The metapopulation of the estuarine species Gammarus tigrinus along the east coast of the United States has been hypothesised to represent two cryptic species divided biogeographically off the coast of North Carolina, USA. This divergence has been attributed to a strong temperature gradient created by the formation of the cold Labrador Current c. 3.0 million years ago. In addition, the northern phylogeographic clade of G. tigrinus has been demonstrated to be invasive in estuarine habitats across a large portion of northern Europe. Recent collections of G. tigrinus from Florida and Maryland, USA, allow for new approaches to test this hypothesis. Using the nuclear 18S and 28S rRNA, and mitochondrial 16S rRNA and cytochrome c oxidase subunit I genes, species delimitation models provide support that the genetic divergence of the northern and southern clades is equivalent to species level. In addition, molecular clock data demonstrate that this phylogeographic divergence coincides with the formation of the Labrador Current. Furthermore, the collections of G. daiberi from Florida, a species with biogeographical and ecological characteristics similar to those of G. tigrinus, provide independent support for the hypothesis. The potential for invasive species to be cryptic highlights the need for accurate identification of taxa to ensure that appropriate biogeographical assessment of potential source populations and mechanisms of dispersal can be made.

Crangonyx sulphurium n. sp., a new species of stygobitic amphipod, is described from three springs in central Florida, U.S.A. The description of the new species brings the number of described stygobitic species of Crangonyx found in the Floridan aquifer to three. The specimens were collected in the spring basins of Wekiwa and Volusia springs and De Leon Springs Cave. Specimens from De Leon Springs Cave were found swimming in and around white microbial mats characteristic of sulfur oxidizing bacteria. Next generation sequencing of microbial mat samples, collected from the spring ten years after the collection of the new species, indicates a heterotrophic and autotrophic community dominated by five phylotypes. The ecology of these three caves may be unique when compared to other caves within the Floridan aquifer.

Crangonyx sulphuriumn. sp., a new species of stygobitic amphipod, is described from three springs in central Florida, U.S.A. The description of the new species brings the number of described stygobitic species of Crangonyx found in the Floridan aquifer to three. The specimens were collected in the spring basins of Wekiwa and Volusia springs and De Leon Springs Cave. Specimens from De Leon Springs Cave were found swimming in and around white microbial mats characteristic of sulfur oxidizing bacteria. Next generation sequencing of microbial mat samples, collected from the spring ten years after the collection of the new species, indicates a heterotrophic and autotrophic community dominated by five phylotypes. The ecology of these three caves may be unique when compared to other caves within the Floridan aquifer.

Extensive blooms of the toxic epiphytic/benthic dinoflagellate Ostreopsis cf. ovata are being reported with increasing frequency and spatial distribution in temperate coastal regions including the Mediterranean. These blooms are of human and environmental health concern due to the production of isobaric palytoxin and a wide range of ovatoxins by Ostreopsis cf. ovata. Bacterial-microalgal interactions are important regulators in algal bloom dynamics and potentially toxin dynamics. This study investigated the bacterial assemblages co-occurring with O. cf. ovata (OA) and from ambient seawaters (SW) during the early and peak phases of bloom development in NW Adriatic Sea. Fractions of the bacterial assemblages co-occurring with O. cf. ovata (OA) and more closely associated to the mucilage layer (LA) embedding O. cf. ovata cells were also reported. In total, 14 bacterial phyla were detected by targeted 454 pyrosequencing of the 16S rRNA gene. The dominant bacterial phyla in the OA assemblages were Proteobacteria and Bacteroidetes; while at the class level, Alphaproteobacteria were the most abundant (83 and 66%, relative abundance, early and peak bloom phases), followed by Flavobacteria (7 and 19%, early and peak phases). Actinobacteria and Cyanobacteria were of minor importance (<5% of the relative bacterial abundance each). Gammaproteobacteria showed a notably presence in OA assemblage only at the early phase of the bloom (genus Haliea, 13%). The Alphaproteobacteria were predominately composed by the genera Ruegeria, Jannaschia and Erythrobacter which represented about half of the total phylotypes' contribution of OA at both early and peak phases of the O. cf. ovata bloom, suggesting interactions between this consortium and the microalga. Moreover, the highest contribution of Ruegeria (30% of the total phylotypes) was observed at the early phase of the bloom in LA assemblage. Microbial assemblages associated with the ambient seawaters while being also dominated by Alphaproteobacteria and Flavobacteria were partially distinct from those associated with O. cf. ovata due to the presence of genera almost not retrieved in the latter assemblages.

