Bacterial diversity in the bacterioneuston (sea surface microlayer): the bacterioneuston through the looking glass

The bacterioneuston is defined as the community of bacteria present within the neuston or sea surface microlayer. Bacteria within this layer were sampled using a membrane filter technique and bacterial diversity was compared with that in the underlying pelagic coastal seawater using molecular ecological techniques. 16S rRNA gene libraries of approximately 500 clones were constructed from both bacterioneuston and the pelagic water samples and representative clones from each library were sequenced for comparison of bacterial diversity. The bacterioneuston was found to have a significantly lower bacterial diversity than the pelagic seawater, with only nine clone types (ecotaxa) as opposed to 46 ecotaxa in the pelagic seawater library. Surprisingly, the bacterioneuston clone library was dominated by 16S rRNA gene sequences affiliated to two groups of organisms, Vibrio spp. which accounted for over 68% of clones and Pseudoalteromonas spp. accounting for 21% of the library. The dominance of these two 16S rRNA gene sequence types within the bacterioneuston clone library was confirmed in a subsequent gene probing experiment. 16S rRNA gene probes specific for these groups of bacteria were designed and used to probe new libraries of 1000 clones from both the bacterioneuston and pelagic seawater DNA samples. This revealed that 57% of clones from the bacterioneuston library hybridized to a Vibrio sp.-specific 16S rRNA gene probe and 32% hybridized to a Pseudoalteromonas sp.-specific 16S rRNA gene probe. In contrast, the pelagic seawater library resulted in only 13% and 8% of 16S rRNA gene clones hybridizing to the Vibrio sp. and Pseudoalteromonas sp. probes respectively. Results from this study suggest that the bacterioneuston contains a distinct population of bacteria and warrants further detailed study at the molecular level.     

Stratification of microorganisms and nutrients in the surface microlayer of small freshwater ponds

Using two different surface microlayer sampling devices (the Harvey and Burzell glass plate and the Garrett screen), two microlayer fractions could be distinguished. The first was from 0 to 50 µm and the second was from 51 to 320 µm. Significantly different (p < 0.05) concentrations of dissolved nutrients between the two microlayer fractions strongly suggests stratification within the surface microlayer. This apparent stratification is also examined for phototrophs, bacteria and other material found within the surface microlayer.Based in part on an M.S. thesis, submitted to the Graduate School of University of Wisconsin - Milwaukee.     

The relationship of individual algal species to the surface microlayer of a small freshwater pond

Surface microlayer and subsurface algal communities were comparedon 14 sample dates over a 5 month period in a small Wisconsinpond. Similarities in species composition indicate that themicrolayer community had many species in common with the subsurfacecommunity. Relative species abundances, however, were differentfrom surface to subsurface. Analysis of the depth distributionof individual species indicates that the response to the uniquephysical/chemical conditions in the microlayer is species specific,with flagellates often in low numbers in the surface microlayeras a result of negative phototaxis; and diatoms often in highnumbers in the surface microlayer, as a result of a specificphysical/chemical affinity. These species specific differencesmay help to explain phytoneuston enrichment found in other studies. ¹Present address: University of Rhode Island, Department ofOceanography, Kingston, R1 02881, USA.²Author to whom reprint requests should be directed.     

Isolation of surfactant-resistant pseudomonads from the estuarine surface microlayer

Bioremediation efforts often rely on the application of surfactants to enhance hydrocarbon bioavailability. However, synthetic surfactants can sometimes be toxic to degrading microorganisms, thus reducing the clearance rate of the pollutant. Therefore, surfactant-resistant bacteria can be an important tool for bioremediation efforts of hydrophobic pollutants, circumventing the toxicity of synthetic surfactants that often delay microbial bioremediation of these contaminants. In this study, we screened a natural surfactant-rich compartment, the estuarine surface microlayer (SML), for cultivable surfactant-resistant bacteria using selective cultures of sodium dodecyl sulfate (SDS) and cetyl trimethylammonium bromide (CTAB). Resistance to surfactants was evaluated by colony counts in solid media amended with critical micelle concentrations (CMC) of either surfactants, in comparison with non-amended controls. Selective cultures for surfactant-resistant bacteria were prepared in mineral medium also containing CMC concentrations of either CTAB or SDS. The surfactantresistant isolates obtained were tested by PCR for the Pseudomonas genus marker gacA gene and for the naphthalene-dioxygenase-encoding gene ndo. Isolates were also screened for biosurfactant production by the atomized oil assay. A high proportion of culturable bacterioneuston was tolerant to CMC concentrations of SDS or CTAB. The gacA-targeted PCR revealed that 64% of the isolates were Pseudomonads. Biosurfactant production in solid medium was detected in 9.4% of tested isolates, all affiliated with genus Pseudomonas. This study shows that the SML is a potential source of surfactant-resistant and biosurfactant-producing bacteria in which Pseudomonads emerge as a relevant group.     

