek, Robert. (1994). DNA primers for amplification of mitochondrial Cytochrome C oxidase subunit I from diverse metazoan invertebrates. Molecular marine biology and biotechnology. 3. 294-9.

Hilsenhoff, William L. (1977), Use of Arthropods to Evaluate Water Quality of Streams. Wisconsin Department of Natural Resources, Technical Bulletin No. 100, Madison, WI

Hilsenhoff, William L. (1982), Using a Biotic Index to Evaluate Water Quality in Streams.

Wisconsin Department of Natural Resources, Technical Bulletin No. 132, Madison, WI Lin, Xiao-Long, et al. “DNA Barcodes and Morphology Reveal Unrecognized Species in Chironomidae (Diptera).” Insect Systematics & Evolution, vol. 49, no. 4, 2018, pp. 329–398.,
doi:10.1163/1876312x-00002172.

MacCafferty, W. P. (1981). Aquatic Entomology. Sudbury, MA: Jones and Bartlett.

Merritt, R. W. (1984). An introduction to the aquatic insects of North America. Dubuque, IA: Kendall Hunt.

Micklos, D. A. (2003). DNA SCIENCE: A First Course. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Micklos, D. A., Nash, B., Hilgert, U. (2013). GENOME SCIENCE: A Practical and Conceptual Introduction to Molecular Genetic Analysis in Eukaryotes. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Michaluk, S. (2019) “A Novel Method of Monitoring the Health of Our Global Fresh Water Supply Using DNA Barcoding of Chironomidae (Diptera).” Mercer Science and Engineering Fair

Monaghan, Kieran A. “Four Reasons to Question the Accuracy of a Biotic Index; the Risk of Metric Bias and the Scope to Improve Accuracy.” PLOS ONE, Public Library of Science, journals.plos.org/plosone/article?id=10.1371/journal.pone.0158383.

NJ Division of Water Monitoring and Standards. From http://www.nj.gov/dep/wms/Sæther, O A. Phylogeny of the Subfamilies of Chironomidae (Diptera). University of Bergen, 25 Dec. 2001.

“Scarcity.” UN-Water, www.unwater.org/water-facts/scarcity/.

Silva, Fabio & Ekrem, Torbjorn & Fonseca-Gessner, Alaide. (2013). DNA barcodes for species delimitation in Chironomidae (Diptera): A case study on the genus Labrundinia. The Canadian Entomologist. 145. 10.4039/tce.2013.44.

Singh, P., Rawal, D. Molecular phylogeny of Einfeldia (Diptera: Chironomidae) inferred from sequencing of mitochondrial cytochrome oxidase subunit 1 (COX1) gene, International Journal of Entomology Research, Volume 1; Issue 6; September 2016; Page No. 11-15

Stein, E. D., White, B. P., Mazor, R. D., Jackson, J. K., Battle, J. M., Miller, P. E.,. Sweeney, B. W. (2014). Does DNA barcoding improve performance of traditional stream bioassessment metrics? Freshwater Science, 33(1), 302-311.doi:10.1086/674782

Taxa Tolerance Values, lakes.chebucto.org/ZOOBENTH/BENTHOS/tolerance.html#Chironomidae.

Pilgrim, E. M., Jackson, S. A., Swenson, S., Turcsanyi, I., Friedman, E., Weigt, L., & Bagley, M.J. (2011). Incorporation of DNA barcoding into a large-scale biomonitoring program: opportunities and pitfalls. Journal of the North American Benthological Society, 30(1), 217-231.
doi:10.1899/10-012.1

“Volunteer Stream Monitoring: A Methods Manual.” United States Environmental Protection Agency, https://nepis.epa.gov/Exe/ZyPDF.cgi/P100MRC3.PDF?Dockey=P100MRC3.PDF

Walton, Brett, and Brett Walton. “Cape Town's Water Panic Was Years in the Making.” CityLab, 17 July 2018, www.citylab.com/environment/2018/07/how-cape-town-got-to-the-brink-of-water-catastrophe/564800/.

“Water Scarcity.” WWF, World Wildlife Fund, www.worldwildlife.org/threats/water-scarcity.

“Water Treatment | Public Water Systems | Drinking Water | Healthy Water | CDC.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, www.cdc.gov/healthywater/drinking/public/water_treatment.html.