Wastewater pollution differently affects the antibiotic resistance gene pool and biofilm bacterial communities across streambed compartments

Wastewater discharges introduce antibiotic residues and antibiotic‐resistant bacteria (ARB) into surface waters. Both inputs directly affect the streambed resistome, either by exerting a selective pressure that favour the proliferation of resistant phenotypes or by enriching the resident communities with wastewater‐associated ARB. Here, we investigated the impact of raw and treated urban wastewater discharges on epilithic (growing on rocks) and epipsammic (growing on sandy substrata) streambed biofilms. The effects were assessed by comparing control and impact sites (i) on the composition of bacterial communities; (ii) on the abundance of twelve antibiotic resistance genes (ARGs) encoding resistance to β‐lactams, fluoroquinolones, sulphonamides, tetracyclines, macrolides and vancomycin, as well as the class 1 integron‐integrase gene (intI1); (iii) on the occurrence of wastewater‐associated bacteria, including putative pathogens, and their potential linkage to target ARGs. We measured more pronounced effects of raw sewage than treated wastewater at the three studied levels. This effect was especially noticeable in epilithic biofilms, which showed a higher contribution of wastewater‐associated bacteria and ARB than in epipsammic biofilms. Comparison of correlation coefficients obtained between the relative abundance of both target ARGs and operational taxonomic units classified as either potential pathogens or nonpathogens yielded significant higher correlations between the former category and genes intI1, sul1, sul2 and ermB. Altogether, these results indicate that wastewater‐associated micro‐organisms, including potential pathogens, contribute to maintain the streambed resistome and that epilithic biofilms appear as sensitive biosensors of the effect of wastewater pollution in surface waters.     

Flow-substrate interactions create and mediate leaf litter resource patches in streams

1. The roles that streambed geometry, channel morphology, and water velocity play in the retention and subsequent breakdown of leaf litter in small streams were examined by conducting a series of field and laboratory experiments. 2. In the first experiment, conditioned red alder (Alnus rubra Bongard) leaves were released individually in three riffles and three pools in a second‐order stream. The transport distance of each leaf was measured. Several channel and streambed variables were measured at each leaf settlement location and compared with a similar number of measurements taken at regular intervals along streambed transects (‘reference locations’). Channel features (such as water depth) and substrate variables (including stone height, stone height‐to‐width ratio, and relative protrusion) were the most important factors in leaf retention. 3. In the second experiment, the role of settlement location and reach type in determining the rate of leaf litter breakdown was examined by placing individual conditioned red alder leaves in exposed and sheltered locations (on the upper and lower edges of the upstream face of streambed stones, respectively) in riffle and pool habitats. After 10 days, percent mass remaining of each leaf was measured. Generally, leaves broke down faster in pools than in riffles. However, the role of exposure in breakdown rate differed between reach types (exposed pool > sheltered pool > sheltered riffle > exposed riffle). 4. In the third experiment, the importance of substrate geometry on leaf litter retention was examined by individually releasing artificial leaves upstream of a series of substrate models of varying shape. Substrates with high‐angle upstream faces (were vertical or close to vertical), and that had high aspect ratios (were tall relative to their width), retained leaves more effectively. 5. These results show that streambed morphology is an important factor in leaf litter retention and breakdown. Interactions between substrate and flow characteristics lead to the creation of detrital resource patchiness, and may partition leaf litter inputs between riffles and pools in streams at baseflow conditions.     

Resolving resource partitioning in parrotfishes (Scarini) using microhistology of feeding substrata

