http://lod.bco-dmo.org/id/dataset/879749 eng; USA utf8 dataset Highest level of data collection, from a common set of sensors or instrumentation, usually within the same research project Biological and Chemical Oceanography Data Management Office (BCO-DMO) Unavailable 508-289-2009 WHOI MS#36 Woods Hole MA 02543 USA info@bco-dmo.org http://www.bco-dmo.org Monday - Friday 8:00am - 5:00pm For questions regarding this resource, please contact BCO-DMO via the email address provided. pointOfContact 2022-09-07 ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data ISO 19115-2:2009(E) Seagrass metrics from from seagrass wasting disease mesocosm experiments conducted at Bodega Marine Laboratory from July-September 2015 2022-10-06 publication 2022-10-06 revision Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA) 2022-11-22 publication https://doi.org/10.26008/1912/bco-dmo.879749.1 A. Randall Hughes Northeastern University principalInvestigator Katherine DuBois University of California-Davis principalInvestigator Melissa Kardish University of California-Davis principalInvestigator Forest Schenck Northeastern University principalInvestigator John J. Stachowicz University of California-Davis principalInvestigator Biological and Chemical Oceanography Data Management Office (BCO-DMO) Unavailable 508-289-2009 WHOI MS#36 Woods Hole MA 02543 USA info@bco-dmo.org http://www.bco-dmo.org Monday - Friday 8:00am - 5:00pm For questions regarding this resource, please contact BCO-DMO via the email address provided. publisher Cite this dataset as: Schenck, F., DuBois, K., Kardish, M., Stachowicz, J. J., Hughes, A. R. (2022) Seagrass metrics from from seagrass wasting disease mesocosm experiments conducted at Bodega Marine Laboratory from July-September 2015. Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version 1) Version Date 2022-10-06 [if applicable, indicate subset used]. doi:10.26008/1912/bco-dmo.879749.1 [access date] Methods and Sampling: &lt;p&gt;&lt;em&gt;Mesocosm experiment.&lt;/em&gt;&amp;nbsp;&lt;br /&gt; We used a substitutive design to test the effects of eelgrass genotypic identity (eight genotypes), diversity (monocultures of 1 genotype vs. polycultures of 4 genotypes), and temperature (ambient or + 3.2° C) on the prevalence and intensity of the wasting disease parasite&amp;nbsp;&lt;em&gt;Labyrinthula&lt;/em&gt;&amp;nbsp;over eight weeks in an array of flow-through 120-Liter mesocosms at the Bodega Marine Laboratory in Bodega Bay, CA. In July 2015, we created ten unique polyculture combinations of four genotypes (4 genotypes per experimental pot) randomly drawn from a pool of eight genotypes; all eight genotypes were also grown in monoculture (1 genotype per pot). We filled pots (8.9 x 8.9 cm) with coarsely sieved sediment collected from Bodega Harbor, and planted 4 shoots of eelgrass per pot, matching the lower range of average field densities reported for Bodega Harbor (Ha and Williams, 2018) to allow for growth during the experiment. Plants were originally collected in Bodega Harbor, CA in 2012, confirmed to be unique genotypes using 11 DNA microsatellite loci developed specifically for&amp;nbsp;&lt;em&gt;Zostera&amp;nbsp;marina&lt;/em&gt;&amp;nbsp;(Abbott et al. 2018), and propagated in separate flow through mesocosms at Bodega Marine Lab. We previously characterized traits of each genotype relating to growth rate, morphology, nutrient content, and chemical defense in common garden experiments at ambient temperature from July 2013 to August 2014 (Abbott et al., 2018). We selected the 8 genotypes used in this experiment to encompass the range of trait values determined for this population of eelgrass measured when the common garden was experiencing marine heatwave conditions (DuBois et al., 2019).&lt;/p&gt; &lt;p&gt;&lt;span style=&quot;background-color:transparent; font-family:arial; font-size:10pt&quot;&gt;We assigned ten pots -- two unique polyculture combinations and each of the eight monocultures -- to each of ten mesocosms, with five mesocosms per temperature treatment (see DuBois et al. 2020 for a diagram of the experimental set up). All mesocosms received sand-filtered flow-through seawater at a rate of approximately 0.8-1.0 liters per minute (L &lt;/span&gt;min&lt;sup&gt;-1&lt;/sup&gt;&lt;span style=&quot;background-color:transparent; font-family:arial; font-size:10pt&quot;&gt;). We allowed the plants to acclimate for one month prior to initiating the temperature treatments. We maintained an ambient temperature treatment by cooling flow-through seawater in a head tank by approximately 1˚C using an Aqua Logic Delta Star in-line titanium chiller. Seawater in the elevated temperature treatment was raised approximately 3˚C above the ambient treatment in a separate header tank using Process Technologies titanium immersion heaters. This level of warming mimicked the 2014 and 2015 extreme warming events in the Northern Pacific, where the mass of unusually warm water called “The Blob” raised summer ocean temperatures three standard deviations above the long-term average (Sanford et al., 2019).&lt;/span&gt;&lt;/p&gt; &lt;div&gt;&lt;span style=&quot;background-color:transparent; font-family:arial; font-size:10pt&quot;&gt;At the end of the experiment (10 weeks), we estimated lesion percent cover of the third rank leaf of the terminal shoot of each transplant (i.e., focal leaf) to measure the signs of wasting disease (Burdick et al., 1993). We recorded lesions as either absent, &amp;lt;1%, &amp;lt;10%, or ≥10% cover. When lesions were ≥10% cover, we also recorded a numerical estimate of lesion percent cover.