intI1 gene abundance from septic tanks in Thailand using validated intI1 primers.

IMPORTANCE: Antimicrobial resistance is a global crisis, and wastewater treatment, including septic tanks, remains an important source of antimicrobial resistance (AMR) genes. The role of septic tanks in disseminating class 1 integron, and by extension AMR genes, in Thailand, where antibiotic use is unregulated remains understudied. We aimed to monitor gene abundance as a proxy to infer potential AMR from septic tanks in Thailand. We evaluated published intI1 primers due to the lack of consensus on optimal Q-PCR primers and the absence of standardization. Our findings confirmed septic tanks are a source of class 1 integron to the environment. We highlighted the significance of intI1 primer choice, in the context of interpretation of risk associated with AMR spread from septic tanks. We recommend the validated set (F3-R3) for optimal intI1 quantification toward the goal of achieving standardization across studies.

Biochemistry shapes growth kinetics of nitrifiers and defines their activity under specific environmental conditions.

Is it possible to find trends between the parameters that define microbial growth to help us explain the vast microbial diversity? Through an extensive database of kinetic parameters of nitrifiers, we analyzed if the dominance of specific populations of nitrifiers could be predicted and explained. We concluded that, in general, higher growth yield (YXS ) and ammonia affinity (a0NH3 ) and lower growth rate (µmax ) are observed for ammonia-oxidizing archaea (AOA) than bacteria (AOB), which would explain their considered dominance in oligotrophic environments. However, comammox (CMX), with the maximum energy harvest per mole of ammonia, and some AOB, have higher a0NH3 and lower µmax than some AOA. Although we were able to correlate the presence of specific terminal oxidases with observed oxygen affinities (a0O2 ) for nitrite-oxidizing bacteria (NOB), that correlation was not observed for AOB. Moreover, the presumed dominance of AOB over NOB in O2 -limiting environments is discussed. Additionally, lower statistical variance of a0O2 values than for ammonia and nitrite affinities was observed, suggesting nitrogen limitation as a stronger selective pressure. Overall, specific growth strategies within nitrifying groups were not identified through the reported kinetic parameters, which might suggest that mostly, fundamental differences in biochemistry are responsible for underlying kinetic parameters.

Reverse transcriptase enzyme and priming strategy affect quantification and diversity of environmental transcripts.

Reverse-transcriptase-quantitative PCR (RT-Q-PCR) and RT-PCR amplicon sequencing, provide a convenient, target-specific, high-sensitivity approach for gene expression studies and are widely used in environmental microbiology. Yet, the effectiveness and reproducibility of the reverse transcription step has not been evaluated. Therefore, we tested a combination of four commercial reverse transcriptases with two priming techniques to faithfully transcribe 16S rRNA and amoA transcripts from marine sediments. Both enzyme and priming strategy greatly affected quantification of the exact same target with differences of up to 600-fold. Furthermore, the choice of RT system significantly changed the communities recovered. For 16S rRNA, both enzyme and priming had a significant effect with enzyme having a stronger impact than priming. Inversely, for amoA only the change in priming strategy resulted in significant differences between the same samples. Specifically, more OTUs and better coverage of amoA transcripts diversity were obtained with GS priming indicating this approach was better at recovering the diversity of amoA transcripts. Moreover, sequencing of RNA mock communities revealed that, even though transcript α diversities (i.e., OTU counts within a sample) can be biased by the RT, the comparison of ß diversities (i.e., differences in OTU counts between samples) is reliable as those biases are reproducible between environments.

Differential ratio amplicons (Ramp ) for the evaluation of RNA integrity extracted from complex environmental samples.

