Plan 9: Research Barnegat Bay— Benthic Invertebrate Community Monitoring & Year 2 Indicator Development for the Barnegat Bay-Little Egg Harbor Estuary - Barnegat Bay Diatom Nutrient Inference Model Assessment of Hard Clams as Indicators of Suspended Particulates in Barnegat Bay Stinging Sea Nettles (Jellyfishes) in Barnegat Bay Assessment of Fishes & Crabs Responses to Human Alteration of Barnegat Bay Baseline Characterization of Phytoplankton and Harmful Algal Blooms Baseline Characterization of Dr. Paul Bologna, Montclair University Zooplankton in Barnegat Bay Project Manager: Joe Bilinski, Division of Science, Research and Environmental Health Multi-Trophic Level Modeling of Barnegat Bay Thomas Belton, Barnegat Bay Research Coordinator Dr. Gary Buchanan, Director—Office of Science Tidal Freshwater & Salt Marsh Wetland Bob Martin, Commissioner, NJDEP Studies of Changing Chris Christie, Governor Ecological Function & Adaptation Strategies Ecological Evaluation of Sedge Island Marine Conservation Zone June, 2015 Impacts of Invasive Sea Nettles (Chrysaora quinquecirrha) and Ctenophores on Planktonic Community Structure and Bloom Prediction of Sea Nettles Using Molecular Techniques. Final Report 2013 Submitted to New Jersey Department of Environmental Protection by Paul Bologna, John Gaynor, Robert Meredith Department of Biology and Molecular Biology Montclair State University Research Support, Data Entry, QAQC Procedures, and Presentation Support by Christie Castellano, Dena Restaino, Idali Rios, Kevin Olsen 1 List of Abbreviations BAY-WIDE SITE ABBREVIATION Metedeconk W MW Metedeconk E ME Silver Bay W SBW Silver Bay E SBE Toms River W TRW Toms River E TRE Forked River W FRW Forked River E FRE Double Creek W DCW Double Creek E DCE Harvey Cedars W HCW Harvey Cedars E HCE Westeconk W WW Westeconk E WE Tuckerton W TW Tuckerton E TE LAGOON SITES ABBREVIATIONS Point Pleasant Lagoon PPL Kettle Creek Lagoon KCL Chadwick Isle Lagoon CIL Toms River Lagoon TRL Cedar Creek Lagoon CCL Harvey Cedars Lagoon HCL Beach Haven Lagoon BHL Tuckerton Lagoon TL List of Appendices Appendix A: CTAB/NaCl DNA Extraction Protocol Appendix B: QAPP as electronic Attachment Appendix C: Summarized Data Files, as electronic Attachment Appendix D: Blast Search Summary 2 Statement of the Problem: Gelatinous zooplankton are increasing in marine ecosystems worldwide as a result of climate change, species introductions, and a number of anthropogenic alterations to coastal food webs that favor jellyfish and ctenophores (Sullivan et al., 2001; Purcell and Decker, 2005; Hay, 2006; Kirby and Beaugrand, 2009; Kirby et al., 2009; Richardson et al., 2009). One important driver of the shift towards greater abundance of gelatinous zooplankton is the construction of hard surfaces such as bulkheads, docks, and other shoreline modifications that provide suitable habitat for scyphozoan polyps (Hoover and Purcell 2009). Another anthropogenic action that favors gelatinous zooplankton is the increase in eutrophication resulting from coastal nutrient loading, which fuels bottom hypoxia in relatively shallow systems. Jellyfish are highly tolerant of low dissolved oxygen conditions and therefore benefit from the impacts of hypoxia on their prey species which are either easier to catch in hypoxic waters or are more concentrated in the overlying normoxic waters. In either situation, jellyfish experience elevated energy intake and reproductive capacity, which ultimately contributes to population growth (Purcell et al., 2001; Grove and Breitburg, 2005; Purcell et al., 2007). Both of these drivers of gelatinous zooplankton increases are prevalent in the Barnegat Bay system. Scope of Project During this project we are evaluating the following Objectives: 1. Assess early stage Chrysaora ephyrae and planktonic larvae through molecular identification of species specific DNA in water samples. 2. Assess the distribution of gelatinous zooplankton and impacts on planktonic community structure. 3. Assess the distribution and density of settling Chrysaora polyps and development of resting podocysts. 4. Assess the diet of Chrysaora through dissection and molecular analysis. Project Methods Objective 1: Assess early stage Chrysaora ephyrae and planktonic larvae through molecular identification of species specific DNA in water samples. Our methods for this objective include sampling triplicate 20 liter water samples from sites in Barnegat Bay. Samples are field sieved through 500 µm and 100 µm filters. Samples are then identified using molecular PCR reactions to identify Chrysaora DNA. This sampling approach allows us to assess early ephyra (500 µm) and larvae and eggs (100 µm). In 2013, we collected 312 water samples (20 liters each) for DNA analysis which were split into 500 µm and 100 µm fractions using stacked filtering units. Of these 312 water samples, 240 were from bay- wide collections and 72 were from lagoonal collections. Detection and analysis of DNA occurred in the detailed methods laid out in the QAPP. Objective 2: Assess the distribution of gelatinous zooplankton and impacts on planktonic community structure. To complete Objective 2, we have divided our sampling approach into monthly bay-wide surveys, and targeted coastal development sampling (i.e., coastal lagoons developments). 