NORTH PACIFIC RESEARCH BOARD SEMIANNUAL PROGRESS REPORT

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1 1. PROJECT INFORMATION NPRB Project Number: 1303 Title: Assessing benthic meiofaunal community structure in the Alaskan Arctic: A high-throughput DNA sequencing approach Subaward period July 1, 2013 Jun 30, 2016 Amount of funding $255,400 Report period Jan 1 June 30, 2014 Report submission date 7 July 2014 Lead Author of Report* Sarah Hardy *Although there may be only one lead author of the report, all PIs and co-pis of the project, as identified in the approved statement of work and listed below, are responsible for the content of the Semiannual Progress report in terms of completeness and accuracy. Principal Investigator(s), Co-Principal Investigators and Recipient Organization(s): PI Sarah Hardy, University of Alaska, Fairbanks ([email protected]) Co-PI Holly Bik, University of California, Davis ([email protected]) Co-PI Arny Blanchard, University of Alaska, Fairbanks ([email protected]) 2. PROJECT OVERVIEW a. Briefly (4-5 sentences) describe both the research purpose and the underlying need for this research. Rapid change is occurring in the Arctic marine environment concurrently with increased human activity, particularly in the form of petroleum resource exploration and development activities. We cannot adequately assess the effects of disturbance impacts from these activities on marine communities without baseline data on the abundance, distribution, and structure of key faunal assemblages. We are conducting the first such surveys for benthic meiofaunal invertebrates in and around the Chukchi and Beaufort lease sale areas using rapid, cost-effective high-throughput DNA sequencing approaches. Despite their importance to monitoring efforts, sediment meiofaunal communities are notoriously difficult to characterize because they lack obvious morphological features used to identify species, and are timeconsuming to work with due to their extremely small size (< 1 mm). Our DNA-based approach provides an alternative to the standard labor-intensive microscopy techniques for assessing community structure and diversity of meiofaunal communities in Arctic sediments. July

2 b. State your hypothesis(es). H 1 : Metagenomic analysis provides a more accurate assessment of meiofaunal community structure than traditional morphological approaches to species identification. H 2 : Environmental characteristics such as grain size and organic matter content govern meiofaunal community structure in the Chukchi and Beaufort Seas. c. List the objective(s) of the research project, exactly as described in your approved Statement of Work. 1. Generate 96 high-quality preparations of environmental DNA from sediment samples collected in the Northeast Chukchi Sea lease sale area, and along transects from 50 to 1000 m depth in both the Alaskan and Canadian portions of the Beaufort Sea shelf and slope. 2. Generate morphological identifications and associated reference gene sequences (full-length 18S ribosomal RNA gene sequences) for a subset of specimens representing the most abundant meiofaunal taxa (with an emphasis on nematodes). 3. Provide baseline surveys of taxonomic diversity, community structure, and relative species abundances for meiofaunal assemblages at our study locations in the Beaufort and Chukchi Seas for use in future monitoring efforts. 4. Compare information on assemblage structure derived from shotgun metagenomic data, environmental 18S rrna amplicon sequencing, and morphological taxonomic approaches to determine whether methods yield similar inferences regarding species richness and taxonomic composition. 5. Evaluate potential environmental drivers of meiofaunal community structure (e.g., sediment organic content, grain size, water mass characteristics). d. Provide a table showing the timeline and milestones for the entire project Tasks J A S O N D J F M A M J Environmental DNA isolation, PCR Microscopy, preserved samples Generation of 18S sequences Progress report Tasks J A 2014 S O N D J F 2015 M A M J Data assembly and analysis Additional sequencing work Manuscript preparation AMSS presentation Progress report Tasks J A S O N D J F M A M J Data QC and submission Manuscript preparation Outreach activities AMSS presentation Final report July