&amp;nbsp; We then collected and preserved the top half of the focal leaf in individual plastic bags sealed with 30 milliliters of silica (Flower Drying Art Silica Gel; Activa) for subsequent DNA extraction and quantitative PCR to estimate &lt;/span&gt;&lt;em&gt;Labyrinthula zosterae&lt;/em&gt;&lt;span style=&quot;background-color:transparent; font-family:arial; font-size:10pt&quot;&gt; cells as a proxy for infection (Bergmann et al., 2011; Bockelmann et al., 2013; Groner et al., 2021).&lt;/span&gt;&lt;/div&gt; &lt;p dir=&quot;ltr&quot;&gt;&lt;span style=&quot;background-color:transparent; font-family:arial; font-size:10pt&quot;&gt;At one-month intervals over the course of the experiment, we measured leaf growth rate of the terminal shoot of each transplant using the “hole-punch” method (Williams and Ruckelshaus, 1993). &lt;/span&gt;&lt;em&gt;Labyrinthula zosterae&lt;/em&gt;&lt;span style=&quot;background-color:transparent; font-family:arial; font-size:10pt&quot;&gt; infection can be affected by plant defenses (Steele et al., 2005; Trevathan-Tackett et al., 2015) and these defenses may trade off with plant growth rate, resulting in a positive relationship between growth rate and infection. Alternatively, &lt;/span&gt;&lt;em&gt;L. zosterae&lt;/em&gt;&lt;span style=&quot;background-color:transparent; font-family:arial; font-size:10pt&quot;&gt; infection can result in reduced leaf growth rate (Graham et al., 2021), leading to a negative relationship. &lt;/span&gt;&lt;em&gt;L. zosterae&lt;/em&gt;&lt;span style=&quot;background-color:transparent; font-family:arial; font-size:10pt&quot;&gt; prevalence can also be affected by plant size (Groner et al., 2016), due to greater leaf-to-leaf contact and resulting increased parasite transmission (Muehlstein, 1992), so we measured the length of the focal leaf of each transplant at the end of the experiment. &lt;/span&gt;&lt;/p&gt; &lt;p&gt;&lt;em&gt;Labyrinthula zosterae&amp;nbsp;DNA extraction and quantitative PCR Assay&lt;/em&gt;&lt;br /&gt; We extracted&amp;nbsp;&lt;em&gt;L. zosterae&lt;/em&gt;&amp;nbsp;DNA from dried leaf tissue using Omega Bio-Tek E.Z. Tissue DNA extraction kits at the Northeastern University Marine Science Center in Nahant, MA. For each sample, we separated the dried leaf tissue into 2-16 mg subsamples and homogenized the tissue in a ball mill (Retsch, Germany) at a frequency of 30 Hz for 5 min (Bockelmann et al. 2013). We lysed ground subsamples individually following the manufacturer’s instructions and added 1 microliter&amp;nbsp;of 500 ng per microliter (ng*uL&lt;sup&gt;-1&lt;/sup&gt;) salmon sperm DNA solution (Invitrogen, USA) to the first subsample of each sample immediately before recombining all subsamples in the spin columns. Salmon sperm DNA was added to enhance extraction efficiency and ensure that even low amounts of target DNA are carried through the filter absorption steps (Bockelmann et al., 2013). We eluted all DNA extractions into 100 uL. Following elution, we used Zymo OneStep-96 PCR Inhibitor Removal kits to clean 50 uL sub-samples of each DNA extraction following the manufacturers instructions. We stored cleaned DNA extractions at -20˚C prior to quantitative PCR.&lt;/p&gt; &lt;p&gt;We used a TaqMan quantitative PCR (qPCR) assay with a forward primer: TTGAACGTAACATT-CGACTTTCGT, reverse primer: ACGCATGAAGCGGTCTTCTT, and MBG probe: TGGACGAGTGTGTTTTG that carries the fluorescence label 6-Fam at the 5’ end and the dark quencher FHQ at the 3’ end (Bio-Rad, USA) developed specifically for&amp;nbsp;&lt;em&gt;L. zosterae&lt;/em&gt;&amp;nbsp;(Bockelmann et al., 2013, Bergmann et al., 2011). We made up qPCR reactions to a 10 uL reaction volume using standard conditions recommended by the manufacturer: 5 uL SsoAdvanced&lt;sup&gt;TM&lt;/sup&gt;&amp;nbsp;Universal Probes Supermix 2x (Bio-Rad, USA), 1 uL template DNA, 0.4 uL 4:1 Primer:Probe Mix (final concentrations of 400 nM forward primer, 400 nM reverse primer, 100 nM probe), and 3.6 uL Milli-Q H&lt;sub&gt;2&lt;/sub&gt;O (Thermofisher, USA). Reactions were run on a CFX96 Real-Time System (Bio-Rad, USA) using the following thermo-cycling program: 3 min at 95˚C, followed by 40 cycles of 15 sec at 95˚C and 1 min at 60˚C. We tested all samples in duplicate and if replicates differed by greater than one cycle threshold (Ct), reactions were rerun in triplicate. We only used the data from reactions in analyses when replicates fell within one Ct. Our lowest detection was 1.76 copies per reaction or 0.15 cells per extraction.&lt;/p&gt; &lt;p&gt;We ran each 96-well plate of qPCR reactions with a set of nine standards: a dilution series of gBlock Gene Fragments (Integrated DNA Technologies, USA) designed based on the highly conserved sequence of the 5.8s ribosomal RNA gene of&amp;nbsp;&lt;em&gt;L. zosterae&lt;/em&gt;&amp;nbsp;known as internal transcribed spacer 1 (ITS) targeted by the TaqMan qPCR assay; an&amp;nbsp;&lt;em&gt;L. zosterae&amp;nbsp;&lt;/em&gt;cell standard consisting of a sample of DNA extracted from a know quantity of pathogenic&amp;nbsp;&lt;em&gt;L. zosterae&amp;nbsp;&lt;/em&gt;cells; and an inhibition control consisting of a half volume of&amp;nbsp;&lt;em&gt;L. zosterae&amp;nbsp;&lt;/em&gt;cell standard and a half volume of a haphazardly selected sample. We ran a total of 31 96-well plates of qPCR reactions with a mean efficiency of 97.4% ± 4.3 and R&lt;sup&gt;2&lt;/sup&gt;&amp;nbsp;0.996 ± 0.004. To convert Ct&amp;nbsp;values to to&amp;nbsp;&lt;em&gt;L. zosterae&lt;/em&gt;&amp;nbsp;cell numbers, four equations were used. The &lt;strong&gt;equations and details are captured in the Supplemental Files section &lt;/strong&gt;of this metadata within the file titled, &quot;Equations for conversion of Ct values to &lt;em&gt;Labyrinthula zosterae&lt;/em&gt;&amp;nbsp;cell numbers.