Toward a Sustainable Fishery for Nunavummiut (TSFN): 0402917N-M AMENDED Research Report 2017 Gjoa Haven HTA and Queen’s University (Supervisor: Virginia K. Walker; M. Sc. Students: Erin Hamilton and Geraint Element; Queen’s University, Department of Biology, Kingston, ON, K7L 3N6) Assessment of Water Microbial Communities and Microplastics in the Canadian Arctic’s Lower Northwest Passage (Kitikmeot Region) Section 1: Collection of Surface Water for Fish Microbiome Study Background Information Both fish slime on the scales as well as the intestines is made up of cells associated with the fish immune system as well as the microbiota or beneficial microorganisms. The microorganisms including symbiotic bacteria contribute to the health of the fish and by extension, the health of fish stocks. It is thought that the microbiomes contribute to fish well-being either through competing with harmful bacteria and therefore excluding them, or through a more complex interaction with the host immune defense response. From an academic view, it is important to characterize the microbiomes of fish to infer the functions these microbes serve to increase understanding of fish immunity, but from a practical view, knowledge of the fish microbiomes and of the waters they live in, will provide insight into the impact of climate change on fish populations that are an important food source to communities in the Northwest Passage. Accompanied by community members from Gjoa Haven, a research team sampled fish and water from fresh and saltwater sites around King William Island, Nunavut (with separate fish sampling permits and animal care permits for this aspect of the work; see application material). Methods (a) Water Sampling: Fish caught by community members in commercial, subsistence and multimesh nets were quickly processed as has been described under other permit reports. Personnel designated for dissection (including trained community members) maintained aseptic technique by spraying down their gloves and all dissection equipment with 70% ethanol between samples (i.e mucosal skin scrape and intestinal samples). Slime samples were obtained by scraping the skin mucous from the left side above the lateral line of the fish (both Arctic char and whitefish, since these were of most interest to community members). Intestinal microbiomes were obtained by dissecting a portion of the intestine, just below the stomach. At each fish sampling site, field personnel filtered 3-4 L of water through nitrocellulose filters (0.22µm) in triplicate, and collected 50 mL of water from the photic zone, and then stored these samples in 50 mL falcon tubes at -20 °C for transport. Once returned to Queen’s University, the water sample filters were stored at -80 °C until microbial analysis by project collaborator Charles Greer (NRC). The small water samples (50 mL) were stored for future Chlorophyll a, bacterial abundance, Particulate Organic Carbon (POC), Dissolved Organic Carbon (DOC), Dissolved Nitrogen (DN, 2- 3- NO3 , NO2), Dissolved Phosphorus (DP, PO4 ), and salinity. (b) On-going Sequence Analysis of Microbiomes In addition to the analysis of water samples, next generation sequencing analysis for both intestinal and skin-associated microbiomes of Arctic char and whitefish are currently being processed. Up to 50 samples obtained from Arctic char and whitefish from a select number of sites within the Lower Northwest Passage, as identified by elders and fishers as of interest for subsistence or possible future commercial harvesting, will be compared. These sequences will then be compared to the water microbial analysis (see above) and allow 1 comparison between the internal and external microbial communities inherent to the fish as well as to their environment, and give insight to the overall health of the fish. Section 2: Collection of Microplastics for Contaminant Research Background Information Microplastics are part of human marine litter. The plastics are small, ranging in size between 5 mm – 0.3 mm and they are of concern since water organisms confuse them with food, eat them and then feel full. Microplastics have spread throughout the oceans of the world because of poor waste management and recycling practices. Some of these plastics float and are found in the top 0-20 m of the water column, called the photic zone. As indicated, they can influence the food chain because they are similar in size to nutritious phytoplankton, and in turn, zooplankton are likely to ingest microplastics, providing a pathway for other pollutants adsorbed to the plastics surface to bioaccumulate