Evaluating Cumulative Ecosystem Response to Restoration Projects in the Lower Columbia River and Estuary, 2009

This is the sixth annual report of a seven-year project (2004 through 2010) to evaluate the cumulative effects of habitat restoration actions in the lower Columbia River and estuary (LCRE). The project, called the Cumulative Effects Study, is being conducted for the U.S. Army Corps of Engineers Portland District (USACE) by the Marine Sciences Laboratory of the Pacific Northwest National Laboratory (PNNL), the Pt. Adams Biological Field Station of the National Marine Fisheries Service (NMFS), the Columbia River Estuary Study Taskforce (CREST), and the University of Washington. The goal of the Cumulative Effects Study is to develop a methodology to evaluate the cumulative effects of multiple habitat restoration projects intended to benefit ecosystems supporting juvenile salmonids in the 235-km-long LCRE. Literature review in 2004 revealed no existing methods for such an evaluation and suggested that cumulative effects could be additive or synergistic. From 2005 through 2009, annual field research involved intensive, comparative studies paired by habitat type (tidal swamp versus marsh), trajectory (restoration versus reference site), and restoration action (tidegate replacement vs. culvert replacement vs. dike breach)

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Sponge symbioses between <i>Xestospongia deweerdtae</i> and <i>Plakortis</i> spp. are not motivated by shared chemical defense against predators

<div><p>The recently described epizoic sponge-sponge symbioses between <i>Xestospongia deweerdtae</i> and two species of <i>Plakortis</i> present an unusual series of sponge interactions. Sponges from the genus <i>Plakortis</i> are fierce allelopathic competitors, rich in cytotoxic secondary metabolites, and yet <i>X</i>. <i>deweerdtae</i> flourishes as an epizoic encrustation on <i>Plakortis deweerdtaephila</i> and <i>Plakortis symbiotica</i>. Our objective in this study was to evaluate the hypothesis that <i>X</i>. <i>deweerdtae</i> grows epizoic to these two species of <i>Plakortis</i> due to a shared chemical defense against predators. We collected free-living individuals of <i>X</i>. <i>deweerdtae</i> and symbiotic pairs from a wide geographical range to generate crude organic extracts and a series of polarity fractions from sponge extract. We tested the deterrency of these extracts against three common coral reef predators: the bluehead wrasse, <i>Thalassoma bifasciatum</i>, the Caribbean sharpnose puffer, <i>Canthigaster rostrata</i>, and the white spotwrist hermit crab, <i>Pagurus criniticornis</i>. While the chemical defenses of <i>P</i>. <i>deweerdtaephila</i> and <i>P</i>. <i>symbiotica</i> are more potent than those of <i>X</i>. <i>deweerdtae</i>, all of the sponge species we tested significantly deterred feeding in all three generalist predators. The free-living form of <i>X</i>. <i>deweerdtae</i> is mostly defended across the region, with a few exceptions. The associated form of <i>X</i>. <i>deweerdtae</i> is always defended, and both species of <i>Plakortis</i> are very strongly defended, with puffers refusing to consume extract-treated pellets until the extract was diluted to 1/256× concentration. Using diode-array high performance liquid chromatography (HPLC) coupled with high-resolution mass spectrometry (LC-MS/IT-TOF), we found two secondary metabolites from <i>P</i>. <i>deweerdtaephila</i>, probably the cyclic endoperoxides plakinic acid I and plakinic acid K, in low concentrations in the associated—but not the free-living—form of <i>X</i>. <i>deweerdtae</i>, suggesting a possible translocation of defensive chemicals from the basibiont to the epibiont. Comparing the immense deterrency of <i>Plakortis</i> spp. extracts to the extracts of <i>X</i>. <i>deweerdtae</i> gives the impression that there may be some sharing of chemical defenses: one partner in the symbiosis is clearly <i>more defended</i> than the other and a small amount of its defensive chemistry may translocate to the partner. However, <i>X</i>. <i>deweerdtae</i> effectively deters predators with its own defensive chemistry. Multiple lines of evidence provide no support for the shared chemical defense hypothesis. Given the diversity of other potential food resources available to predators on coral reefs, it is improbable that the evolution of these specialized sponge-sponge symbioses has been driven by predation pressure.</p></div

Results from serial dilution feeding assays with Caribbean sharpnose puffers (<i>Canthigaster rostrata</i>), using tissue extract from the free-living form of <i>Xestospongia deweerdtae</i> from three geographic locations.

<p>Horizontal axis shows a progression from concentrated (2×) to dilute (1/4×), and parentheses indicate the number of replicate individual sponges tested. Vertical axis and other details as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174816#pone.0174816.g001" target="_blank">Fig 1</a>.</p

Collection locations for sponge material and assay predators employed in this study.

<p>Each collection took place in June or July of the year specified, except for samples from Puerto Rico, which were collected in October.</p

Results from serial dilution feeding assays with Caribbean sharpnose puffers (<i>Canthigaster rostrata</i>), using tissue extract from the associated form of <i>Xestospongia deweerdtae</i> (top panel), a 1:1 Mix of tissue from both sponges in the association (middle panel), and <i>Plakortis</i> spp. (bottom panel).

<p>Horizontal axis shows a progression from concentrated (2×) to dilute (1/512×), and parentheses indicate the number of replicate individual sponges tested. Vertical axis and other details as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174816#pone.0174816.g001" target="_blank">Fig 1</a>.</p

Comparative high performance liquid chromatography (HPLC) chromatograms of crude extracts of sponge tissue dissolved in MeOH (UV absorbance at 235 nm).

<p>Neither Main Compound was detected in crude extracts of free-living <i>Xestospongia deweerdtae</i>, but both appear in crude extracts of associated <i>X</i>. <i>deweerdtae</i>, and <i>Plakortis deweerdtaephila</i>. Peaks labeled with mass values from subsequent experiment by TOF-MS. Mass spectra and UV absorbance are archived in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174816#pone.0174816.s003" target="_blank">S1 Fig</a>.</p

Proportional masses of Main Compounds from crude sponge extract, λ max. 235, using LC-DAD-MS.

<p>Proportional masses of Main Compounds from crude sponge extract, λ max. 235, using LC-DAD-MS.</p

Results of feeding assays with white spotwrist hermit crabs (<i>Pagurus criniticornis</i>), in which crude organic tissue extracts from sponges, incorporated at a 1× volumetric concentration into artificial food pellets, were offered to hermit crabs.

<p>Bars indicate the mean number of pellets eaten (out of 10 pellets offered) for multiple individuals of the given sponge at the given site. Error bars show standard error. In every case, 10/10 control pellets were consumed. The dotted line indicates a threshold for statistical significance as determined by a modified Fisher’s Exact Test; a sample is significantly deterrent (<i>p</i> < 0.05) if 6 or fewer pellets are consumed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174816#pone.0174816.ref044" target="_blank">44</a>]. The horizontal axis specifies species and growth form used to generate each extract: XDFL = <i>Xestospongia deweerdtae</i> free-living; XDA = <i>X</i>. <i>deweerdtae</i> associated; PL = <i>Plakortis deweerdtaephila</i>. The number of replicate individual sponges tested from each site is shown as a number overlaid on the bar. Tabulated data are archived in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174816#pone.0174816.s001" target="_blank">S1 Table</a>.</p