Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Scientists and laypeople interested in the effects of nanoparticles on fish and microbiota. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During year two, this project employed a postdoctoral researcher that supervised two undergraduate research assistants that participated in conducting the exposure aspects of the experiments. These undergraduate assistants learned about fish husbandry, administering feed treatments, conducting experiments with fish, and sample collection. Students successfully completed the Institutional Animal Care and Use training requirements for participation in research investigations with fish. In addition, to participating in the conduct of the experiments, undergraduate assistants participated in processing of samples in the laboratory including extraction of DNA from samples of trout intestine. How have the results been disseminated to communities of interest?Results have not been disseminated because analyses are ongoing; however, project details and activities are listed on the project website (http://epaquatic.org/rbt-gut-nano/). What do you plan to do during the next reporting period to accomplish the goals?Having completed the actual animal experiments in 2015-2017, the goalwill be to finish laboratory analyses of samples, undertake statistical analysis of the findings and begin work on publication of our findings.

Impacts
What was accomplished under these goals? Experiments were conducted as part of NSF CBET project "Effects of ingestion of manufactured nanoparticles on endogenous microbiota and pathogen resistance in rainbow trout" in January-March 2017 to explore unanswered questions from the exposure experiments carried out in 2015 and 2016. The goal of these experiments is to measure the effects of manufactured NPs on the microbiome in the environmentally relevant, commercially significant, and scientifically important rainbow trout Oncorhynchus mykiss. Short-term dietary exposures were conducted in project year 1(2015) to determine range of nanoparticle concentrations affecting the gut microbial community. Chronic exposure experiments were then conducted in project year 2 (2016) to evaluate changes in gut microbial community, immune system function, and outcome of disease challenge with a live pathogen. In 2016, we found a high degree of variation among fish within the same treatment (i.e., differences in gut microbiota), and we believed that treatment effects could be resolved more clearly by reducing variation by repeated sampling of feces from the same individual fish. The primary research objective of year 3 (2017) was to conduct NP dietary exposure in juvenile rainbow trout and to collect sequential fecal samples from individual fish for evaluation of changes in gut microbial community over time. We exposed trout via their feed to 50 mg/kg, 500 mg/kg and 2000 mg/kg AgNP, CuNP, AgNO3 or CuSO4 for 2 weeks. The individual fish where tagged with subcutaneous injections of colored elastomers and fecal samples were taken on 4 occasions during the period of exposure. Extraction and sequencing of gut microbiome DNA and histopathological sectioning of the gut tissue have been conducted and analysis of the data is currently ongoing.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Brande-Lavridsen, N., T. Vishnivetskaya, R.J. Strange, D.E. Williams, K. Ritter-Gordy, and T. Henry. 2017. Effects of ingestion of antimicrobial nanoparticles on gut health in rainbow trout. Society of Environmental Toxicology and Chemistry Europe (SETAC Europe) 27th Annual Meeting, Brussels.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Scientists and laypeople interested in the effects ofnanoparticles on fish and microbiota. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During year two, this project employed a postdoctoral researcher that supervised three undergraduate research assistants that participated in conducting the exposure aspects of the experiments. These undergraduate assistants learned about fish husbandry, administering feed treatments, conducting experiments with fish, and sample collection. Students successfully completed the Institutional Animal Care and Use training requirements for participation in research investigations with fish. In addition, to participating in the conduct of the experiments, undergraduate assistants participated in processing of samples in the laboratory including extraction of DNA from samples of trout intestine. How have the results been disseminated to communities of interest?Results have not been disseminated because analyses are ongoing; however, project details and activities are listed onthe project website (http://epaquatic.org/rbt-gut-nano/). What do you plan to do during the next reporting period to accomplish the goals?The primary goal of the next project period is to use the results of year one experiments to direct conduct of chronic exposure of rainbow trout to selected nanoparticle treatments. The endpoints of chronic exposure will include changes in gut microbiota and host (fish) response including expression of immune-related genes, histological analyses of internal tissues, and assessment of changes in gut physiology. Chronic exposures will begin in early 2017.