Parrotfishes (Scarini) are considered key agents in coral reef health and recovery, but the drivers of parrotfish–coral dynamics remain contentious. The prevailing view of parrotfishes as ecosystem engineers is based on the perceived removal of algal turf, macroalgae and sediment, but these are effects of feeding, not causes. The recent proposal that most parrotfishes are ‘microphages’ that target microscopic photoautotrophs (particularly cyanobacteria) identifies the need to resolve dietary targets at a microscopic scale. Here, we investigate parrotfish dietary targets by posing the following two questions: (1) are microscopic photoautotrophs the most consistent and dominant elements of the prey community, and (2) do the prey community and substratum taphonomy vary between parrotfish species? In order to identify and quantify dietary targets, five parrotfish species were followed until focused feeding was observed at Lizard Island on the Great Barrier Reef, Australia. Feeding sites were photographed in situ and extracted as substratum bite cores. Cores were analysed microscopically to identify and quantify all epilithic photoautotrophs. Endolithic photoautotrophs accessible to excavating parrotfish were also investigated by vacuum-embedding cores with epoxy resin followed by decalcification to expose endolith microborings. The dominant functional groups of epilithic biota on the cores were tufted cyanobacteria, turfing algae and crustose coralline algae (CCA). The only consistent feature across all cores was the high density of filamentous cyanobacteria, supporting the view that these parrotfishes target microphotoautotrophs. Macroalgae was absent or a minor component on cores, supporting the hypothesis that parrotfishes avoid larger algae. The microchlorophyte Ostreobium was the dominant photoautotrophic euendolith (true borer) in the cores of the excavating parrotfish Chlorurus microrhinos. Significant differences in CCA coverage, turf height and substrate taphonomy were found among the five parrotfish species, suggesting that interspecific resource partitioning is based on successional stage of feeding substrata.

     

Ecological Effects of Serial Impoundment on the Cotter River, Australia

This study examines the ecological effects of serial impoundments (three dams) on a rocky upland stream in southeastern Australia. Physical, chemical and biological changes were quantified and interpreted within a three-level hierarchy of effects model developed previously by Petts [1984, Impounded Rivers. John Wiley and Sons, New York] and the Australian Rivers Assessment System (AUSRIVAS) to predict pre-dam biota. First-order effects were decreased median monthly discharges and floods of lesser magnitude following construction of the dams. No effect on water characteristics (pH, electrical conductivity and major ions) was evident. The second-order effect on channel morphology was a decrease in bank-full cross-sectional area by up to 75% because of reduced flows. At all sites, the predominantly cobble streambed was armoured and generally highly stable. The discharge required to initiate movement of the streambed surface sediments (38.9 m³ s⁻¹) was 40% less frequent since construction of the dams, implying alteration to the natural disturbance regime for benthic biota. Benthic algal growth appeared more prolific at sites directly below dams. Fewer macroinvertebrate taxa than expected and modified assemblages within 1 km of all three dams were third-order effects. Compared to reference conditions, macroinvertebrate samples from the sites directly below the dams had relatively more Chironomidae larvae, Oligochaeta and Acarina, and fewer of the more sensitive taxa, Plecoptera, Ephemeroptera, Trichoptera and Coleoptera. Biological recovery to the macroinvertebrate assemblage was evident within 4 km downstream of the second dam.     

Temperate bioerosion: ichnodiversity and biodiversity from intertidal to bathyal depths (Azores)

In the temperate Azores carbonate factory, a substantial fraction of the calcareous skeletal components is recycled by a remarkable biodiversity of biota producing bioerosion traces (incipient trace fossils). To study this biodiversity, experimental carbonate substrates were exposed to colonisation by epilithic and endolithic organisms along a bathymetrical gradient from 0 to 500 m depth, during 1 and 2 years of exposure. The overall bioerosion ichnodiversity is very high and comprises 56 ichnotaxa and ichnoforms attributed to cyanobacteria, chlorophytes, fungi, other micro-chemotrophs, macroborers, grazers and epilithic attachment scars. In the intertidal, hydrodynamic force, partial emersion and strong temperature fluctuations lead to the lowest ichnospecies richness. This contrasts with the highest ichnodiversity found at 15 m under the most favourable environmental conditions. Towards aphotic depths, a gradual depletion in ichnodiversity is observed, most probably because of the restricted light availability and a slowdown in ichnocoenosis development. Analysis of similarity (ANOSIM), in combination with non-metrical multidimensional scaling (NMDS), was used to highlight variability in the relative abundance of traces among depths, substrate orientations and exposure times. Ichnodiversity and abundance of traces decrease significantly with depth and are higher on up-facing versus down-facing substrates, whereas differences between years were not as pronounced. This study demonstrates that statistical methods of biodiversity analysis are not per se restricted to biotaxa but may well be applied also to ichnotaxa. In the analysis of trace fossil assemblages, this approach supports the recognition of diversity patterns and their relation to environmental gradients.     