&quot;&lt;/p&gt; &lt;p&gt;We used a pure culture of the pathogenic &lt;em&gt;L. zosterae &lt;/em&gt;isolate 316b provided by D. Martin in 2015 to make our &lt;em&gt;L. zosterae&lt;/em&gt; cell standard (Martin et al., 2016; GenBank: KU559372.1). We cultured &lt;em&gt;L. zosterae &lt;/em&gt;cells on serum seawater agar media (Muehlstein et al., 1991). We scraped&lt;strong&gt; &lt;/strong&gt;cells from an actively growing edge of &lt;em&gt;L. zosterae&lt;/em&gt; culture into serum seawater liquid media (D. Martin pers. com.). We mixed the liquid media +&amp;nbsp;&lt;em&gt;L. zosterae&lt;/em&gt; cell slurry vigorously on a bench top vortex for 30 sec and aliquoted immediately into three replicate subsamples for cell counts and extraction. In order to break up cell clumps for ease of counting, we added Tween80 (Sigma-Aldrich, USA) to a final concentration of 1:100 into the two subsamples used for cell counts, and mixed for 30 sec. We counted cells of four replicate aliquots per subsample on a hemocytometer. We calculated cell concentration by averaging over all replicates. Prior to DNA extraction, we centrifuged the third replicate &lt;em&gt;L. zosterae&lt;/em&gt; cell solution at 6,000 g for 10 min and drew off the supernatant without disturbing the cell pellet. We then added a ~4 mg section of dried healthy &lt;em&gt;Zostera. marina&lt;/em&gt; tissue to the cell pellet to account for possible interference of&amp;nbsp;&lt;em&gt;Zostera&amp;nbsp;marina&amp;nbsp;&lt;/em&gt;compounds in the extraction process. To extract &lt;em&gt;L. zosterae &lt;/em&gt;DNA, we followed the DNA extraction and inhibitor removal protocols outlined above.&lt;/p&gt; &lt;p&gt;We designed the gBlock double stranded DNA fragments (Integrated DNA Technologies, USA) using published sequences of the ITS region of the &lt;em&gt;L. zosterae&lt;/em&gt; genome (GenBank: JN121409-13).&lt;/p&gt; &lt;p&gt;5’-CTGTGATCTCTGAAAATACTTGTTT&lt;br /&gt; (1)&lt;u&gt;TTGAACGTAACATTCGACTTTCGT&lt;/u&gt;CGATT TTG&lt;br /&gt; (2)&lt;u&gt;TGGACGAGTGTGTTTTGT &lt;/u&gt;AAACCTACCC&lt;br /&gt; (3)&lt;u&gt;AAGAAGACCGCTTCATGCGT &lt;/u&gt;GTCGCTGACTAATGAAACAAACAAA-3’&lt;/p&gt; &lt;p&gt;The gBlock fragment sequences were a total length of 130 bp (base pairs), which included target regions for the forward (1) and reverse (3) primers and the MGD probe (2), underlined above, as well as 25 base pairs of additional sequence on both the 5’ and 3’ ends to increase fragment stability. We diluted gBlock fragments in Milli-Q H&lt;sub&gt;2&lt;/sub&gt;O (Thermofisher, USA) to seven concentrations: 2.24e&lt;sup&gt;1&lt;/sup&gt;, 1.12e&lt;sup&gt;2&lt;/sup&gt;, 5.61e&lt;sup&gt;2&lt;/sup&gt;, 2.81e&lt;sup&gt;3&lt;/sup&gt;, 1.40e&lt;sup&gt;4&lt;/sup&gt;, 7.02e&lt;sup&gt;4&lt;/sup&gt;, 7.02e&lt;sup&gt;5&lt;/sup&gt; copies/µL and included this dilution series in each qPCR run as a standard curve (Bergmann et al., 2011). The range of the gBlock dilution curve: approx. 1-60,000 cells/extraction encompassed the range of most &lt;em&gt;L. zosterae&lt;/em&gt; values observed in our samples: 0.15-450,000 cells/extraction or 1.84e&lt;sup&gt;2&lt;/sup&gt;-5.52e&lt;sup&gt;8&lt;/sup&gt; copies/extraction.&lt;/p&gt; &lt;p&gt;&lt;span style=&quot;color:rgb(0, 0, 0); font-family:times new roman,serif; font-size:16px&quot;&gt;&lt;strong&gt;Life Sciences Identifiers (LSID) for taxonomic names:&lt;/strong&gt;&lt;/span&gt;&lt;br /&gt; Zostera marina (urn:lsid:marinespecies.org:taxname:145795)&lt;br /&gt; Labyrinthula zosterae (urn:lsid:marinespecies.org:taxname:395093)&lt;br /&gt; Labyrinthula (urn:lsid:marinespecies.org:taxname:119090)&lt;/p&gt; Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-1652320 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=1652320 completed A. Randall Hughes Northeastern University 781-581-7370 430 Nahant Rd. Nahant MA 01908 USA ann.hughes@northeastern.edu pointOfContact Katherine DuBois University of California-Davis Bowdoin College Schiller Coastal Studies Center 6775 College Station Brunswick ME 04011-8479 USA khoering@gmail.com pointOfContact Melissa Kardish University of California-Davis 707-875-1968 University of California Davis Bodega Marine Lab P.O.Box 247, 2099 Westshore Road Bodega Bay CA 94923 USA mrkardish@ucdavis.edu pointOfContact Forest Schenck Northeastern University 30 Emerson Avenue Gloucester MA 01930 USA forest.schenck@mass.gov pointOfContact John J. Stachowicz University of California-Davis 530-752-1113 Department of Evolution and Ecology 1 Shields Ave Davis CA 95616 USA jjstachowicz@ucdavis.edu pointOfContact asNeeded Dataset Version: 1 Unknown start_date end_date bin pot shoot diversity temperature genotype length growth_rate lesion lesion_group lesion_cover mass cells_per_extraction cells_per_mg