Negative staining of freshwater bacterioneuston sampled directly with electron microscope specimen support grids

A technique for observation of surface microlayer bacteria (bacterioneuston) is described, utilizing direct sampling of the air-water interface with carbon-stabilized electron microscope specimen support grids, followed by negative staining and transmission electron microscopy. The method resulted in excellent preservation of forms of microcolonial association, regular surface arrays, surface appendages, and prosthecae in the bacterioneuston of a freshwater pond.     

The mechanism of self-organization in a surface water microlayer utilizing thermocapillary convection

The formation of helical macrostructures in a surface-water layer has been experimentally studied. The thermocapillary Marangoni convection bringing about self-organizing helical and dissipative structures in a thin subsurface layer of cooling water with a free surface was demonstrated in an experiment for the first time. The most likely candidates that allow the motion in the corresponding basic experiments were found. These candidates are thermocapillary cells that result from Marangoni convection. The described mathematical tools for modeling the dynamics of thermocapillary Marangoni diffusion are the nonlinear equations of heat diffusion and the equations of self-organization (nonlinear differential equations of a parabolic type). The results of a computational experiment confirmed the mechanisms of self-organization in a surface-water layer.     

Relation between bacterial activity in the surface microlayer and estuarine hydrodynamics

Bacterial communities of the surface microlayer (SML) of the estuary Ria de Aveiro (Portugal) were characterized in terms of abundance and activity during a 2-year survey at two sites with distinct hydrodynamic properties (marine and brackish water zones). The hydrodynamic conditions were simulated using a bidimensional numerical model and related to the microbiological observations. The pattern of variation of bacterial biomass productivity (BBP) was distinct between the two sampling sites. At the outer site, BBP was significantly lower at the SML, whereas at the inner site, it was significantly enhanced at the SML. Although the total bacterial abundance was similar in the SML and underlying water (UW), the fraction of cells attached to particles was significantly higher at the SML (two to three times). The integration of microbiological results with environmental and hydrological variables shows that strong currents in the marine zone promote the vertical mixing, inhibiting the establishment of an SML bacterial community distinct from that of UW. In contrast, in the brackish water zone, lower current velocities provide conditions for enhancing the bacterial activity in the enriched SML. Estuarine dynamics influence the distribution and activity of microorganisms at the SML and in the water column, with anticipated impacts for the carbon cycle in the estuarine environment.     

Prey selection and diel feeding of the freshwater jellyfish, Craspedacusta sowerbyi

1. The diet of the invasive freshwater jellyfish, Craspedacusta sowerbyi (Lankester), was assessed by analysing its stomach contents. 2. The medusae ingested various zooplankton and benthic prey in the 0.1–3.0 mm size range. The selectivity indices for prey showed that larger zooplankton (0.4–1.4 mm) and active prey such as copepods were preferred; small potential prey, and the loricate rotifer Keratella cochlearis in particular, were almost never consumed. Measurements suggest that spacing between the tentacle branches could determine prey selection. Another possibility is that some prey are too small or slow to activate nematocysts after collision with the predator. Prey > 1.4 mm can probably escape or are otherwise too large to be handled. 3. The medusae of Craspedacusta medusae stayed in deep water during daytime and migrated upwards at night. The greatest number of prey in freshwater jellyfish stomachs was detected at night. 4. Calculation of consumption rates, based on mean stomach contents and digestion times of 4–5 h, showed that, on average, one medusa takes 190 zooplankton prey day^??1 at a mean prey density of about 100 L^??1. At the observed medusae density of 1 m^??3, the cropping rate was about 0.2 zooplankton prey L^??1 day^??1.     

