Quantifying environmental reservoirs of fecal indicator bacteria associated with sediment and submerged aquatic vegetation.

Elevated concentrations of fecal indicator bacteria (FIB) in aquatic sediments and vegetation have prompted concern that environmental reservoirs of FIB disrupt the correlation between indicator organisms, pathogens and human health risks. FIB numbers, however, are typically normalized to volume of water or mass of substrate. Because these reservoirs tend to differ greatly in magnitude within and between water bodies, direct comparison between water column and benthic population sizes can be problematic. Normalization to a set volume of water or mass of substrate, e.g. cfu (100 ml)(-1) or cfu(100 g)(-1), can give a false picture of the relative contributions of various reservoirs to FIB numbers across the ecosystem, and of the potential for FIBs to trigger health advisories as they pass from one reservoir to another. Here, we normalized enterococci concentrations from water, sediment and submerged aquatic vegetation (SAV) to land surface area (m(2) ) to compare their relative importance in the entire system. SAV-associated enterococci comprised only 0-18% of the entire population, even though they displayed the highest concentrations of enterococci per unit mass. The largest proportion of the enterococci population was in the water column (4-77%) or sediments (20-95%), depending on the volume of each substrate available at a site and FIB concentrations within them. Models indicated that large shifts in the relative size of FIB populations in each substrate can result from changes in per cent SAV cover, water depth and depth of sediment colonization. It follows that high concentrations of FIB in sediments or SAV do not necessarily signify large environmental reservoirs of FIB that can affect the water column. Comprehensive analyses that include FIB measurements from water, SAV and sediment normalized to land surface area offer a more balanced perspective on total FIB numbers contained in various matrices of an aquatic system.

The effects of submerged aquatic vegetation on the persistence of environmental populations of Enterococcus spp.

Enterococcus spp. are utilized worldwide as faecal indicator bacteria, but certain strains exhibit extended survival in environmental habitats and the factors influencing their persistence are poorly understood. We used flowing freshwater mesocosms to explore the effect of submerged aquatic vegetation (SAV) on the persistence of natural enterococci populations from a subtropical lake. The highest mean densities of culturable enterococci over 2 weeks occurred in SAV [8.6 x 10(2) colony-forming units (cfu) per 100 g wet weight], followed by sediments (1.3 x 10(2) cfu per 100 g) and water (18 cfu per 100 ml). However, due to relative differences in the total mass of each substrate in the entire system (water > sediments > SAV), SAV-associated enterococci represented only a minor proportion of the total population. Vegetated mesocosms harboured significantly higher mean cfu per mesocosm and cfu densities in sediments compared with their unvegetated counterparts, suggesting that SAV indirectly facilitates persistence in aquatic habitats. Populations were dominated (> 96%) by a single Enterococcus casseliflavus strain according to BOX-PCR genotyping, which did not change over the 10-month study and strongly suggests bacterial replication in the lake. The presence of such strains in the environment may represent highly competitive, naturalized and reproducing indicator bacteria populations that are not directly related to pollution events.

A Novel µCT Analysis Reveals Different Responses of Bioerosion and Secondary Accretion to Environmental Variability.

Corals build reefs through accretion of calcium carbonate (CaCO3) skeletons, but net reef growth also depends on bioerosion by grazers and borers and on secondary calcification by crustose coralline algae and other calcifying invertebrates. However, traditional field methods for quantifying secondary accretion and bioerosion confound both processes, do not measure them on the same time-scale, or are restricted to 2D methods. In a prior study, we compared multiple environmental drivers of net erosion using pre- and post-deployment micro-computed tomography scans (µCT; calculated as the % change in volume of experimental CaCO3 blocks) and found a shift from net accretion to net erosion with increasing ocean acidity. Here, we present a novel µCT method and detail a procedure that aligns and digitally subtracts pre- and post-deployment µCT scans and measures the simultaneous response of secondary accretion and bioerosion on blocks exposed to the same environmental variation over the same time-scale. We tested our method on a dataset from a prior study and show that it can be used to uncover information previously unattainable using traditional methods. We demonstrated that secondary accretion and bioerosion are driven by different environmental parameters, bioerosion is more sensitive to ocean acidity than secondary accretion, and net erosion is driven more by changes in bioerosion than secondary accretion.