cells latitude longitude flow through tanks Aqua Logic Delta Star in-line titanium chiller Retsch Mixer Mill 400 Process Technologies titanium immersion heater Bio-Rad CFX96 Real-Time System theme None, User defined date tank sample identification treatment incubation or cultivation temperature (not ambient water temp) sample type length growth flag percent coverage mass cell_concentration latitude longitude featureType BCO-DMO Standard Parameters Aquarium Aquarium chiller Homogenizer Immersion heater qPCR Thermal Cycler instrument BCO-DMO Standard Instruments otherRestrictions otherRestrictions Access Constraints: none. Use Constraints: Please follow guidelines at: http://www.bco-dmo.org/terms-use Distribution liability: Under no circumstances shall BCO-DMO be liable for any direct, incidental, special, consequential, indirect, or punitive damages that result from the use of, or the inability to use, the materials in this data submission. If you are dissatisfied with any materials in this data submission your sole and exclusive remedy is to discontinue use. CAREER: Linking genetic diversity, population density, and disease prevalence in seagrass and oyster ecosystems https://osprey.bco-dmo.org/project/709942 CAREER: Linking genetic diversity, population density, and disease prevalence in seagrass and oyster ecosystems <p><em>NSF Award Abstract:</em><br /> Disease outbreaks in the ocean are increasing, causing losses of ecologically important marine species, but the factors contributing to these outbreaks are not well understood. This 5-year CAREER project will study disease prevalence and intensity in two marine foundation species - the seagrass Zostera marina and the Eastern oyster Crassostrea virginica. More specifically, host-disease relationships will be explored to understand how genetic diversity and population density of the host species impacts disease transmission and risk. This work will pair large-scale experimental restorations and smaller-scale field experiments to examine disease-host relationships across multiple spatial scales. Comparisons of patterns and mechanisms across the two coastal systems will provide an important first step towards identifying generalities in the diversity-density-disease relationship. To enhance the broader impacts and utility of this work, the experiments will be conducted in collaboration with restoration practitioners and guided by knowledge ascertained from key stakeholder groups. The project will support the development of an early career female researcher and multiple graduate and undergraduate students. Students will be trained in state-of-the-art molecular techniques to quantify oyster and seagrass parasites. Key findings from the surveys and experimental work will be incorporated into undergraduate courses focused on Conservation Biology, Marine Biology, and Disease Ecology. Finally, students in these courses will help develop social-ecological surveys and mutual learning games to stimulate knowledge transfer with stakeholders through a series of workshops.</p> <p>The relationship between host genetic diversity and disease dynamics is complex. In some cases, known as a dilution effect, diversity reduces disease transmission and risk. However, the opposite relationship, known as the amplification effect, can also occur when diversity increases the risk of infection. Even if diversity directly reduces disease risk, simultaneous positive effects of diversity on host density could lead to amplification by increasing disease transmission between infected and uninfected individuals. Large-scale field restorations of seagrasses (Zostera marina) and oysters (Crassostrea virginica) will be utilized to test the effects of host genetic diversity on host population density and disease prevalence/intensity. Additional field experiments independently manipulating host genetic diversity and density will examine the mechanisms leading to dilution or amplification. Conducting similar manipulations in two marine foundation species - one a clonal plant and the other a non-clonal animal - will help identify commonalities in the diversity-density-disease relationship. Further, collaborations among project scientists, students, and stakeholders will enhance interdisciplinary training and help facilitate the exchange of information to improve management and restoration efforts. As part of these efforts, targeted surveys will be used to document the perceptions and attitudes of managers and restoration practitioners regarding genetic diversity and its role in ecological resilience and restoration.