Photosynthetic capacity at the surface microlayer during the mixing period

The pbytoplankton biomass and primary production of the seasurface microlayer (upper 3 mm) and the bulk water (1, 10, 20,30 and 40 m) were investigated in Saronicos Gulf, Aegean Sea,during the period of November-December 1987. The experimentswere performed by using newly developed sampling and incubationin situ techniques regarding the surface microlayer. In thewell mixed water column, maximum assimilation ratios rangingfrom 2.63 to 10.97 mg C mg Chla^–1 h^–1 were recordedat 1 m depth and these values were reduced to {small tilde}45%at the surface microlayer regardless of the uniform distributionof chlorophyll a in these layers. The 1% light level variedbetween 20 and 40 m depth and the assimilation ratio at thislevel averaged 12% in relation to the maximum value at 1 m depth.The problems associated with the static bottle incubations andthe type of glass of the bottles used are discussed with respectto the photosynthetic measurements at the surface microlayer.     

Dissolved organic carbon and bacterial populations in the gelatinous surface microlayer of a Norwegian fjord mesocosm

The sea surface microlayer is the interfacial boundary layer between the marine environment and the troposphere. Surface microlayer samples were collected during a fjord mesocosm experiment to study microbial assemblage dynamics within the surface microlayer during a phytoplankton bloom. Transparent exopolymer particles were significantly enriched in the microlayer samples, supporting the concept of a gelatinous surface film. Dissolved organic carbon and bacterial cell numbers (determined by flow cytometry) were weakly enriched in the microlayer samples. However, the numbers of Bacteria 16S rRNA genes (determined by quantitative real-time PCR) were more variable, probably due to variable numbers of bacterial cells attached to particles. The enrichment of transparent exopolymer particles in the microlayer and the subsequent production of a gelatinous biofilm have implications on air–sea gas transfer and the partitioning of organic carbon in surface waters.     

A survey on bacteria inhabiting the sea surface microlayer of coastal ecosystems

Bacterial populations inhabiting the sea surface microlayer from two contrasted Mediterranean coastal stations (polluted vs. oligotrophic) were examined by culturing and genetic fingerprinting methods and were compared with those of underlying waters (50 cm depth), for a period of two years. More than 30 samples were examined and 487 strains were isolated and screened. Proteobacteria were consistently more abundant in the collection from the pristine environment whereas Gram-positive bacteria (i.e., Actinobacteria and Firmicutes) were more abundant in the polluted site. Cythophaga-Flavobacter-Bacteroides (CFB) ranged from 8% to 16% of total strains. Overall, 22.5% of the strains showed a 16S rRNA gene sequence similarity only at the genus level with previously reported bacterial species and around 10.5% of the strains showed similarities in 16S rRNA sequence below 93% with reported species. The CFB group contained the highest proportion of unknown species, but these also included Alpha- and Gammaproteobacteria. Such low similarity values showed that we were able to culture new marine genera and possibly new families, indicating that the sea-surface layer is a poorly understood microbial environment and may represent a natural source of new microorganisms. Genetic fingerprinting showed, however, no consistent differences between the predominant bacterial assemblages from surface microlayer and underlying waters, suggesting that the presence of a stable and abundant neustonic bacterial community is not a common trait of coastal marine environments.     

Seasonal and diel relationships between the isotopic compositions of dissolved and particulate organic matter in freshwater ecosystems

A study of the isotopic composition of organic matter was conducted in a freshwater marsh over seasonal and diel time scales to determine the sources of dissolved organic matter (DOM) and the processes leading to its formation. Bulk C and N isotopic compositions of the bacterial fraction (0.2–0.7 m) and particulate organic matter (POM; 0.7–10 m) were compared on a seasonal basis with the change in ¹³C of DOM. The bulk isotopic data support the idea that DOM was, in part, derived from the breakdown of larger organic matter fractions. The bacterial fraction and POM were compositionally similar throughout the year, based on a comparison of the ¹³C of individual amino acids in each fraction. Annual variation in the ¹³C of amino acids in DOM was greater relative to the variation in larger fractions indicating that microbial reworking was an important factor determining the proteinaceous component of DOM. The ¹³C enrichment of serine and leucine in each organic matter fraction suggested microbial reworking was an important factor determining organic matter composition during the most productive times of year. Changes in the bulk ¹³C of DOM were more significant over daily, relative to seasonal, time scales where values ranged by 6 and followed changes in chlorophyll a concentrations. Although bulk ¹³C values for POM ranged only from –29 to –28 during the same diel period, the ¹³C of alanine in POM ranged from –30 to –22. Alanine is directly synthesized from pyruvate and is therefore a good metabolic indicator. The ¹³C of individual amino acids in DOM revealed the diel change in the importance of autotrophic versus heterotrophic activity in influencing DOM composition. Diel changes in the ¹³C of phenylalanine, synthesized by common pathways in phytoplankton and bacteria, were similar in both DOM and POM. The diel change in ¹³C of isoleucine and valine, synthesized through different pathways in phytoplankton and bacteria, were distinctly different in DOM versus POM. This disparity indicated a decoupling of the POM and DOM pools, which suggests a greater source of bacterial-derived organic matter at night. The results of this study demonstrate the use of the isotopic composition of individual amino acids in determining the importance of microbial reworking and autotrophic versus heterotrophic contributions to DOM over both diel and seasonal time scales.     