Vibrio cholerae is a human pathogenic marine bacterium inhabiting coastal regions and is vectored into human food and water supplies via attachment to particles including detritus, phytoplankton, and zooplankton. Particle colonization by the pathogen is inhibited by an antagonistic interaction with the particle-associated Vibrionales bacterium SWAT3, a producer of the antibiotic andrimid. By analyzing the individual movement behaviors of V cholerae exposed to a gradient of andrimid in a microfluidics device, we show that the pathogen has a concentration dependent avoidance response to sub-lethal concentrations of the pure antibiotic and to the metabolites produced by a growing colony of SWAT3-wild-type. This avoidance behavior includes a 25% increase in swimming speeds, 30% increase in run lengths, and a shift in the direction of the bacteria away from the andrimid source. Consequently, these behavioral shifts at low concentrations of andrimid would lead to higher diffusivity and result in the dispersion of bacteria away from the competitor and source of the antibiotic. Such alterations in motility were not elicited in response to a non-andrimid-producing SWAT3 mutant, suggesting andrimid may be a negative effector of chemotaxis for V cholerae. The behavioral response of colonizing bacteria to sub-inhibitory concentrations of competitor-produced antibiotics is one mechanism that can influence microbial diversity and interspecific competition on particles, potentially affecting human health in coastal communities and element cycling in the ocean.

Microbial mats are laminae of self-sustaining microbial communities with a high level of competition for resources. We tested the hypothesis that chemically mediated antagonism is a potential mechanism for structuring the bacterial community. In the co-culturing assay, 57% of the isolates expressed antagonistic behavior toward one or more isolates and 5% of the isolates inhibited more than 80% of the isolates. We observed greater levels of antagonism between isolates from adjacent laminae than within. The bacterial isolate library derived from the mat was predominately Gram-positive, and inhibition within this group was greater than against the few Gram-negative isolates. Microdiversity of 16S rRNA gene was observed for Bacillus marisflavi isolates, which represented 23 of the 75 isolates in the library. Within this and other groups, the patterns of inhibition and sensitivity varied greatly, suggesting rapid gain and loss of the ability to produce antagonistic secondary metabolites and resistance toward such molecules. Our observations are consistent with the hypothesis that antagonistic interactions are a potential mechanism in addition to physiochemical properties that regulate the vertical distribution of aerobic heterotrophic bacteria in hypersaline microbial mats.

Pharmaceuticals are common chemical contaminants in estuaries receiving effluent from wastewater and sewage treatment facilities. The purpose of this research was to examine benthic microalgal (BMA) community responses to sublethal exposures to tylosin, a common and environmentally persistent antibiotic. Bioassays, using concentrations of 0.011–218μmoltylosinl−1, were performed on intertidal muddy sediments from North Inlet Estuary, SC. Exposure to tylosin resulted in a reduction in total BMA biomass and primary productivity. Furthermore, exposure seemed to retard diatom growth while having a minimal effect on cyanobacteria biomass. Estuarine systems receiving chronic inputs of trace concentrations of tylosin, as well as other antibiotics, may experience significant reductions in BMA biomass and primary productivity. Given the well-documented role of BMA in the trophodynamics of estuaries, these impacts will likely be manifested in higher trophic levels with possible impairments of the structure and function of these sensitive systems.