Relative effects of gamete compatibility and hydrodynamics on fertilization in the green sea urchin Strongylocentrotus droebachiensis.

Intraspecific variation in gamete compatibility among male/female pairs causes variation in the concentration of sperm required to achieve equivalent fertilization levels. Gamete compatibility is therefore potentially an important factor controlling mating success. Many broadcast-spawning marine invertebrates, however, also live in a dynamic environment where hydrodynamic conditions can affect the concentration of sperm reaching eggs during spawning. Thus flow conditions may moderate the effects of gamete compatibility on fertilization. Using the green sea urchin Strongylocentrotus droebachiensis as a model system, we assessed the relative effects of gamete compatibility (the concentration of sperm required to fertilize 50% of the eggs in specific male/female pairs; F50) and the root-mean-square of total velocity (urms; 0.01-0.11 m s(-1)) on fertilization in four locations near a spawning female (water column, wake eddy, substratum, and aboral surface) in both unidirectional and oscillatory flows. Percent fertilization decreased significantly with increasing urms at all locations and both flow regimes. However, although gamete compatibility varied by almost 1.5 orders of magnitude, it was not a significant predictor of fertilization for most combinations of position and flow. The notable exception was a significant effect of gamete compatibility on fertilization on the aboral surface under unidirectional flow. Our results suggest that selection on variation in gamete compatibility may be strongest in eggs fertilized on the aboral surface of sea urchins and that hydrodynamic conditions may add environmental noise to selection outcomes.

Patterns in temporal variability of temperature, oxygen and pH along an environmental gradient in a coral reef.

Spatial and temporal environmental variability are important drivers of ecological processes at all scales. As new tools allow the in situ exploration of individual responses to fluctuations, ecologically meaningful ways of characterizing environmental variability at organism scales are needed. We investigated the fine-scale spatial heterogeneity of high-frequency temporal variability in temperature, dissolved oxygen concentration, and pH experienced by benthic organisms in a shallow coastal coral reef. We used a spatio-temporal sampling design, consisting of 21 short-term time-series located along a reef flat-to-reef slope transect, coupled to a long-term station monitoring water column changes. Spectral analyses revealed sharp gradients in variance decomposed by frequency, as well as differences between physically-driven and biologically-reactive parameters. These results highlight the importance of environmental variance at organismal scales and present a new sampling scheme for exploring this variability in situ.

Effects of oscillatory flow on fertilization in the green sea urchin Strongylocentrotus droebachiensis.

Broadcast spawning invertebrates that live in shallow, high-energy coastal habitats are subjected to oscillatory water motion that creates unsteady flow fields above the surface of animals. The frequency of the oscillatory fluctuations is driven by the wave period, which will influence the stability of local flow structures and may affect fertilization processes. Using an oscillatory water tunnel, we quantified the percentage of eggs fertilized on or near spawning green sea urchins, Strongylocentrotus droebachiensis. Eggs were sampled in the water column, wake eddy, substratum and aboral surface under a range of different periods (T = 4.5-12.7 s) and velocities of oscillatory flow. The root-mean-square wave velocity (rms(u(w))) was a good predictor of fertilization in oscillatory flow, although the root-mean-square of total velocity (rms(u)), which incorporates all the components of flow (current, wave and turbulence), also provided significant predictions. The percentage of eggs fertilized varied between 50-85% at low flows (rms(u(w)) <0.02 m s(-1)), depending on the location sampled, but declined to below 10% for most locations at higher rms(u(w)). The water column was an important location for fertilization with a relative contribution greater than that of the aboral surface, especially at medium and high rms(u(w)) categories. We conclude that gametes can be successfully fertilized on or near the parent under a range of oscillatory flow conditions.

