Marina Ermakova 1 , Lora McGuinness 2 , and Lee J. Kerkhof 2

1. Assessing top-down and bottom-up control mechanisms structuring marine microbial communities Marina Ermakova1, Lora McGuinness2, and Lee J. Kerkhof2 1 Dept. of Molecular Biology and Biochemistry 2 Institute of Marine and Coastal Sciences, Rutgers University New Brunswick, NJ Introduction This experiment focuses on the factors affecting the diversity of microbial populations in coastal marine sediments. Top-down and bottom-up mechanisms of control were studied by setting up experiments where samples contain environments with different levels of predator exclusion and food sources. Specifically, a mixture of anoxic and sterile sediment were placed in packets of 3 different mesh pore sizes (1, 100, and 250 microns) and exposed to 3 different carbon levels (ambient, 36mg agarose, and 36mg agarose amended with 3 mg tryptone). The level of tryptone was chosen to be above the ambient levels and to be enough to be significant in comparison to agarose levels. The mesh is known to cause predator exclusion from an experiment conducted the previous summer. The anoxic sediment was used because the microbial life forms living in it would die once brought into an oxic enviromnent, thus allowing other life forms to colonize it. Results Terminal restriction fragments (bp) T0 No Addition T1 1 µ mesh, agarose plug 1 µ mesh, agarose plug T2 Fluorescence T3 1 µ mesh, agarose plug Sorensen's cluster with alternating colors indicating groupings 1 µ mesh, agarose plug T4 Experimental Design: T0 No Addition 100 µ mesh, no plug T1 T2 100 µ mesh, no plug 100 µ mesh, no plug T3 Bray-Curtis cluster with alternating colors indicating groupings Conclusion 100 µ mesh, no plug T4 The clusters appear to be structured by temporal effects rather than predator exclusion or carbon sources. There are no apparent clusters based on mesh size. There are some sub-clusters that group carbon sources together. Additionally, the clusters seem to be more coherent for the later time points. Temporal effects seem to be more significant to microbial diversity than either addition of carbon sources or predator exclusion for the time frame in this study. The clusters also imply that carbon sources are more significant than predator exclusion. T0 No Addition T1 250 µ mesh, tryptone plug Future Direction T2 250 µ mesh, tryptone plug Follow up experiments may have a longer timeline and more mesh sizes to test these variables more in depth. Additionally, a different slow release matrix from agarose should be used. This matrix should diffuse a carbon source over the course of weeks rather than days and should not be a carbon source itself. Left: The diffusion of red food dye out of 6% agarose plugs when enclosed in sediment was teated. The picture shows plugs removed from water at different time points. Right: These bags of mesh containing sediment and a carbon treatment were suspended in a marine environment for 1,2,3, or 4 weeks. T3 250 µ mesh, tryptone plug Methods DNA was extracted from sediment samples of each treatment. T-RFLP analysis was used to fingerprint the microbial communities, as described in Scala and Kerkhof (1998). References DNA fingerprints of samples Lewin-Jacus, Joshua, L. McGuinness, and L. J. Kerkhof. The Effects of Grazer Exclusion on Sand Colonization by Microbial Communities. American Society of Microbiology; 2010; San Diego, California. Scala, D. J., and L. J. Kerkhof.1998. Nitrous oxide reductase (nosZ) genespecific PCR primers for detection of denitrifiers and three nosZ genes from marine sediments. FEMS Microbiol. Lett. 162: 61–68. Acknowledgements We would like to thank RUMFS for facilitating the deployment of the experiment and the RIOS program for funding.