The success of many enteric bacteria is hinged on the ability to tolerate environmental stress such as extreme acidity. The acid stress response (ASR) has been investigated in many enteric bacteria and has been shown to involve variable expression of a broad spectrum of genes involved in transcriptional regulation, metabolism, colonization and virulence; representing a linkage between acid tolerance and pathogenicity. Though the majority of ASR studies have been conducted in laboratory conditions and from the perspective of pathogenicity, the role of environmental reservoirs on acid adaptation has recently emerged as an important aspect of pathogenic microbial ecology. This mini-review profiles ASR in three opportunistic enteric pathogens and synthesizes recent work pertaining to the study of this dynamic response.

Aims: To investigate with high geographical resolution the small-scale spatial and temporal distribution of the pathogen Vibrio vulnificus throughout the water column in a temperate Texas bay where numerous V. vulnificus infections had been reported by the regional media the previous summer.Methods and Results: Surface and bottom water samples were collected from 19 sites between April 2005 and October 2006 from Matagorda Bay, TX. Physicochemical parameters were measured and V. vulnificus were analysed using quantitative polymerase chain reaction (Q-PCR) as a means of overcoming constraints of traditional culturing techniques. V. vulnificus was detected through out the year, although it's temporal and spatial distribution was patchy. V. vulnificus abundances at individual sites ranged from <10 to >1.1 x 10(3) cells ml(-1). No statistically reliable predictive model related to the physicochemical parameters could be developed for this pathogen.Conclusions: his study demonstrates that year round detection of V. vulnificus while likely in the viable but nonculturable (VBNC) state during the winter months and emphasizes why physicochemical factors are insufficient metrics for robust regression modelling of this pathogen.Significance and Impact of the Study: This study provides an effective new tool, Q-PCR, to study environmental distribution of V. vulnificus and that in the light of the patchy distribution observed, new reliable approaches and a mechanistic understanding of pathogen ecology need to be considered to effectively model the aquatic distribution of V. vulnificus.

To gain new insights into the variability of particulate organic carbon (POC) fluxes and to better understand the factors controlling the POC/234Th ratios in suspended and sinking particulate matter, we investigated the relationships between POC/234Th ratios and biochemical composition (uronic acids, URA; total carbohydrates, TCHO; acid polysaccharides, APS; and POC) of suspended and sinking matter from the Gulf of Mexico in 2005 and 2006. Our data show that URA/POC in sediment traps (STs), APS/POC in the suspended particles, and turnover times of particulate 234Th in the water column and those of bacteria in STs inside eddies usually increased with depth, whereas particulate POC/234Th (10–50μm) and the sediment-trap parameters (POC flux, POC/234Th ratio, bacterial biomass, and bacterial production) decreased with depth. However, this trend was not the case for most biological parameters (e.g., phytoplankton and bacterial biomass) or for the other parameters at the edges of eddies or at coastal-upwelling sites.In general, the following relationships were observed: 1) 234Th/POC ratios in STs were correlated with APS flux, and these ratios in the 10–50μm suspended particles also correlated with URA/POC ratios; 2) neither URA fluxes nor URA/POC ratios were significantly related to bacterial biomass; 3) the sum of two uronic acids (G2, glucuronic, and galacturonic acid, which composed most of the URA pool) was positively related to bacterial biomass; and 4) the POC/234Th ratios in intermediate-sized particles (10–50μm) were close to those in sinking particles but much lower than those in >50μm particles. The results indicate that acid polysaccharides, though a minor fraction (~1%) of the organic carbon, act more likely as proxy compound classes that might contain the more refractory 234Th-binding biopolymer, rather than acting as the original 234Th “scavenger” compound. Moreover, these acid polysaccharides, which might first be produced by phytoplankton and then modified by bacteria, also influence the on-and-off “piggy-back” processes of organic matter and 234Th, thus causing additional variability of the POC/234Th in particles of different sizes.