</p> Seagrass and Oyster Ecosystems largerWorkCitation project eng; USA biota oceans -123.06572 -123.06572 38.31753 38.31753 2015-07-01 2015-09-14 Coastal New England 0 BCO-DMO catalogue of parameters from Seagrass metrics from from seagrass wasting disease mesocosm experiments conducted at Bodega Marine Laboratory from July-September 2015 Biological and Chemical Oceanography Data Management Office (BCO-DMO) Unavailable 508-289-2009 WHOI MS#36 Woods Hole MA 02543 USA info@bco-dmo.org http://www.bco-dmo.org Monday - Friday 8:00am - 5:00pm For questions regarding this resource, please contact BCO-DMO via the email address provided. pointOfContact http://lod.bco-dmo.org/id/dataset-parameter/881989.rdf Name: start_date Units: unitless Description: <p>Start date of the mesocosm warming experiment</p> http://lod.bco-dmo.org/id/dataset-parameter/881990.rdf Name: end_date Units: unitless Description: <p>End date of the mesocosm warming experiment</p> http://lod.bco-dmo.org/id/dataset-parameter/881991.rdf Name: bin Units: unitless Description: <p>Unique identifier number assigned to each of the ten 120 L mesocosms at the Bodega Marine Lab supplied with flow-through seawater</p> http://lod.bco-dmo.org/id/dataset-parameter/881992.rdf Name: pot Units: unitless Description: <p>Unique identifier number assigned to each of the 100 pots (8.9 x 8.9 cm) planted with Zostera marina</p> http://lod.bco-dmo.org/id/dataset-parameter/881993.rdf Name: shoot Units: unitless Description: <p>Identifier number assigned to shoots from each genotype within each bin; shoots 1-4 of each genotype planted in monoculture and shoot 5 planted in polyculture</p> http://lod.bco-dmo.org/id/dataset-parameter/881994.rdf Name: diversity Units: unitless Description: <p>Descriptor of the genotypic diversity treatment applied to Zostera marina plant communities; mono = one genotype; poly = four genotypes</p> http://lod.bco-dmo.org/id/dataset-parameter/881995.rdf Name: temperature Units: unitless Description: <p>Descriptor of the temperature treatment applied to seawater where ambient = cooled flow-through seawater by approximately 1 degree Celsius; elevated = heated flow-through seawater by approximately 3 degrees Celsius</p> http://lod.bco-dmo.org/id/dataset-parameter/881996.rdf Name: genotype Units: unitless Description: <p>Descriptor of the genotype of each Zostera marina plant</p> http://lod.bco-dmo.org/id/dataset-parameter/881997.rdf Name: length Units: centimeters (cm) Description: <p>Length of the third rank leaf of the terminal shoot (i.e. focal leaf) of each Zostera marina plant</p> http://lod.bco-dmo.org/id/dataset-parameter/881998.rdf Name: growth_rate Units: centimeters per day (cm/day) Description: <p>Growth rate of the focal leaf of each Zostera marina plant</p> http://lod.bco-dmo.org/id/dataset-parameter/881999.rdf Name: lesion Units: unitless Description: <p>Binomial code for the presence of lesions characteristic of wasting disease on focal leaf of each Zostera marina plant. 1 = lesions present; 0 = no lesions present.</p> http://lod.bco-dmo.org/id/dataset-parameter/882000.rdf Name: lesion_group Units: unitless Description: <p>Categorical descriptor of the percent cover of lesion characteristic of wasting disease on the focal leaf. 0; &lt;1%; &lt;10%; or _10% cover</p> http://lod.bco-dmo.org/id/dataset-parameter/882001.rdf Name: lesion_cover Units: unitless Description: <p>Numerical descriptor of the percent cover of lesions characteristic of wasting disease on the focal leaf; for lesions &lt;10% numerical percent covers were converted from categorical estimates using the following method: 0 = 0; &lt;1% = 0.5; &lt;10% = 5; for lesions _10% cover numerical percent cover estimates were recorded during sampling</p> http://lod.bco-dmo.org/id/dataset-parameter/882002.rdf Name: mass Units: milligrams (mg) Description: <p>The mass of the tissue segment of the focal leaf of each Zostera marina plant from which Labyrinthula zosterae DNA was extracted</p> http://lod.bco-dmo.org/id/dataset-parameter/882003.rdf Name: cells_per_extraction Units: cells Description: <p>The number of Labyrinthula zosterae cells detected by quantitative-PCR from the extracted tissue segment of the focal leaf of each Zostera marina plant</p> http://lod.bco-dmo.org/id/dataset-parameter/882004.rdf Name: cells_per_mg Units: cells Description: <p>The number Labyrinthula zosterae cells detected by quantitative-PCR from the extracted tissue segment of the focal leaf of each Zostera marina plant per milligram of extracted Zostera marina tissue</p> http://lod.bco-dmo.org/id/dataset-parameter/882005.rdf Name: cells Units: unitless Description: <p>Binomial code for the presence of <em>Labyrinthula zosterae</em> cells detected by quantitative-PCR from the extracted tissue segment of the focal leaf of each <em>Zostera marina</em> plant. 1 = cells present; 0 = no cells present.</p> http://lod.bco-dmo.org/id/dataset-parameter/882006.rdf Name: latitude Units: decimal degrees Description: <p>Latitude of Bodega Marine Laboratory (Bodega Bay, CA)</p> http://lod.bco-dmo.org/id/dataset-parameter/882007.rdf Name: longitude Units: decimal degrees Description: <p>Longitude of Bodega Marine Laboratory (Bodega Bay, CA)</p> GB/NERC/BODC > British Oceanographic Data Centre, Natural Environment Research Council, United Kingdom Biological and Chemical Oceanography Data Management Office (BCO-DMO) Unavailable 508-289-2009 WHOI MS#36 Woods Hole MA 02543 USA info@bco-dmo.org http://www.bco-dmo.org Monday - Friday 8:00am - 5:00pm For questions regarding this resource, please contact BCO-DMO via the email address provided. pointOfContact 45270 https://darchive.mblwhoilibrary.org/bitstream/1912/29522/1/dataset-879749_mesocosm-warming-experiment-seagrass-metrics__v1.tsv download https://doi.org/10.26008/1912/bco-dmo.879749.1 download onLine dataset &lt;p&gt;&lt;em&gt;Mesocosm experiment.