2020 MSEF Senior Division Grand Prize Winner Research Plan

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.