3 3. PROGRESS SUMMARY a. Describe report period progress. Graduate student Alexis Walker began work on the project in Jan 2014, and completed her first semester in the MS program with As in all classes. Alexis has also identified two prospective committee members and begun drafting her thesis proposal. Major progress was made in generation of environmental DNA preparations and sequencing. A total of 60 high-quality preparations of environmental DNA were generated and sequenced (parallel DNA extractions representing raw sediment and meiofauna fractions from 30 sites in both the Chukchi and Beaufort Seas). The resulting sequence datasets were subsequently generated using the Illumina MiSeq and HiSeq platforms: 18S rrna amplicons from all 30 sites, 16S rrna amplicons from all 30 sites, and shotgun metagenomes from a subset of 15 sites (representing both raw sediment and extracted meiofauna fractions). This effort involved sieving bulk sediment samples to concentrate meiofaunal organisms, DNA extraction, amplification (for 18S samples), and submission for sequencing. Data has now been received for all sequencing runs: two Illumina MiSeq runs (18S and 16S rrna amplicons) and one Illumina HiSeq run (metagenome dataset). Raw sequence data has been downloaded and checked for quality. We are now conducting preliminary data analysis in the QIIME pipeline ( beginning with OTU clustering, taxonomic assignments for environmental sequences, and alpha/beta diversity analyses to compare species assemblages between sites. Additional samples will be collected in August 2014, and processing and sequencing of those samples is planned for spring (Objective 1, 3, 4, and 5) Formalin-preserved samples (in addition to those processed during the last reporting period) from the Chukchi and Beaufort Seas were processed and sorted to examine morphology-based community structure. Samples were submitted to a nematode taxonomist (Jo Sharma) for morphological identifications. (Objectives 3 and 4). Additional environmental data (pigment concentrations, lipid content) for Beaufort Sea samples was also generated during this reporting period as part of a separate project (PI Hardy), and is now available for use here (Objective 5). We are currently exploring methods for generating the 18S reference sequences for vouchered meiofaunal specimens, but have not begun this work yet. We are working to identify suitable material that can be used for this work, and how best to coordinate with taxonomic experts to provide good morphological identifications for reference specimens prior to sequencing. (Objective 2) b. Describe preliminary results. Analysis of sequence data is still in the very early stages, and Co-PI Bik is working with graduate student Alexis Walker as she gains experience with computational biology tools and command line software such as QIIME. We have carried out preliminary OTU clustering and identified some taxa from the environmental 18S rrna dataset. In addition, nematodes from a subset of the Chukchi Sea samples have been identified by a taxonomic expert and will be used in analysis of community structure. c. Describe any concerns you may have about your project s progress. No problems were encountered during the reporting period. July

4 d. Poster and oral presentations at scientific conferences or seminars A poster was presented at the Alaska Marine Science Symposium in Jan 2014: SM Hardy, HM Bik, AM Walker, Assessing benthic meiofaunal community structure in the Alaskan Arctic: A high-throughput DNA sequencing approach e. Education and outreach We contracted a curriculum developer to work with us in developing an educational activity related to the project, and substantial progress has been made on this task. We have developed a series of five activities/lessons that can be used as a unit or, with the exception of the final assessment activity, can be used independently. The activities are written for multiple levels beginning with middle school (grades 7-8). Additional materials and lesson planning are included to provide a more detailed understanding of the concepts, making the unit useful both for high-level learners in middle school, and general level learners in high school biology (grades 9-10). This format provides science teachers in grades 7-10 the ability to engage in differentiated instruction within their classroom to meet the individual needs of their students. The overall goals for all students in grades 7-10 are to understand and be able to explain: The importance of soil and sediment microbes and the role they play in ecosystems What DNA is, and how it is being used to identify species The importance of gathering identification/baseline data regarding all species in an ecosystem in order to understand ecosystem change The lesson activity sequence is as follows: 1. Students collect their own sediment or soil locally, look for and try to identify the main groups of macro and microscopic organisms present. (Guiding question: What are microbes?) 2. Students attempt to identify the flaw in a marine food web that does not contain decomposers. (Guiding question: Why are microbes important?) 3. Students determine that DNA can be used to identify species, and extract DNA that is visible to the naked eye from an onion. (Guiding question: What is DNA?) 4. Students will learn basic methods of DNA sequencing, and will use an online database to identify an unknown organisms based on its DNA sequences. (Guiding question: How is DNA used to identify species?) 5. The final assessment activity, if students have engaged in the whole lesson sequence, is to write the introduction to a project proposal from the perspective of a research scientist. This written assessment needs to cover what (in general) microbial research they are going to conduct, why it is important to gain information this topic, and what techniques they are going to use in their research. A middle school and a high school teacher have both agreed to test these activities during the fall 2014 semester. 4. PROGRESS STATUS We have made good progress toward our objectives during this reporting period. Analysis of all samples currently in hand is well underway, and data analysis is beginning according to schedule. We will collect July

5 additional samples in the Beaufort Sea this summer and will process them in the lab in preparation for DNA extraction and additional sequencing runs. By the end of the next reporting period (i.e., in the next 6 months), we should have all supporting environmental data in hand, and will have made significant progress analyzing the 16S/18S rrna amplicons and metagenome data currently in hand, as well as completed morphological identifications on nematodes from all samples designated for this purpose. Data analysis should be at the stage of testing H 1 (addressing Objective 4) in time for preparation of a presentation for AMSS The outreach activity should also be near completion, having been tested in two pilot classrooms this fall. We would thus be able to make final modifications and post materials online in spring July