&lt;/em&gt;&amp;nbsp;&lt;br /&gt; We used a substitutive design to test the effects of eelgrass genotypic identity (eight genotypes), diversity (monocultures of 1 genotype vs. polycultures of 4 genotypes), and temperature (ambient or + 3.2° C) on the prevalence and intensity of the wasting disease parasite&amp;nbsp;&lt;em&gt;Labyrinthula&lt;/em&gt;&amp;nbsp;over eight weeks in an array of flow-through 120-Liter mesocosms at the Bodega Marine Laboratory in Bodega Bay, CA. In July 2015, we created ten unique polyculture combinations of four genotypes (4 genotypes per experimental pot) randomly drawn from a pool of eight genotypes; all eight genotypes were also grown in monoculture (1 genotype per pot). We filled pots (8.9 x 8.9 cm) with coarsely sieved sediment collected from Bodega Harbor, and planted 4 shoots of eelgrass per pot, matching the lower range of average field densities reported for Bodega Harbor (Ha and Williams, 2018) to allow for growth during the experiment. Plants were originally collected in Bodega Harbor, CA in 2012, confirmed to be unique genotypes using 11 DNA microsatellite loci developed specifically for&amp;nbsp;&lt;em&gt;Zostera&amp;nbsp;marina&lt;/em&gt;&amp;nbsp;(Abbott et al. 2018), and propagated in separate flow through mesocosms at Bodega Marine Lab. We previously characterized traits of each genotype relating to growth rate, morphology, nutrient content, and chemical defense in common garden experiments at ambient temperature from July 2013 to August 2014 (Abbott et al., 2018). We selected the 8 genotypes used in this experiment to encompass the range of trait values determined for this population of eelgrass measured when the common garden was experiencing marine heatwave conditions (DuBois et al., 2019).&lt;/p&gt; &lt;p&gt;&lt;span style=&quot;background-color:transparent; font-family:arial; font-size:10pt&quot;&gt;We assigned ten pots -- two unique polyculture combinations and each of the eight monocultures -- to each of ten mesocosms, with five mesocosms per temperature treatment (see DuBois et al. 2020 for a diagram of the experimental set up). All mesocosms received sand-filtered flow-through seawater at a rate of approximately 0.8-1.0 liters per minute (L &lt;/span&gt;min&lt;sup&gt;-1&lt;/sup&gt;&lt;span style=&quot;background-color:transparent; font-family:arial; font-size:10pt&quot;&gt;). We allowed the plants to acclimate for one month prior to initiating the temperature treatments. We maintained an ambient temperature treatment by cooling flow-through seawater in a head tank by approximately 1˚C using an Aqua Logic Delta Star in-line titanium chiller. Seawater in the elevated temperature treatment was raised approximately 3˚C above the ambient treatment in a separate header tank using Process Technologies titanium immersion heaters. This level of warming mimicked the 2014 and 2015 extreme warming events in the Northern Pacific, where the mass of unusually warm water called “The Blob” raised summer ocean temperatures three standard deviations above the long-term average (Sanford et al., 2019).&lt;/span&gt;&lt;/p&gt; &lt;div&gt;&lt;span style=&quot;background-color:transparent; font-family:arial; font-size:10pt&quot;&gt;At the end of the experiment (10 weeks), we estimated lesion percent cover of the third rank leaf of the terminal shoot of each transplant (i.e., focal leaf) to measure the signs of wasting disease (Burdick et al., 1993). We recorded lesions as either absent, &amp;lt;1%, &amp;lt;10%, or ≥10% cover. When lesions were ≥10% cover, we also recorded a numerical estimate of lesion percent cover.&amp;nbsp; We then collected and preserved the top half of the focal leaf in individual plastic bags sealed with 30 milliliters of silica (Flower Drying Art Silica Gel; Activa) for subsequent DNA extraction and quantitative PCR to estimate &lt;/span&gt;&lt;em&gt;Labyrinthula zosterae&lt;/em&gt;&lt;span style=&quot;background-color:transparent; font-family:arial; font-size:10pt&quot;&gt; cells as a proxy for infection (Bergmann et al., 2011; Bockelmann et al., 2013; Groner et al., 2021).&lt;/span&gt;&lt;/div&gt; &lt;p dir=&quot;ltr&quot;&gt;&lt;span style=&quot;background-color:transparent; font-family:arial; font-size:10pt&quot;&gt;At one-month intervals over the course of the experiment, we measured leaf growth rate of the terminal shoot of each transplant using the “hole-punch” method (Williams and Ruckelshaus, 1993). &lt;/span&gt;&lt;em&gt;Labyrinthula zosterae&lt;/em&gt;&lt;span style=&quot;background-color:transparent; font-family:arial; font-size:10pt&quot;&gt; infection can be affected by plant defenses (Steele et al., 2005; Trevathan-Tackett et al., 2015) and these defenses may trade off with plant growth rate, resulting in a positive relationship between growth rate and infection. Alternatively, &lt;/span&gt;&lt;em&gt;L. zosterae&lt;/em&gt;&lt;span style=&quot;background-color:transparent; font-family:arial; font-size:10pt&quot;&gt; infection can result in reduced leaf growth rate (Graham et al., 2021), leading to a negative relationship. &lt;/span&gt;&lt;em&gt;L. zosterae&lt;/em&gt;&lt;span style=&quot;background-color:transparent; font-family:arial; font-size:10pt&quot;&gt; prevalence can also be affected by plant size (Groner et al., 2016), due to greater leaf-to-leaf contact and resulting increased parasite transmission (Muehlstein, 1992), so we measured the length of the focal leaf of each transplant at the end of the experiment. &lt;/span&gt;&lt;/p&gt; &lt;p&gt;&lt;em&gt;Labyrinthula zosterae&amp;nbsp;DNA extraction and quantitative PCR Assay&lt;/em&gt;&lt;br /&gt; We extracted&amp;nbsp;&lt;em&gt;L. zosterae&lt;/em&gt;&amp;nbsp;DNA