Diversity of assimilatory nitrate reductase genes from plankton and epiphytes associated with a seagrass bed.

Assimilatory nitrate reductase gene fragments were isolated from epiphytes and plankton associated with seagrass blades collected from Tampa Bay, Florida, USA. Nitrate reductase genes from diatoms (NR) and heterotrophic bacteria (nasA) were amplified by polymerase chain reaction (PCR) using two sets of degenerate primers. A total of 129 NR and 75 nasA clones from four clone libraries, two from each of epiphytic and planktonic components, were sequenced and aligned. In addition, genomic DNA sequences for the NR fragment were obtained from Skeletonema costatum and Thalassiosira weissflogii diatom cultures. Rarefaction analysis with an operational taxonomic unit cut-off of 6% indicated that diversity of the NR and nasA clone libraries were similar, and that sequencing of the clone libraries was not yet saturated. Phylogenetic analysis indicated that 121 of the 129 NR clones sequenced were similar to diatom sequences. Of the eight non-diatom sequences, four were most closely related to the sequence of Chlorella vulgaris. Introns were found in 8% of the Tampa Bay NR sequences; introns were also observed in S. costatum, but not T. weissflogii. Introns from within the same clone library exhibited close similarity in nucleotide sequence, position and length; the corresponding exon sequences were unique. Introns from within the same component were similar in position and length, but not in nucleotide sequence. These findings raise questions about the function of introns, and mechanisms or time evolution of intron formation. A large cluster of 14 of the 75 nasA sequences was similar to sequences from Vibrio species; other sequences were closely related to sequences from Alteromonas, alpha-proteobacteria and Marinomonas-like species. Biogeographically consistent patterns were observed for the nasA Tampa Bay sequences compared with sequences from other locations: for example, Tampa Bay sequences were similar to those from the South Atlantic Bight, but not the Barents Sea. The Tampa Bay NR clone libraries contained sequences that exhibited phylogenetic similarity with sequences from coastal New Jersey and Monterey Bay, USA. For both NR and nasA, the sequences formed phylogenetic clusters containing nitrate reductase gene fragments that were common to both plankton and epiphyte components, and sequences that were unique to just one component. The implication that some organisms may be differentially represented in epiphytic versus planktonic components of the community suggests that local environmental conditions may have ramifications for regulation of nitrate assimilation processes, community composition, and ecosystem function.

Assessment of sperm chemokinesis with exposure to jelly coats of sea urchin eggs and resact: a microfluidic experiment and numerical study.

Specific peptides contained within the extracellular layer, or jelly coat, of a sea urchin egg have been hypothesized to play an important role in fertilization, though separate accounting of the effects of chemoattraction, chemokinesis, sperm agglomeration and the other possible roles of the jelly coat have not been reported. In the present study, we used a microfluidic device that allowed determination of the differences in the diffusion coefficients of sperm of the purple sea urchin Arbacia punctulata subjected to two chemoattractants, namely the jelly coat and resact. Our objectives were twofold: (1) to experimentally determine and compare the diffusion coefficients of Arbacia punctulata spermatozoa in seawater, jelly coat solution and resact solution; and (2) to determine the effect of sea urchin sperm diffusion coefficient and egg size on the sperm-egg collision frequency using stochastic simulations. Numerical values of the diffusion coefficients obtained by diffusing the spermatozoa in seawater, resact solution and jelly coat solution were used to quantify the chemotactic effect. This allowed direct incorporation of known enlargements of the egg, and altered sperm diffusion coefficients in the presence of chemoattractant, in the stochastic simulations. Simulation results showed that increase in diffusion coefficient values and egg diameter values increased the collision frequency. From the simulation results, we concluded that type of sperm, egg diameter and diffusion coefficient are significant factors in egg fertilization. Increasing the motility of sperm appears to be the prominent role of the jelly coat.