Reliability and reproducibility of transcriptomics-based studies are dependent on RNA integrity. In microbial ecology, microfluidics-based techniques, such as the Ribosomal Integrity Number (RIN), targeting rRNA are currently the only approaches to evaluate RNA integrity. However, the relationship between rRNA and mRNA integrity is unknown. Here, we present an integrity index, the Ratio Amplicon, Ramp , adapted from human clinical studies, to directly monitor mRNA integrity from complex environmental samples. We show, in a suite of experimental degradations of RNA extracted from sediment, that while the RIN generally reflected the degradation status of RNA the Ramp mapped mRNA degradation better. Furthermore, we examined the effect of degradation on transcript community structure by amplicon sequencing of 16S rRNA, amoA and glnA transcripts. We successfully sequenced transcripts for all three targets even from highly-degraded RNA samples. While RNA degradation changed the community structure of the mRNA profiles, no changes were observed for the 16S rRNA transcript profiles. Since both RT-Q-PCR and sequencing results were obtained, even from highly degraded samples, we strongly recommend evaluating RNA integrity prior to downstream processing to ensure meaningful results. For this, both the RIN and Ramp are useful, with the Ramp better evaluating mRNA integrity in this study.

Community and functional shifts in ammonia oxidizers across terrestrial and marine (soil/sediment) boundaries in two coastal Bay ecosystems.

Terrestrial-marine boundaries are significant sites of biogeochemical activity with delineated gradients from land to sea. While niche differentiation of ammonia-oxidizing archaea (AOA) and bacteria (AOB) driven by pH and nitrogen is well known, the patterns and environmental drivers of AOA and AOB community structure and activity across soil-sediment boundaries have not yet been determined. In this study, nitrification potential rate, community composition and transcriptional activity of AOA and AOB in soil, soil/sediment interface and sediments of two coastal Bays were characterized using a combination of field investigations and microcosm incubations. At DNA level, amoA gene abundances of AOA were significantly greater than AOB in soil, while in sediments AOB were significantly more abundant than AOA, but at the soil/sediment interface there were equal numbers of AOA and AOB amoA genes. Microcosm incubations provided further evidence, through qPCR and DGGE-sequencing analysis of amoA transcripts, that AOA were active in soil, AOB in sediment and both AOA and AOB were active at the soil/sediment interface. The AOA and AOB community composition shifted across the coastal soil-interface-sediment gradient with salinity and pH identified as major environmental drivers.

Survival, Biofilm Formation, and Growth Potential of Environmental and Enteric Escherichia coli Strains in Drinking Water Microcosms.

UNLABELLED: Escherichia coli is the most commonly used indicator for fecal contamination in drinking water distribution systems (WDS). The assumption is that E. coli bacteria are of enteric origin and cannot persist for long outside their host and therefore act as indicators of recent contamination events. This study investigates the fate of E. coli in drinking water, specifically addressing survival, biofilm formation under shear stress, and regrowth in a series of laboratory-controlled experiments. We show the extended persistence of three E. coli strains (two enteric isolates and one soil isolate) in sterile and nonsterile drinking water microcosms at 8 and 17°C, with T90 (time taken for a reduction in cell number of 1 log10 unit) values ranging from 17.4 ± 1.8 to 149 ± 67.7 days, using standard plate counts and a series of (reverse transcription-)quantitative PCR [(RT-)Q-PCR] assays targeting 16S rRNA, tuf, uidA, and rodA genes and transcripts. Furthermore, each strain was capable of attaching to a surface and replicating to form biofilm in the presence of nutrients under a range of shear stress values (0.6, 2.0, and 4.4 dynes [dyn] cm(-2); BioFlux system; Fluxion); however, cell numbers did not increase when drinking water flowed over the biofilm (P > 0.05 by t test). Finally, E. coli regrowth within drinking water microcosms containing polyethylene PE-100 pipe wall material was not observed in the biofilm or water phase using a combination of culturing and Q-PCR methods for E. coli The results of this work highlight that when E. coli enters drinking water it has the potential to survive and attach to surfaces but that regrowth within drinking water or biofilm is unlikely. IMPORTANCE: The provision of clean, safe drinking water is fundamental to society. WDS deliver water to consumers via a vast network of pipes. E. coli is used as an indicator organism for recent contamination events based on the premise that it cannot survive for long outside its host. A key public health concern therefore arises around the fate of E. coli on entering a WDS; its survival, ability to form a biofilm, and potential for regrowth. In particular, if E. coli bacteria have the ability to incorporate and regrow within the pipe wall biofilm of a WDS, they could reinoculate the water at a later stage. This study sheds light on the fate of environmental and enteric strains of E. coli in drinking water showing extended survival, the potential for biofilm formation under shear stress, and importantly, that regrowth in the presence of an indigenous microbial community is unlikely.