from dried leaf tissue using Omega Bio-Tek E.Z. Tissue DNA extraction kits at the Northeastern University Marine Science Center in Nahant, MA. For each sample, we separated the dried leaf tissue into 2-16 mg subsamples and homogenized the tissue in a ball mill (Retsch, Germany) at a frequency of 30 Hz for 5 min (Bockelmann et al. 2013). We lysed ground subsamples individually following the manufacturer’s instructions and added 1 microliter&amp;nbsp;of 500 ng per microliter (ng*uL&lt;sup&gt;-1&lt;/sup&gt;) salmon sperm DNA solution (Invitrogen, USA) to the first subsample of each sample immediately before recombining all subsamples in the spin columns. Salmon sperm DNA was added to enhance extraction efficiency and ensure that even low amounts of target DNA are carried through the filter absorption steps (Bockelmann et al., 2013). We eluted all DNA extractions into 100 uL. Following elution, we used Zymo OneStep-96 PCR Inhibitor Removal kits to clean 50 uL sub-samples of each DNA extraction following the manufacturers instructions. We stored cleaned DNA extractions at -20˚C prior to quantitative PCR.&lt;/p&gt; &lt;p&gt;We used a TaqMan quantitative PCR (qPCR) assay with a forward primer: TTGAACGTAACATT-CGACTTTCGT, reverse primer: ACGCATGAAGCGGTCTTCTT, and MBG probe: TGGACGAGTGTGTTTTG that carries the fluorescence label 6-Fam at the 5’ end and the dark quencher FHQ at the 3’ end (Bio-Rad, USA) developed specifically for&amp;nbsp;&lt;em&gt;L. zosterae&lt;/em&gt;&amp;nbsp;(Bockelmann et al., 2013, Bergmann et al., 2011). We made up qPCR reactions to a 10 uL reaction volume using standard conditions recommended by the manufacturer: 5 uL SsoAdvanced&lt;sup&gt;TM&lt;/sup&gt;&amp;nbsp;Universal Probes Supermix 2x (Bio-Rad, USA), 1 uL template DNA, 0.4 uL 4:1 Primer:Probe Mix (final concentrations of 400 nM forward primer, 400 nM reverse primer, 100 nM probe), and 3.6 uL Milli-Q H&lt;sub&gt;2&lt;/sub&gt;O (Thermofisher, USA). Reactions were run on a CFX96 Real-Time System (Bio-Rad, USA) using the following thermo-cycling program: 3 min at 95˚C, followed by 40 cycles of 15 sec at 95˚C and 1 min at 60˚C. We tested all samples in duplicate and if replicates differed by greater than one cycle threshold (Ct), reactions were rerun in triplicate. We only used the data from reactions in analyses when replicates fell within one Ct. Our lowest detection was 1.76 copies per reaction or 0.15 cells per extraction.&lt;/p&gt; &lt;p&gt;We ran each 96-well plate of qPCR reactions with a set of nine standards: a dilution series of gBlock Gene Fragments (Integrated DNA Technologies, USA) designed based on the highly conserved sequence of the 5.8s ribosomal RNA gene of&amp;nbsp;&lt;em&gt;L. zosterae&lt;/em&gt;&amp;nbsp;known as internal transcribed spacer 1 (ITS) targeted by the TaqMan qPCR assay; an&amp;nbsp;&lt;em&gt;L. zosterae&amp;nbsp;&lt;/em&gt;cell standard consisting of a sample of DNA extracted from a know quantity of pathogenic&amp;nbsp;&lt;em&gt;L. zosterae&amp;nbsp;&lt;/em&gt;cells; and an inhibition control consisting of a half volume of&amp;nbsp;&lt;em&gt;L. zosterae&amp;nbsp;&lt;/em&gt;cell standard and a half volume of a haphazardly selected sample. We ran a total of 31 96-well plates of qPCR reactions with a mean efficiency of 97.4% ± 4.3 and R&lt;sup&gt;2&lt;/sup&gt;&amp;nbsp;0.996 ± 0.004. To convert Ct&amp;nbsp;values to to&amp;nbsp;&lt;em&gt;L. zosterae&lt;/em&gt;&amp;nbsp;cell numbers, four equations were used. The &lt;strong&gt;equations and details are captured in the Supplemental Files section &lt;/strong&gt;of this metadata within the file titled, &quot;Equations for conversion of Ct values to &lt;em&gt;Labyrinthula zosterae&lt;/em&gt;&amp;nbsp;cell numbers.&quot;&lt;/p&gt; &lt;p&gt;We used a pure culture of the pathogenic &lt;em&gt;L. zosterae &lt;/em&gt;isolate 316b provided by D. Martin in 2015 to make our &lt;em&gt;L. zosterae&lt;/em&gt; cell standard (Martin et al., 2016; GenBank: KU559372.1). We cultured &lt;em&gt;L. zosterae &lt;/em&gt;cells on serum seawater agar media (Muehlstein et al., 1991). We scraped&lt;strong&gt; &lt;/strong&gt;cells from an actively growing edge of &lt;em&gt;L. zosterae&lt;/em&gt; culture into serum seawater liquid media (D. Martin pers. com.). We mixed the liquid media +&amp;nbsp;&lt;em&gt;L. zosterae&lt;/em&gt; cell slurry vigorously on a bench top vortex for 30 sec and aliquoted immediately into three replicate subsamples for cell counts and extraction. In order to break up cell clumps for ease of counting, we added Tween80 (Sigma-Aldrich, USA) to a final concentration of 1:100 into the two subsamples used for cell counts, and mixed for 30 sec. We counted cells of four replicate aliquots per subsample on a hemocytometer. We calculated cell concentration by averaging over all replicates. Prior to DNA extraction, we centrifuged the third replicate &lt;em&gt;L. zosterae&lt;/em&gt; cell solution at 6,000 g for 10 min and drew off the supernatant without disturbing the cell pellet. We then added a ~4 mg section of dried healthy &lt;em&gt;Zostera. marina&lt;/em&gt; tissue to the cell pellet to account for possible interference of&amp;nbsp;&lt;em&gt;Zostera&amp;nbsp;marina&amp;nbsp;&lt;/em&gt;compounds in the extraction process. To extract &lt;em&gt;L. zosterae &lt;/em&gt;DNA, we followed the DNA extraction and inhibitor removal protocols outlined above.