Die-Away Kinetic Analysis of the Capacity of Epilithic and Planktonic Bacteria from Clean and Polluted River Water To Biodegrade Sodium Dodecyl Sulfate

The capacities of epilithic and planktonic river bacterial populations to degrade sodium dodecyl sulfate (SDS) in samples taken at two times during 1987 from one clean and four polluted sites in a South Wales river were estimated in die-away tests under simulated environmental conditions. There was a relatively slow disappearance of SDS in die-away tests for both planktonic and epilithic populations taken from the clean source site, as compared with those taken from the downstream polluted sites, for which the rate of biodegradation was accelerated, sometimes after an apparent initial lag period. The kinetic components contributing to the die-away curves were quantified by nonlinear regression analysis in which the experimental data were fitted to a variety of possible kinetic models. All samples except for one from the polluted sites best fitted a model which describes the biodegradation of SDS at concentrations well below its K_m by bacteria whose growth is exponential and unaffected by the addition of a test substrate. Each sample from the clean source site fitted a different model, but there was generally little or no growth on endogenous carbon. A consideration of the numerical values of constants derived from the modeling of epilithic and planktonic populations from polluted sites indicated clearly that the biodegradative capacity of epilithic bacterial populations towards SDS is more stable than that of planktonic bacterial populations.     

Epilithic Algae Distribution Along a Chemical Gradient in a Naturally Acidic River, Río Agrio (Patagonia, Argentina)

The epilithic algae distribution along a pH gradient and the relationship between the chemical gradient and biomass development were studied in Río Agrio, a naturally acidic river located in Patagonia (Argentina). The epilithic community was monitored during the summer of three consecutive years in sites located above and below the entrance of tributaries. The epilithic community showed differences between sites based on the chemical composition of the water and the precipitates that appear on the streambed of the river. The lowest biomass, diversity, and number of species were found at the most extreme part of the river in terms of pH (ca. 2) and element concentrations. Euglena mutabilis was the dominant species in this section of the river. As pH increased (ca. 3), the community changed to be dominated by filamentous green algae (Ulothrix spp., Mougeotia sp., Klebsormidium sp.) showing luxuriant growths in terms of biomass. With the inflow of a neutral tributary, the pH of Río Agrio increased above 3, and the precipitates of orange-red iron hydroxides appeared. The algal community was not affected by these precipitates or the low P concentrations, along the next 30 km of river downstream from this site. The apparent physical stress that the precipitates impose on algae is in fact a dynamic reservoir of P because diel cycle of Fe could be promoting precipitation and redissolution processes that binds and releases P from these precipitates. Where the pH increased above 6, precipitates of aluminum hydroxides appeared. At this site, the epilithic biomass and density decreased, some algae species changed, but the diversity and the number of species in general remained consistent with the upstream values. The physical stress of the Al precipitates on the algae is added to the chemical stress that represents the sequestering of P in these precipitates that are not redissolved, resulting P a limiting nutrient for algae growth.     

Are the oral teeth of combtooth bennies (Salariini: Teleostei) merely combs?

Careful analysis of gut contents has resulted in the reclassification of several species of grazing fish as detritivores, shifting the trophodynamic assignment of many prominent reef grazers. Combtoothed blennies, which are among the most numerous grazing fish of the Great Barrier Reef, have been shown to target the detrital component of the epilithic algal matrix (EAM). It has been suggested that blennies have specialized dental morphology that allows them to comb through fronds of algae, collecting detritus, while leaving the algal component intact. In this study, we analysed the capability of a common reef flat blenny, Salarias fasciatus, to remove algae by (i) examining oral morphology for evidence of wear and adaptations for abrasion, and (ii) a short-term EAM feeding experiment. Examination of S. fasciatus teeth with scanning electron microscopy (SEM) showed evidence of macrowear (changes in tooth height or shape), microwear (surface chips and striae), and tooth replacement that suggests abrasion on the substrate. Energy Dispersive X-ray Spectrometry (EDS) revealed that oral teeth incorporate mineral iron. When fed artificial substrata with a developed EAM layer S. fasciatus removed 57% of photosynthetic material and 38.5% of organic biomass. Although studies have found that blenny gut contents consist predominantly of detritus, blennies are still likely to contribute to the removal of algae on coral reefs.     