Surface associated microbes have historically been difficult to accurately and effectively enumerate. In the current study, we propose a rapid and simple method for estimating abundance of surface associated microbial cells by fluorescence of SYBRGreen stained bacteria and in vivo chlorophyll a fluorescence of benthic diatoms in 24 and 48-well microtiter plates. The effectiveness of this high-throughput technique is demonstrated by assessing sensitivity of a clinical strain of Vibrio cholerae, a benthic bacterial isolate and the benthic microalgae Cylindrotheca closterium to three antibiotics — tylosin, lincomycin and ciproflaxacin. We report on the significant linear relationships between spectral chl a fluorescence and cell abundance and between microalgal growth rates derived from cell counts and fluorescence. Additionally, we provide a simplified and improved method for preparation of a silica gel matrix (SGM), which is an ideal plating media for fluorescence applications. These findings indicate that spectrofluorometry is an inexpensive tool for rapidly estimating abundance of surface associated microbiota and can be employed for assessing antibiotic sensitivity.

To better understand the inter-relationships between particulate organic carbon (POC) fluxes and phytoplankton and bacteria biomass and production, we compared POC fluxes determined in sediment traps and approaches based on size-fractionated (1–10, 10–50, 50–150 and > 150 μm) suspended particulate 234Th and POC concentrations in oligotrophic sections of the Gulf of Mexico during August 2005 and May 2006 and in the oligotrophic northwestern Pacific Ocean during 2009. In 2005, the sediment trap POC flux near the bottom of the euphotic zone (120 m) ranged from 71 to 94 mg C m− 2 day− 1, while 234Th-derived POC fluxes using POC/234Th ratios in the 10–50 µm and 50–150 µm varied from 71 to 150 mg C m− 2 day− 1. In 2006, the sediment trap POC flux at 120 m ranged from 24 to 67 mg C m− 2 day− 1, while the 234Th-derived POC fluxes in the 10–50 µm fraction were comparable or somewhat higher, ranging from 71 to 119 mg C m− 2 day− 1. The POC fluxes in 2006, calculated by using POC/234Th ratios in the 1–10 µm and the 50–150 µm fractions were much higher, ranging from 847 to 1369 mg C m− 2 day− 1. Correlations with biological and chemical parameters support a likely mechanism of sinking aggregates of haptophytes (0.2–20 µm) of higher density held together by Th-complexing and uronic acid containing exopolymeric substances. The observations that 234Th (and POC) is mainly associated with medium-sized (10–15 µm) suspended particles rather than larger (50–150 µm) ones may be caused by the use of a one-filter method and standard filtration and processing procedures that were applied here for collecting suspended particles. This then raises the question of what constitutes representative material from the ocean that settles on the characteristic time scale of 234Th. As a comparison, size-fractionated trap-collected particles in the oligotrophic northwestern Pacific Ocean showed that the 10–50 μm fraction contained the largest proportion of POC (22–41%), followed by the 50–150 μm (22–37%), the > 150 μm (15–27%), and the 1–10 μm (17–23%) fraction. The partitioning of 234Th in trap-collected particles was slightly different from that of POC, with the 1–10 μm fraction representing the largest proportion (27–48%) of 234Th flux. Together, the < 50 μm particles contributed, on average, 52 ± 6% of POC, which suggests that the POC/234Th ratios traditionally derived from large (> 50 µm) pump-collected particles may not accurately reflect the majority of sinking particles. Therefore, estimated POC fluxes may be significantly biased using a conventional 234Th based approach, i.e., using POC/234Th ratios from a single filter obtained from large (> 50 µm) pump-collected particles.