Sex-specific metabolic changes in the annual reproductive cycle of a freshwater catfish

Sex-specific enzymatic and other biochemical changes were studied in the annual reproductive cycle of the freshwater catfish, Clarias batrachus. Citrate synthase (CS) activity of brain, liver and skeletal muscle was maximum in spawning and minimum in postspawning showing a sharp decline in aerobic capacity after spawning. Similar CS activity in remaining phases of the annual reproductive cycle reflects similar energy need during regressed, preparatory and prespawning phases. Glucose 6-phosphate dehydrogenase (G6-PDH) activity declined in spawning and postspawning indicating a possible decrease in lipid and nucleic acid syntheses. The subsequent increase in G6-PDH activity with onset of resting and maintenance of the increased level throughout preparatory and prespawning phases shows restoration of biosynthetic activity. Higher activity of G6-PDH in female than male may be to satisfy a greater biosynthetic need of female reproduction and breeding. The decreased RNA content of tissues showed reduction in protein synthesis capacity during spawning and subsequent increase through postspawning until resting phase. The RNA content of brain and liver was higher in female than male during preparatory and prespawning, which may be associated with higher protein synthesis requirement of female for preparation of reproductive activities. The requirement based sex related changes in metabolism of catfish may be enzyme, tissue or reproductive phase-specific.     

To migrate, or not to migrate: partial diel horizontal migration of fish in a temperate freshwater reservoir

The diel horizontal migration (DHM) of fish between the inshore and offshore zones of the ?ímov Reservoir (Czech Republic, deep, stratified, meso-eutrophic) was investigated by a combination of horizontal and vertical hydroacoustic surveys at 3-h intervals over 48 h and day/night purse seining in August 2007. An overwhelming majority of fish were aggregated within the epilimnetic layer. Considering only the horizontal surveys, cyclic diel fish movements between inshore and offshore habitats were apparent, while the total fish biomass remained constant between day and night. A higher fish biomass was detected in the offshore zone during daytime by both hydroacoustics and purse seining. In contrast, a higher fish biomass was recorded at night in the inshore zone. Bream Abramis brama, roach Rutilus rutilus, and perch Perca fluviatilis dominated the daytime offshore fish assemblage whereas bleak Alburnus alburnus prevailed at night. Bream and roach decreased in abundance at night while perch completely disappeared from the offshore habitat. The diel differences in size distributions and direct catches suggested the population-wide horizontal offshore migration of bleak and inshore migration of all perch during dusk. On the other hand, partial inshore migration of bream and roach adults was observed during dusk (52 and 80%, respectively). The different proportions of offshore residents among species and size classes suggested that differences in size, and, therefore, predation vulnerability, contributed to the observed migration patterns.     

Influence of the surface microlayer on nutrients,chlorophyll and algal diversity of a small eutrophic bog pond

Nutrients, chlorophyll, phaeophytin and algal abundances were investigated in the surface microlayer and at subsurface depths in a small eutrophic bog pond. Nutrient levels were consistently higher in the microlayer while algal abundance was sometimes higher but sometimes lower in the microlayer than at near subsurface depths. Algal diversity values were strongly influenced by the depth of flagellate blooms, and in contrast to previous studies, diversity in the microlayer was higher than at near subsurface depths. These results are discussed in terms of weather parameters, affinity of algal species for the surface and differences between microlayer ecology in shallow and deep water systems.     