The effect of wind exposure and filamentous algae on the distribution of surf zone macroinvertebrates in Lake Esrom,Denmark

Distribution and composition of the epilithic macroinvertebrate fauna was studied on 21 different sites situated in the surf zone of Lake Esrom. A relative degree-of-exposure (windinduced wave activity) was calculated for each station from the specific number of wind days, wind velocity and a measured wind fetch. Macroinvertebrate abundance averaged 20 200 ind. m^–2 stone bottom, and was composed primarily of six taxa (70% of the mean densities). The distribution patterns of the 48 most common taxa were compared to exposure, the amount of filamentous algae and shore slope, by regression, cluster analysis and ordination analysis (PCA and RDA). The abundance of 25 taxa was significantly correlated to the amount of filamentous algae, and a protectional effect of algae growth was suggested for the exposed sites. Seventeen taxa showed significant difference in abundance between sheltered western stations and exposed eastern stations. Stations with a gentle slope were characterised by sand covered stones and a fauna that qualitatively was similar to the fauna at greater depth in the littoral. Redundancy analysis (RDA) showed filamentous algae and slope to be the most important factors controlling the species distribution, whereas the gradient of exposure in Lake Esrom was too short to explain the species tolerance range.     

Isolation of Marine Bacteria by In Situ Culture on Media-Supplemented Polyurethane Foam

Polyurethane foam (PUF) supplemented with various agar media was used in situ to trap marine bacteria and it consequently provided a substrate on which they could be cultivated while exposed to natural seawater in the coral reef area. The bacterial population on the PUF blocks was analyzed by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. Changing the composition of the cultivation medium in the PUF blocks and selecting different sampling sites resulted in different bacteria being detected on the PUF blocks. For example, iron-utilizing (IU) bacteria, siderophore-producing (SP) bacteria, and petroleum-degrading (PD) bacteria were isolated from PUF blocks and it was discovered that IU and SP contained iron and PD contained hydrocarbon. This method opens up the possibility for isolating novel and useful marine bacteria.     

Interactions between detritivores and microfungi during the leaf detritus decomposition in a volcanic lake (Lake Vico – central Italy)

The role of biota in the mass loss of Phragmites australis (Cav.) Trin. ex Steud was studied in the littoral belt of a central Italy volcanic lake. The research focussed on the feeding interactions between detritivores and decomposing fungi as drivers of the leaf litter decomposition. The litterbag technique was used to assess the leaf mass loss, the number of colonizing fungi and the patterns of leaf colonization by detritivores during 40 days of submersion in 16 sampling sites. Cores of bottom sediment were collected to estimate the organic content and ergosterol concentration as measure of fungal mass. The rate of leaf mass loss showed significant variability among the sampling sites and was non-linearly related to the quantity of organic depositions onto the lake bottom, peaking at about 40% of the dry matter. The rate was also positively correlated with the density of detritivore mass relative to the leaf unit mass, which increased with time. On the 20th day of litterbag immersion, when 40% of the initial leaf litter remained, we observed the best accordance between the two measures as well as the lowest difference in the detritivore mass density among sampling sites. In the absence of animals, the decomposition rate was positively related to the number of fungi on the decaying litter. The feeding activity of detritivores changed both the species richness and composition of the fungal community on the litter. The substrate reduction due to intense animal feeding appeared to limit the ability of fungi to regrow after grazing. As a result, an inverse relationship between the number of fungi and the decomposition rate was observed.     

The impact of physical disturbance on stream communities: lessons from road culverts

This study examined the impact of physical disturbance from long-established road culverts on stream macroinvertebrate communities. Three streams within a 6 km section of highway on the Avalon Peninsula, Newfoundland, Canada, were sampled. Streams had the entire upstream watershed and at least 100 m downstream of the road with natural boreal forest/barren vegetation and all had, within the sampled reaches, similar physical streambed characteristics. The fauna on stones from riffles was sampled at two upstream and three downstream sites, i.e., from 50 m above to about 100 m below the road in each stream. A total of 33 taxa were identified among the streams, with differences limited to a few rare taxa. The sample site communities did not significantly differ from each other with respect to the taxa present. Total macroinvertebrate abundance by site, for combined data of all streams, indicated the site at the exit of culvert plunge pool (site 3) had significantly elevated abundances. Analysis of individual taxa showed this was primarily due to very high numbers of Simulium spp. The other most notable changes were a decrease in numbers of Hydropsyche spp. and Elmidae below the road. The abundances of the remaining taxa were more variable among all sites. The study indicated that long-standing point source physical disturbance primarily impacted taxa abundance rather than community present/absent data, which will recolonize the disturbed zone by downstream drift. The differences in abundance are probably the result of the cleaning of substrate by abrasion, movement of substrate and reduction of detritus during each spate. Handling editor: D. Dudgeon     