and biomagnify through the food-chain. This is similar to the manner that other toxins bioaccumulate in predators. It is important to assess the abundance of microplastics within the lower Northwest Passage to assist in determining the impacts of increased shipping traffic, exploration, and tourism in the Arctic. Since the human population of the Arctic is low, it is expected that microplastic pollution may also be low, but this depends on the additional effect of ocean currents. To assist in determining the origin of any microplastics found in the Canadian Arctic within this study, we are also assessing the microbial diversity of both the microplastics and the water column in which they are sampled. Biofilms can form and adhere to the surface of plastics, and it may be possible to determine whether the plastics have a different microbial population in comparison to the waters in which they are found. Methods Aboard the R/V Martin Bergmann (Arctic Research Foundation) samples were collected from Cambridge Bay, Western and Eastern Queen Maud Gulf, Simpson Strait, Gjoa Haven Bay, Rasmussen Basin, and Chantrey Inlet within the Kitikmeot region of Nunavut. Reduced water sampling consisted of a ~30 minute net tow, in which the cod-end of the net was retrieved and debris ranging between 0.3 mm-5.6 mm was collected and subsequently frozen at -20 °C. For microbial comparison, surface samples were collected using the ship's underway pump and 500-750 mL was then filtered through 45 mm 0.22 µm pore-sized Millipore GV filters, which were then frozen at -20 °C short-term, and at -80 °C long-term. In addition, triplicate bulk water samples were obtained from a 4L Niskin bottle consisting of 1 L each and taken at a depth of 10 m or 20 m, depending on bottom depth. In addition, water samples were collected similar to that described in Section 1, and the filters were transported to Queen's University for storage prior to processing by Dr. Charles Greer (NRC). All microplastic samples are to be analyzed at Queen’s University. Future Work In January 2018, microplastic samples will be counted and analyzed for microbial abundance. Any net tow and/or bulk microplastic will have their accompanying filtered water samples sent for sequencing to compare the microbial diversity present in each. Microplastics may act as vectors for pathogens in the marine environment, and this will be determined after sequencing. 2 Appendix Figures: Fig. 2. 1. A picture of the 5.6 mm sieve, also with the 0.3 mm sieve below with some debris caught on it. While processing cod-end samples from net tows, debris would sometimes get caught on the 5.6mm top sieve, and was discarded. Pictured above, a zooplankton animal was caught on the sieve. This zooplankton appears to have blue inclusions (see arrows) inside since the organism was rinsed thoroughly with filtered sea-water to remove anything sticking to it before this photo was taken, so it is possible that what is left has been ingested by the organism. 3 Table summaries for collection sites and each method used in 2017: Table summaries for water collection sites in 2017: Table 1.1: Summary of water collection sites for microbial analysis under TSFN fishing project (2017). Location Name (Site) GPS Coordinates Port Perry (TSFN 1) 69°33’ 28.76”N, 97°26’ 13.89”W Murchison River 1 (TSFN 8) 68° 34’ 1.20”N, 93° 22’ 37.40”W Murchison River 2 (TSFN 14) 68°25’ 35.50”N, 93°19’ 11.60”W Lady Franklin Lake (TSFN 15) 67°19’ 5.34”N, 96°46’ 11.04”W Back River 1 (TSFN 13) 66°57’ 30.70”N, 95°18’5.20”W Back River 2 (TSFN 18) 67° 9’ 17.35”N, 95° 21’ 21.41”W Table 2.1: Microplastic sampling information from the Side Net Tow procedure (2017). Location Name GPS Tow In GPS Tow Out Flowmeter Flowmeter Flowmeter Total In Out Difference time in water (min) Cambridge Bay 69°6’49.5”N, 69°6’49.5”N, 168240 187806 19566 18:05 (B1) 105°3’33.7”W 105°3’33.7”W Queen Maud 68°12’46.5”N, 68°13’38.45”N, 187830 229684 41854 30:18 West (Ellice River 103°57’51.1”W 103°57’51.135”W 1) Queen Maud East 68°10’44.5”N, 68°10’49.7”N, 229688 272100 42412 29:30 (QME) 100°17’14.5”W 100°14’57.1”W Simpson Strait 68°32’26.5”N, 68°32’22.9”N, 298072 363705 65633 31:48 (SS3) 97°27’43.7”W 97°25’52.7”W Gjoa Haven Bay 68°36’22.3”N, 68°37’41.2”N, 363734 416475 52741 28:52 95°53’29.7”W 95°52’49.1”W Chantrey Inlet 67°44’48.7”N, 67°45’13.2”N, 416515 468158 51643 32:31 (CI3) 95°48’1.3”W 95°47’1.3”W Rasmussen Basin 68°21’43.8”N, 68°22’44.4”N, 467173 513830 46657 31:25 (CI1) 95°13’23.6”W 95°14’2.1”W Table 2.2: Microbial surface sample locations to compliment the net tow microplastic data.