Role of aquatic surface microlayer in the dynamics of nutrients and organic compounds in lakes,with implications for their ecotones

The origin and mechanisms of formation of aquatic surface microlayers are reviewed, and processes within the layer responsible for enrichment of inorganic and organic substances, particles, and microorganisms are discussed. In a study of the chemical and biological composition of microlayers in three lakes of different levels of productivity, surface slicks were sampled with a revolving cylinder coated with hydrophilic Teflon. DOC, POC, DON and various forms of phosphorus were enriched in the microlayer compared to those in the subsurface water. In eutrophic and oligotrophic lakes the DOC:DON ratio shows that the slicks were more influenced by allochtonous sources than was the subsurface water, which indicates that processes in the ecotone influence the microlayer composition.     

The effect on Aphis fabae of diel changes in their food quality

ABSTRACT. Marked diurnal changes in total soluble carbohydrates were found in broad bean leaves, with a maximum of c. 11% of dry weight at 18.00 hours to a minimum of c. 4% at 06.00 hours. If reflected in the phloem sap, this would mean that aphids receive different food from plants by night and by day. The growth rate of Aphis fabae was therefore compared on broad bean plants under light and darkness. Although the aphids showed the same mean gross growth rate under both conditions, those in the dark had a higher final percentage moisture content, their dry weight increase being negligible. Counts of honeydew droplets on a 'honeydew clock' indicated a reduction in excretion during the dark. Measurements of haemolymph concentration of Acyrthosiphon pisum were obtained by comparison of haemolymph vapour pressure with that of known sucrose solutions, using capillary tubes. Haemolymph samples collected at the end of the day showed a higher (mean 11.53 atm) and more variable osmotic pressure than those obtained at the end of the night (mean 8.62 atm). The more dilute sap in the plant at night may benefit the aphid by allowing it to compensate for osmotic stress incurred during daytime feeding. These results suggest that the diurnal change in total soluble carbohydrates in leaves of the broad bean is reflected in the phloem sap.     

Parasite-induced changes in the diet of a freshwater amphipod: field and laboratory evidence

Trophically transmitted parasites are likely to strongly influence food web-structure. The extent to which they change the trophic ecology of their host remains nevertheless poorly investigated and field evidence is lacking. This is particularly true for acanthocephalan parasites whose invertebrate hosts can prey on other invertebrates and contribute to leaf-litter breakdown. We used a multiple approach combining feeding experiments, neutral lipids and stable isotopes to investigate the trophic ecology of the freshwater amphipod Gammarus roeseli parasitized by the bird acanthocephalan Polymorphus minutus. Infected compared to uninfected amphipods consumed as many dead isopods, but fewer live isopods and less leaf material. Infection had no influence on the total concentration of neutral lipids. Contrary to what we expected based on laboratory findings, the nitrogen isotope signature, which allows for the estimation of consumer's trophic position, was not influenced by infection status. Conversely, the carbon isotope signature, which is used to identify food sources, changed with infection and suggested that the diet of infected G. roeseli includes less perilithon (i.e. fixed algae on rocks, stones) but more terrestrial inputs (e.g. leaf material) than that of uninfected conspecifics. This study shows evidence of changes in the trophic ecology of P. minutus-infected G. roeseli and we stress the need to complement feeding experiments with field data when investigating top-down effects of infection in an opportunistic feeder which adapts its diet to the available food sources.     

Long-term changes in strontium-90 concentrations within a freshwater predator-prey system

It proved possible to determine the levels of Sr-90 in the opercular bones of individual pike, Esox Iucius , and in pooled samples of bones from perch, Perca fluviatilis . Results from both species from Windermere demonstrated that Sr-90 levels rose from below the detection limits in the 1940s to a peak in the 1960s, followed by a decline in the subsequent two decades. This decline was slower than would have been expected from the decline in northern hemisphere Sr-90 fallout, indicating the likelihood of recycling within the environment. Sr-90 levels were consistently lower in pike than in perch, their main prey fish. Thus, there is no concentration of Sr-90 up this part of the aquatic food chain. Tracking Sr-90 in bones taken in successive years from ages 3 to 8 for a single cohort of pike showed that the quantity of Sr-90 was closely related to opercular bone (and hence fish) weight. No significant increase in Sr-90 concentration in the bone with increasing age was demonstrated.