&lt;/p&gt; &lt;p&gt;We designed the gBlock double stranded DNA fragments (Integrated DNA Technologies, USA) using published sequences of the ITS region of the &lt;em&gt;L. zosterae&lt;/em&gt; genome (GenBank: JN121409-13).&lt;/p&gt; &lt;p&gt;5’-CTGTGATCTCTGAAAATACTTGTTT&lt;br /&gt; (1)&lt;u&gt;TTGAACGTAACATTCGACTTTCGT&lt;/u&gt;CGATT TTG&lt;br /&gt; (2)&lt;u&gt;TGGACGAGTGTGTTTTGT &lt;/u&gt;AAACCTACCC&lt;br /&gt; (3)&lt;u&gt;AAGAAGACCGCTTCATGCGT &lt;/u&gt;GTCGCTGACTAATGAAACAAACAAA-3’&lt;/p&gt; &lt;p&gt;The gBlock fragment sequences were a total length of 130 bp (base pairs), which included target regions for the forward (1) and reverse (3) primers and the MGD probe (2), underlined above, as well as 25 base pairs of additional sequence on both the 5’ and 3’ ends to increase fragment stability. We diluted gBlock fragments in Milli-Q H&lt;sub&gt;2&lt;/sub&gt;O (Thermofisher, USA) to seven concentrations: 2.24e&lt;sup&gt;1&lt;/sup&gt;, 1.12e&lt;sup&gt;2&lt;/sup&gt;, 5.61e&lt;sup&gt;2&lt;/sup&gt;, 2.81e&lt;sup&gt;3&lt;/sup&gt;, 1.40e&lt;sup&gt;4&lt;/sup&gt;, 7.02e&lt;sup&gt;4&lt;/sup&gt;, 7.02e&lt;sup&gt;5&lt;/sup&gt; copies/µL and included this dilution series in each qPCR run as a standard curve (Bergmann et al., 2011). The range of the gBlock dilution curve: approx. 1-60,000 cells/extraction encompassed the range of most &lt;em&gt;L. zosterae&lt;/em&gt; values observed in our samples: 0.15-450,000 cells/extraction or 1.84e&lt;sup&gt;2&lt;/sup&gt;-5.52e&lt;sup&gt;8&lt;/sup&gt; copies/extraction.&lt;/p&gt; &lt;p&gt;&lt;span style=&quot;color:rgb(0, 0, 0); font-family:times new roman,serif; font-size:16px&quot;&gt;&lt;strong&gt;Life Sciences Identifiers (LSID) for taxonomic names:&lt;/strong&gt;&lt;/span&gt;&lt;br /&gt; Zostera marina (urn:lsid:marinespecies.org:taxname:145795)&lt;br /&gt; Labyrinthula zosterae (urn:lsid:marinespecies.org:taxname:395093)&lt;br /&gt; Labyrinthula (urn:lsid:marinespecies.org:taxname:119090)&lt;/p&gt; Specified by the Principal Investigator(s) &lt;p&gt;&lt;strong&gt;BCO-DMO Processing:&lt;/strong&gt;&lt;br /&gt; - Imported data from source file &quot;Schenck_etal_mesocosm_warming_data.csv&quot; into the BCO-DMO data system.&amp;nbsp;Data file imported using missing data identifier&amp;nbsp;&quot;NA&quot;.&lt;br /&gt; - Added columns for start and end date in year-month-day format&lt;br /&gt; - Added conventional header with dataset name, PI name, version date.&lt;br /&gt; - Modified parameter (column) names to conform with BCO-DMO naming conventions.&lt;/p&gt; Specified by the Principal Investigator(s) asNeeded 7.x-1.1 Biological and Chemical Oceanography Data Management Office (BCO-DMO) Unavailable 508-289-2009 WHOI MS#36 Woods Hole MA 02543 USA info@bco-dmo.org http://www.bco-dmo.org Monday - Friday 8:00am - 5:00pm For questions regarding this resource, please contact BCO-DMO via the email address provided. pointOfContact flow through tanks flow through tanks PI Supplied Instrument Name: flow through tanks Instrument Name: Aquarium Instrument Short Name:Aquarium Instrument Description: Aquarium - a vivarium consisting of at least one transparent side in which water-dwelling plants or animals are kept Aqua Logic Delta Star in-line titanium chiller Aqua Logic Delta Star in-line titanium chiller PI Supplied Instrument Name: Aqua Logic Delta Star in-line titanium chiller PI Supplied Instrument Description:We maintained an ambient temperature treatment by cooling flow-through seawater in a head tank by approximately 1˚C using an Aqua Logic Delta Star in-line titanium chiller.  Instrument Name: Aquarium chiller Instrument Short Name:Aquarium chiller Instrument Description: Immersible or in-line liquid cooling device, usually with temperature control. Retsch Mixer Mill 400 Retsch Mixer Mill 400 PI Supplied Instrument Name: Retsch Mixer Mill 400 PI Supplied Instrument Description:For each sample, we separated the dried leaf tissue into 2-16 mg subsamples and homogenized the tissue in a ball mill (Retsch, Germany)  Instrument Name: Homogenizer Instrument Short Name:Homogenizer Instrument Description: A homogenizer is a piece of laboratory equipment used for the homogenization of various types of material, such as tissue, plant, food, soil, and many others. Process Technologies titanium immersion heater Process Technologies titanium immersion heater PI Supplied Instrument Name: Process Technologies titanium immersion heater PI Supplied Instrument Description:Seawater in the elevated temperature treatment was raised approximately 3˚C above the ambient treatment in a separate header tank using Process Technologies titanium immersion heaters  Instrument Name: Immersion heater Instrument Short Name:Immersion heater Instrument Description: Submersible heating element for water tanks and aquaria. Bio-Rad CFX96 Real-Time System Bio-Rad CFX96 Real-Time System PI Supplied Instrument Name: Bio-Rad CFX96 Real-Time System PI Supplied Instrument Description:The focal leaf was stored for subsequent DNA extraction and quantitative PCR to estimate Labyrinthula zosterae cells as a proxy for infection. Software: Bio-Rad CFX Manager Software (version 3.1) Instrument Name: qPCR Thermal Cycler Instrument Short Name:qPCR Instrument Description: An instrument for quantitative polymerase chain reaction (qPCR), also known as real-time polymerase chain reaction (Real-Time PCR).