3 a. Monthly assessment in Barnegat Bay We have established 16 sampling sites in Barnegat Bay which we sampled monthly between May and September 2013 (Fig. 1). This provides essential data regarding the spatial and temporal distribution throughout Barnegat Bay. A combined sampling program of lift nets and plankton tows were used to assess the distribution of adult and juvenile sea nettles, as well as other zooplankton comprising the pelagic community. These data provide the broad based distribution of organisms and were used to identify potential relationships among various zooplankton groups (e.g., food web interactions: sea nettles vs. comb jellies, copepods, fish larvae, etc…). b. Targeted coastal development sampling Sea nettle polyps are known to settle on various hard substrates, and increasing coastal development has increased the amount of available surface for settling larvae. As these polyps are the ultimate source of new adults, we sampled developed regions in Barnegat Bay to gain an understanding of the distribution within these communities. Eight sites were sampled in June, July, and August using lift nets and plankton nets to assess adult and juvenile sea nettles (Fig. 1). Water samples (Objective 1) were also collected at these locations to assess ephyra and competent larvae, which are generated and/or preparing to settle in these lagoonal communities. Our eight sampling regions from north to south included Point Pleasant, Kettle Creek, Chadwick Island, Toms River, Cedar Creek, Harvey Cedars, Beach Haven West, and Tuckerton Creek (Fig. 1). These sites cover all of Barnegat Bay and provide information about the presence of Chrysaora within these communities and potential export of ephyrae into Barnegat Bay. Objective 3: Assess the distribution and density of settling Chrysaora polyps and development of resting podocysts. As part of the Targeted Coastal Development sampling, we deployed small settling plates within these coastal developments to assess the settlement distribution patterns. Plates will be deployed in June and July and retrieved during the following sampling period (July and August) in these same communities. Since this is the likely source of sea nettle populations, this will allow us to identify and quantify polyp settlement and distribution. Additionally, a short-term broad settlement plate experiment was carried out to get a larger spatial distribution during the summer. Objective 4. Assess the diet of Chrysaora through dissection and molecular analysis. 4a. Field-Based Diet Assessment Juvenile and adult sea nettles were collected within Barnegat Bay including near shore and open water. Individuals were collected in June, July, and August to provide a temporal assessment of diet and samples were collected from several regions within the bay to provide spatial assessment of potential diet. Field collected individuals were measured and immediately preserved in ethanol to stop digestion and allow the oral cavity and oral arms to be investigated for prey items. All food items present were identified to lowest possible taxonomic level and enumerated. Prey selection from samples was then compared to available prey collected in the plankton tows to assess whether selection was occurring or whether feeding was indiscriminant. This was done in relation to month of collection and location in the Bay. Some of the dissected organisms were used for DNA sequencing, using PCR to amplify and detect the 16S rDNA gene 4 of Mnemiopsis leidyi in Chrysaora quinquecirrha guts. A sub-sample of dissected prey taxa was isolated and subsequently analyzed using the Next Generation Sequencing Protocol described below in the Metagenomic Analysis. 4b. Molecular Analysis of the Gut Contents of Chrysaora Often, gut contents of organisms can be substantially degraded due to digestion and limits the assessment of their impacts on prey species, because identification is relatively impossible using tradition techniques. Through molecular techniques, we can collect gastrovascular cavity contents via syringe and assay them against known DNA markers for specific organisms. In particular, the potential declines associated with hard clams and blue crabs may be due in part to the rise and dominance of these pelagic