Recurring seasonal dynamics of microbial communities in stream habitats

Recurring seasonal patterns of microbial distribution and abundance in three third-order temperate streams within the southeast Pennsylvania Piedmont were observed over 4 years. Populations associated with streambed sediments and rocks (epilithon) were identified using terminal restriction length polymorphism (tRFLP) and sequencing of 16S rRNA genes selectively amplified with primers for the bacterial domain. Analyses of the relative magnitudes of tRFLP peak areas by using nonmetric multidimensional scaling resolved clear seasonal trends in epilithic and sediment populations. Oscillations between two dominant groups of epilithic genotypes, explaining 86% of the seasonal variation in the data set, were correlated with temperature and dissolved organic carbon. Sequences affiliated with epilithic phototrophs (cyanobacteria and diatom chloroplasts), a Rhodoferax sp., and a Bacillus species clustered in the summer, whereas sequences most closely related to "Betaproteobacteria" (putative Burkholderia sp.), and a putative cyanobacterium clustered in the fall/spring. The sediment genotypes also clustered into two groups, and these explained 85% of seasonal variation but correlated only with temperature. A summer tRFLP pattern was characterized by prevalence of "Betaproteobacteria," "Gammaproteobacteria," and a Bacillus sp., whereas the winter/spring pattern was characterized by phylotypes most closely related to "Firmicutes," "Gammaproteobacteria," and "Nitrospirae." A close association between these headwater streams and their watersheds was suggested by the recovery of sequences related to microbial populations provisionally attributed to not only freshwaters but also terrestrial habitats.     

Habitat selection by two streamside plethodontid salamanders

Summary A series of experiments was conducted to test the hypothesis that habitat selection by the salamanders Desmognathus monticola and Desmognathus quadramaculatus is influenced differently by interspecific interference than by intraspecific interference. Individuals of neither species were influenced differently by the presence of heterospecifics than by the presence of conspecifics in their activity away from cover sites, in selection of different substrate moisture levels, in selection of different substrate textures, nor in selection of microhabitats relative to the streambed. Both species generally selected substrates with the highest moisture and the coarsest texture. Selection of different cover sizes by D. monticola depended on microhabitat, with larger rocks being selected most frequently when salamanders were farther from the streambed. D. quadramaculatus was seldom found away from the streambed and did not discriminate among the cover sizes available. The strong affinity of D. quadramaculatus for streambed microhabitat, compared with the much more variable microhabitat selection of D. monticola, resulted in spatial separation of individuals of the two species most of the time. Results of this study do not support the hypothesis that interspecific inference is more important than intraspecific interference in the microhabitat distributions of D. monticola and D. quadramaculatus.     

Histochemical quantification of dopa-oxidase (tyrosinase) activity in epidermal melanocytes

Summary The graphical method of Lineweaver-Burk was applied to the histochemical system to obtain quantitative information on the dopa-oxidase activity in human epidermal melanocytes. In vitro experiment showed that the incubation time necessary for yielding a certain amount of dopa-melanin is related to the amount of enzyme present. In the case of histochemical experiment, the time required for the first appearance of dopa-melanin deposition in melanocytes at various substrate concentrations was plotted against the reciprocal of the substrate concentration. This technique made it possible to compare the dopa-oxidase activity at various sites of the tissue.     

Histochemical quantification of dopa-oxidase (tyrosinase) activity in epidermal melanocytes.

The graphical method of Lineweaver-Burk was applied to the histochemical system to obtain quantitative information on the dopa-oxidase activity in human epidermal melanocytes. In vitro experiment showed that the incubation time necessary for yielding a certain amount of dopa-melanin is related to the amount of enzyme present. In the case of histochemical experiment, the time required for the first appearance of dopa-melanin deposition in melanocytes at various substrate concentrations was plotted against the reciprocal of the substrate concentration. This technique made it possible to compare the dopa-oxidase activity at various sites of the tissue.     