Microbial mats in Bahamian hypersaline lagoons are affected by the combination of salinity fluctuations and external nutrient inputs, both of which are seasonally variable. The purpose of this study was to examine the singular and combined effects of salinity and nutrient (N+P) stress on primary production, extracellular enzyme activity, and the composition of the photoautotroph community in this episodically varying extreme environment. Anoxygenic phototrophic bacteria were able to increase their relative abundance when nutrients were supplied under hypersaline conditions (300 g l(-1)). When salinities were lowered (38 g l(-1)) and nutrients added, extracellular enzyme activity (aminopeptidase, alpha-glucosidase, and beta-glucosidase), rates of oxygenic photosynthesis, and phototroph biomass increased in the oxic surface layers of the mat. Once salinity stress had been lowered, oxygenic photosynthesis allowed the proliferation of Cyanobacteria, heterotrophic activity, and a corresponding reduction in the abundance of anoxygenic phototrophic bacteria. On reduction of nutrient stress, mat phototrophs responded by increasing biomass (using either anoxygenic or oxygenic photosynthesis, or both). In this hypersaline system, seasonal as well as short-term (days) variations in environmental conditions may promote structural changes in the mat community which alter the rates of major processes such as oxygenic photosynthesis and heterotrophy, and illustrate the cyclic behavior of microbial dormancy and proliferation in this extreme environment. Cycles in nutrient input and salinity are primary forcing factors for the maintenance of a dynamic and diverse (both structurally and functionally) benthic microbial community in a small hypersaline lagoon, Salt Pond, on San Salvador Island, Bahamas.

The biochemical composition of dissolved organic phosphorus (DOP) in the ocean is dominated by phosphoesters (C-O-P and C-O-P-O-C bonds), which are hydrolyzed by a diverse group of alkaline phosphatases (PhoA, PhoD, PhoX), and by phosphonates (C-P bond), which are degraded by C-P lyases and hydrolases. We designed a bioinformatics pipeline and a statistical approach to recover and analyze the alkaline phosphatase and phosphonate utilization genes from a metagenomic database derived from water samples collected from 7 depths (between 10 and 4000 m) in the oligotrophic North Pacific Subtropical Gyre. The alkaline phosphatase genes phoD and phoX were more abundant than phoA in the euphotic zone (10-130 m) and in deep waters (500-4000 m). The C-P lyase genes were most abundant in the euphotic zone at 70 m and were rare in deep water (>= 500 m) where phosphate concentrations were relatively high. These observations indicate that phosphonates are utilized primarily as a phosphorus source by bacterial C-P lyases; this is consistent with the observation that C-P lyase genes are part of the pho regulon which is expressed upon phosphorus limitation. In contrast, C-P hydrolase and alkaline phosphatase genes were often more abundant in deep waters, indicating that DOP serves mainly as a carbon and energy source in phosphate-rich deep waters which are depleted in bioavailable dissolved organic matter (DOM). The observed differences in depth distributions and presumed functions of C-P lyase and hydrolase genes indicate variability in the chemical composition of phosphonates between the euphotic zone and deep waters.

A significant proportion of microphytobenthos (MPB) production is in the form of intraand extracellular carbohydrates.We measured changes in MPB biomass, associated carbohydrates, and relative exoenzyme activity in an intertidal biofilm over the course of daylight tidal emersion.Two experiments were carried out in North Inlet (South Carolina, USA) during June 2007.MPB biomass accumulated in the upper 5 mm of sediment during daylight emersion, and there was a loss of biomass after tidal flooding of the sediment, with a 50 ± 21% (mean ± SD) decrease in sediment chl a content.Six carbohydrate fractions were measured in the sediment, and there were significant positive correlations between chl a and all carbohydrate fractions, indicating that the MPB was the major source of carbohydrates in the sediment, despite significant local primary production by Spartina alterniflora.Rates of relative glycosidase activity followed the pattern: β-glucosidase > α-glucosidase > (β-xylosidase = β-galactosidase = α-galactosidase).Glucosidase enzymes dominated the hydrolysis of carbohydrates, contributing 70 ± 6% of the measured total relative exoenzyme activity.There were significant positive correlations between the total and hot water-extracted carbohydrates in the sediment with relative α-glucosidase activity.Relative α-galactosidase activity was positively correlated with the saline-extractable and saline-extractable low molecular weight fractions.In addition to βglucosidase, other exoenzymes play a significant role in the remineralization of exopolymers in MPBdominated intertidal sediments.

