My primary goals for this pilot study are to (1) characterize the spatial and temporal patterns in the food web structure of pitcher plant invertebrate communities through repeated sampling over the growing season, (2) pilot methods for future field experiments, and (3) collect, isolate, identify, and culture microinvertebrates for lab experiments at my home institution. The University of Michigan Biological Station has hosted research on pitcher plant inquiline communities for decades, providing a foundation for my questions about the mechanisms that maintain diversity in food webs. Goal 1: To sample food webs within a pitcher, I will stir the fluid to homogenize the distribution of invertebrates and draw small (<5 mL) aliquots of fluid as samples. I will identify and estimate the abundance of all invertebrates within subsamples using a video microscope and neural network algorithm trained to identify invertebrates by their swimming behavior (bemovi; http://bemovi.info/). I will sterilize my stirring and sampling instruments between each pitcher to prevent cross contamination. To characterize spatial turnover in food web structure, I will use a spatially nested sampling design composed of four levels (from largest to smallest): Populations, Patches, Plants, and Pitchers. I will identify 3-5 replicate units at each level of the hierarchy (e.g., 3-5 Pitchers per Plant and 3-5 Populations) and separate units by a distance according to the scale. Populations will be separated by >5 km, Patches within Populations will be separated by 50-100 m, and Plants within Patches will be separated by 1-5 m. By sampling in this way, I will be able to determine the spatial scale at which food web structure turns over. I will mark each pitcher with a small wire tag and GPS. I will determine the temporal turnover in food web structure in two ways. First, I will resample the marked pitchers from the nested sampling design described above each week from June-August. This will provide information on the dynamics of the food web over time. Second, I will use the full range of Pitcher ages on each Plant following the aging methods of Fish & Hall (1978). Here, older Pitchers have had more time for the process of community assembly to play out. This should lead to more similar food web structure and greater structural stability over time. Goal 2: I hope to be able to experimentally assemble food webs in real and artificial pitchers in the field. As a step towards this goal, I need to know whether I can control the introduction of species to pitchers. In a small number of plants (ca. 25), I will drain the fluid from pitcher leaves and sterilize the inner surface of the pitcher using an insecticidal soap following Trzcinski et al. (2003). I will rinse and refill the pitcher with microwave sterilized rain water, taking care to collect all fluid contaminated with insecticide for proper disposal. I will monitor succession in pitchers that are fully open to the environment as well as with a variety of cage/screen designs that exclude some or all invertebrates based on body size. I will test whether I can attach screens to cover pitchers using a fabric glue that has worked well on other plant species (Aleene’s Ok to Wash It; Petry et al. 2013). Similarly, I will place 50 mL centrifuge tubes filled with sterilized rainwater and dead insects near natural pitchers to monitor colonization. Goal 3: In late August, I will collect and isolate each microinvertebrate species from each Population to begin lab cultures for follow-up experiments that will measure species interaction strength and artificially assemble food webs that are predicted to exhibit a range of stabilities. I will follow standard isolation protocols (Altermatt et al. 2015) that require approximately the same small volume of pitcher fluid as one of the above described sampling events. I will bring these cultures back to Princeton, and I hope to be able to use them as stocks for establishing future field experiments at UMBS. Candidate field sites: Iverness Mud Lake Bog, Livingston Bog, Bryant’s Bog, Grass Bay, Waldron Fen, Gates Bog, Smith’s Bog, Linné Bog, Penny Lake, Dingman Marsh, Malony Lake References: Altermatt, F., Fronhofer, E. A., Garnier, A., Giometto, A., Hammes, F., Klecka, J., … Petchey, O. L. (2015). Big answers from small worlds: a user’s guide for protist microcosms as a model system in ecology and evolution. Methods in Ecology and Evolution, 6(2), 218–231. doi: 10.1111/2041-210X.12312 Fish, D., & Hall, D. W. (1978). Succession and stratification of aquatic insects inhabiting the leaves of the insectivorous pitcher plant, Sarracenia purpurea. The American Midland Naturalist, 99(1), 172–183. doi: 10.2307/2424941 Petry, W. K., Perry, K. I., Fremgen, A., Rudeen, S. K., Lopez, M., Dryburgh, J., & Mooney, K. A. (2013). Mechanisms underlying plant sexual dimorphism in multi-trophic arthropod communities. Ecology, 94(9), 2055–2065. doi: 10.1890/12-2170.1 Trzcinski, M. K., Walde, S. J., & Taylor, P. D. (2003). Colonisation of pitcher plant leaves at several spatial scales. Ecological Entomology, 28(4), 482–489. doi: 10.1046/j.1365-2311.2003.00530.x