Impacts
What was accomplished under these goals? The primary research objective of year 2 was to conduct chronic NP dietary exposure in juvenile rainbow trout and to collect samples for evaluation of changes in gut microbial community, immune system function, and outcome of disease challenge with live pathogen. After the short-term dietary exposures conducted in project year 1, six feed treatments (50 mg/kg feed of each Cu-NPs, Ag-NPs, C60, Ag-bulk, Cu bulk, and carbon black) were selected for chronic exposure experiments in project year 2. An eight-week chronic exposure experiment was completed that included the independent variables feed treatment (six different treatments plus control) and pathogen challenge (challenge versus not challenged). The experiment was completed including the difficult task of pathogen challenge in which we hit our target of 30% mortality of fish in pathogen challenge treatments (positive controls). Samples of fish were collected for gut microbiota assessment, gene expression analysis (intestine, liver, spleen), whole blood smear (differential cell counts), histopathology (intestine, liver, and spleen), and tissue metals concentrations (Ag, and Cu). Upon completion of the experiments processing of the various sample types has been underway. DNA was extracted from intestine samples for sequencing (~60 samples) and determination of microbial species and abundances. Samples have been submitted for sequencing following PCR amplification of target sequences. Samples for histopathology were prepared in preparation for examination, and proportional blood cell counts determined in prepared blood smear slides. Gene expression protocols are underway for measurement of immune response related genes. The eight-week chronic dietary exposure experiment was successful. No fish died due to dietary treatment; however, exposure to Y. ruckeri (bacterial pathogen of rainbow trout) cause ~30% mortality in positive control. A 30% mortality in the positive control fish was excellent because this demonstrates that our pathogen strain is sufficiently virulent and that an effective challenge was imposed on the fish fed the dietary treatments. Numerous samples have been generated that we are now processing.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Ritter-Gordy, K. 2016. Elucidating the Toxic Effects of Silver and Copper Nanoparticles on Oncorhynchus mykiss, 20th Exhibition of Undergraduate Research and Creative Achievement (EUR?CA), April 11-15, Knoxville, TN, USA. Brande-Lavridsen, N., T. Vishnivetskaya, R. Strange, D. Williams, K. Ritter-Gordy and T. Henry. 2016. Effects of ingestion of antimicrobial nanoparticles on endogenous microbiota and pathogen resistance in rainbow trout. SETAC, November 6-10, Orlando, FL, USA.

Progress 10/23/14 to 09/30/15

Outputs
Target Audience:Scientists and laypeople interested in the effects of nanoparticles on fish and microbiota. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project employed three undergraduate assistants that participated in conducting the exposure aspects of the experiments. These undergraduate assistants learned about fish husbandry, administering feed treatments, conducting experiments with fish, and sample collection. Students successfully completed the Institutional Animal Care and Use training requirements for participation in research investigations with fish. In addition, to participating in the conduct of the experiments, undergraduate assistants participated in processing of samples in the including extraction of DNA from samples of trout intestine. How have the results been disseminated to communities of interest?Results have not been disseminated because analyses are ongoing; however, project details and activities are listed on the project website (http://epaquatic.org/rbt-gut-nano/). What do you plan to do during the next reporting period to accomplish the goals?The primary goal of the next project period is to use the results of year one experiments to direct conduct of chronic exposure of rainbow trout to selected nanoparticle treatments. The endpoints of chronic exposure will include changes in gut microbiota and host (fish) response including expression of immune-related genes, histological analyses of internal tissues, and assessment of changes in gut physiology. Chronic exposures will begin in early 2016.

Impacts
What was accomplished under these goals? Fish feeds (pellets) were prepared with eight different experimental treatments that included different NPs (Cu-NPs, Ag-NPs, C60-NPs) and bulk controls (CuSO4, AgNO3, carbon black), and un-amended control feed. Each NP feed and bulk control feed was prepared at three concentrations (5, 50, and 500 mg/kg feed). Feed preparations were analyzed to determine the amount of metals present and variance among feed pellets. Three 14-day exposure experiments were conducted with juvenile rainbow trout to obtain samples for assessment of changes in gut microbiota relative to concentration of treatment (i.e., 0, 5, 50, 500 mg/kg feed) and duration of exposure (0, 1, 3, 7, 14 days). Each experiment consisted of a nanoparticle type (e.g., Cu-NPs), the bulk control (e.g., CuSO4), and un-amended control. Fish from each treatment and control were sampled from each tank on each sampling day. Each fish was measured and the intestine was sampled and preserved (-80°C) prior to DNA extraction for next generation sequencing (NGS). A total community genomic DNA (gDNA) was extracted from 100 samples and among these 77 gDNA preparations were selected for amplicon sequencing using TruSeq LT and Illumina MiSeq approach. During the initial processing of the raw sequencing data using Mothur MiSeq SOP the sequences from 16 samples gave error and were removed from further analyses. Paired sequences 150 bp x 150 bp from 61 fish gut samples were further analyzed using Mothur MiSeq SOP. Raw sequences from 61 fish were uploaded to public database. Preparation of feed pellets with metal nanoparticles and bulk controls was successful and pellets contained metals concentrations at an acceptable level relative to nominal (expected) concentrations. Assessment of the presence of NPs and distribution within pellets is on-going. There was no fish mortality related to experimental treatments (mortality <5%), which indicated that our intention of selecting sub-lethal treatment levels was successful. We were able to sample the intestine of fish and extract microbial DNA from samples in preparation for NGS.

Publications