With the ban of tributyl tin and its analogs and the fact that copper and its derivatives are under legislative pressure, we must consider alternatives for the control of biofouling. So-called fouling-release coatings are one potential solution, but they do not control microfouling well. Compounds derived from macrobiota that appear to be fouling-free appear to be suitable molecules for investigation as components of antifouling coatings. If a molecule is to be active against a variety of organisms, it is important that it inhibit some universal metabolic process. Ideally, such a molecule would interfere with the surface sensing process itself and the events that result from the reception of that signal. Cell signaling in all eukaryotes is mediated by changes in the internal Ca2+ concentration. Therefore, a molecule that interferes with Ca-mediated events would be an ideal candidate to inhibit cellular adhesion and thus fouling. Using an image analysis-directed assay with diatoms and Ca-fluorophores to detect Ca fluxes, we report how 2-n-pentyl-4-quinolinol, D-600, and trans, trans-2,4-decadienal influence diatom adhesion and motility. All three molecules show activity as antifoulants.

Bacterial alkaline phosphatases (APases) are important enzymes in organophosphate utilization in the ocean. The subcellular localization of APases has significant ecological implications for marine biota but is largely unknown. The extensive metagenomic sequence databases from the Global Ocean Sampling Expedition provide an opportunity to address this question. A bioinformatics pipeline was developed to identify marine bacterial APases from the metagenomic databases, and a consensus classification algorithm was designed to predict their subcellular localizations. We identified 3,733 bacterial APase sequences (including PhoA, PhoD, and PhoX) and found that cytoplasmic (41%) and extracellular (30%) APases exceed their periplasmic (17%), outer membrane (12%), and inner membrane (0.9%) counterparts. The unexpectedly high abundance of cytoplasmic APases suggests that the transport and intracellular hydrolysis of small organophosphate molecules is an important mechanism for bacterial acquisition of phosphorus (P) in the surface ocean. On average, each marine bacterium possessed at least one suite of uptake of glycerol phosphate (ugp) genes (e.g., ugpA, ugpB, ugpC, ugpE) for dissolved organic phosphorus (DOP) transport, but only half of them had ugpQ, which hydrolyzes transported DOP, indicating that cytoplasmic APases play a role in hydrolyzing transported DOP. The most abundant heterotrophic marine bacteria, α- and γ-Proteobacteria, might hydrolyze DOP outside the cytoplasmic membrane, but the former could also transport and hydrolyze DOP in the cytoplasm. The abundant extracellular APases could provide bioavailable P for organisms that cannot directly access organophosphates, and thereby increase marine biological productivity and diversity.

The degradation in performance of submerged marine structures through their colonization by micro‐ and macrobiota is well known. Historically, toxic surface coatings have been used to control both types of fouling. Legislative requirements worldwide now require alternative non‐ or minimally toxic coatings to be used. Here we report on the potential for synthetic 2‐pentyl‐4‐quinolinol (PQ), an antibiotic from an Alteromonas sp., as a candidate antifoulant. Hundred micromolar PQ reduces the growth of four marine diatoms that usually grow as biofilms. Furthermore, PQ inhibits the initial adhesive process in Amphora coffeaeformis in a logarithmic dose‐dependent manner, but not the adhesion of a Navicula sp., indicating differing adhesive processes in these two diatoms. Treatment of established biofilms with PQ causes a rapid and complete loss of motility in both Amphora and Navicula and eventually, as seen by Sytox Green staining, cell death, and lysis. We conclude that PQ, or one of its chemical analogues, holds promise as an antifoulant molecule for inclusion in marine surface coating. © 2007 Wiley Periodicals, Inc. Environ Toxicol 22: 275–280, 2007.

Chemical studies of a Bacillus endophyticus isolated from a Bahamian hypersaline microbial mat led to the isolation of bacillamides B and C, new tryptamide thiazole metabolites. Bioassay-guided fractionation using a HPLC-UV-MS bioassay technique enabled the detection of these trace fermentation products, and their total structures were elucidated by combined spectroscopic techniques.