Quantification of DNA strand breaks and abasic sites by oxime derivatization and accelerator mass spectrometry: application to gamma-radiation and peroxynitrite

We report a highly sensitive method to quantify abasic sites and deoxyribose oxidation products arising in damaged DNA. The method exploits the reaction of aldehyde- and ketone-containing deoxyribose oxidation products and abasic sites with [(14)C]methoxyamine to form stable oxime derivatives, as originally described by Talpaert-Borle and Liuzzi [Reaction of apurinic/apyrimidinic sites with [(14)C]methoxyamine. A method for the quantitative assay of AP sites in DNA, Biochim. Biophys. Acta 740 (1983) 410-416]. The sensitivity of the method was dramatically improved by the application of accelerator mass spectrometry to quantify the (14)C, with a limit of detection of 1 lesion in 10(6) nucleotides in 1 microg of DNA. The method was validated using DNA containing a defined quantity of abasic sites, with a >0.95 correlation between the quantities of abasic sites and those of methoxyamine labels. The original applications of this and similar oxyamine derivatization methods have assumed that abasic sites are the only aldehyde-containing DNA damage products. However, deoxyribose oxidation produces strand breaks and abasic sites containing a variety of degradation products with aldehyde and ketone moieties. To assess the utility of methoxyamine labeling for quantifying strand breaks and abasic sites, the method was applied to plasmid DNA treated with gamma-radiation and peroxynitrite. For gamma-radiation, there was a 0.99 correlation between the quantity of methoxyamine labels and the quantity of strand breaks and abasic sites determined by a plasmid nicking assay; the abasic sites comprised less than 10% of the radiation-induced DNA damage. Studies with peroxynitrite demonstrate that the method, in conjunction with DNA repair enzymes that remove damaged bases to produce aldehydic sugar residues or abasic sites, is also applicable to quantifying nucleobase lesions in addition to strand break products. Compared to other abasic site quantification techniques, the modified method offers the advantage of providing a straightforward and direct measurement of aldehyde- and ketone-containing strand breaks and abasic sites, with the potential for direct labeling in cells prior to DNA isolation.     

Decomposition of Sarcocornia quinqueflora on an Iron-Smelting Slag Substrate

Restoration of salt marsh habitat is becoming more common in Australia. However, little is known about restoring salt marshes on substrates contaminated by slag from iron smelting, which could affect microbial activity. This study, conducted near Newcastle, Australia, compares initial C, N, and P mass and decomposition of Sarcocornia quinqueflora (glasswort or samphire) from (1) a restoration site with a slag‐and‐mud substrate, (2) the restoration site's donor marsh, and (3) other nearby sites sampled to provide information on background variability. A litterbag technique with a 180‐day incubation period was used to quantify total, C, N, and P mass losses from decomposition. Although there were significant differences between sites in initial N mass and loss of C and P over the period of our study, the presence of slag did not slow decomposition rates as measured using litterbags. Further work is needed to assess other aspects of wetland structure and function on slag substrates.     

Trace elements in the Upper Fly River, Papua New Guinea

SUMMARY. Measurements were made of calcium, magnesium and eight trace elements (Cu, Zn, Fe, Mn, Ni, Pb, Cd, Co) in waters, sediment and biota at eight sites in the Upper Fly River, Papua New Guinea. Determinations from streams draining an area rich in copper ore showed relatively low values for soluble copper and this was attributed to the small amount of ore exposed to weathering, heavy rainfall and high pH of water associated with the limestone bedrock. More iron was transported in a soluble form down a headwater tributary (Alice River) than in the Fly River but higher iron concentrations were associated with particulate matter in the Fly than in the Alice. The geochemistry of the catchment and the distance from the headwaters were important influences on the amount of particulate iron and its distribution between soluble and particulate forms.
Levels of metals in the biota generally reflected the background concentrations of metals encountered at each site. Concentrations of Cu, Cd, and Zn were lower in starved invertebrates compared with unstarved individuals but no consistent changes were observed in Fe or Mn content.