A rapid culture independent methodology to quantitatively detect and identify common human bacterial pathogens associated with contaminated high purity water.

BACKGROUND: Water and High Purity Water (HPW) distribution systems can be contaminated with human pathogenic microorganisms. This biocontamination may pose a risk to human health as HPW is commonly used in the industrial, pharmaceutical and clinical sectors. Currently, routine microbiological testing of HPW is performed using slow and labour intensive traditional microbiological based techniques. There is a need to develop a rapid culture independent methodology to quantitatively detect and identify biocontamination associated with HPW. RESULTS: A novel internally controlled 5-plex real-time PCR Nucleic Acid Diagnostics assay (NAD), was designed and optimised in accordance with Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines, to rapidly detect, identify and quantify the human pathogenic bacteria Stenotrophomonas maltophilia, Burkholderia species, Pseudomonas aeruginosa and Serratia marcescens which are commonly associated with the biocontamination of water and water distribution systems. The specificity of the 5-plex assay was tested against genomic DNA isolated from a panel of 95 microorganisms with no cross reactivity observed. The analytical sensitivities of the S. maltophilia, B. cepacia, P. aeruginosa and the S. marcescens assays are 8.5, 5.7, 3.2 and 7.4 genome equivalents respectively. Subsequently, an analysis of HPW supplied by a Millipore Elix 35 water purification unit performed using standard microbiological methods revealed high levels of naturally occurring microbiological contamination. Five litre water samples from this HPW delivery system were also filtered and genomic DNA was purified directly from these filters. These DNA samples were then tested using the developed multiplex real-time PCR NAD assay and despite the high background microbiological contamination observed, both S. maltophilia and Burkholderia species were quantitatively detected and identified. At both sampling points the levels of both S. maltophilia and Burkholderia species present was above the threshold of 10 cfu/100 ml recommended by both EU and US guidelines. CONCLUSIONS: The novel culture independent methodology described in this study allows for rapid (<5 h), quantitative detection and identification of these four human pathogens from biocontaminated water and HPW distribution systems. We propose that the described NAD assay and associated methodology could be applied to routine testing of water and HPW distribution systems to assure microbiological safety and high water quality standards.

Low-abundant but highly transcriptionally active uncharacterised Nitrosomonas drive ammonia-oxidation in the Brouage mudflat, France.

Exploring differences in nitrification within adjacent sedimentary structures of ridges and runnels on the Brouage mudflat, France, we quantified Potential Nitrification Rates (PNR) alongside amoA genes and transcripts. PNR was lower in ridges (≈1.7 fold-lower) than runnels, despite higher (≈1.8 fold-higher) ammonia-oxidizing bacteria (AOB) abundance. However, AOB were more transcriptionally active in runnels (≈1.9 fold-higher). Sequencing of amoA genes and transcripts revealed starkly contrasting profiles with transcripts from ridges and runnels dominated (≈91 % in ridges and ≈98 % in runnels) by low abundant (≈4.6 % of the DNA community in runnels and ≈0.8 % in ridges) but highly active phylotypes. The higher PNR in runnels was explained by higher abundance of this group, an uncharacterised Nitrosomonas sp. cluster. This cluster is phylogenetically similar to other active ammonia-oxidizers with worldwide distribution in coastal environments indicating its potential, but previously overlooked, contribution to ammonia oxidation globally. In contrast DNA profiles were dominated by highly abundant but low-activity clusters phylogenetically distinct from known Nitrosomonas (Nm) and Nitrosospira (Ns). This cluster is also globally distributed in coastal sediments, primarily detected as DNA, and often classified as Nitrosospira or Nitrosomonas. We therefore propose to classify this cluster as Ns/Nm. Our work indicates that low abundant but highly active AOB could be responsible for the nitrification globally, while the abundant AOB Ns/Nm may not be transcriptionally active, and as such account for the lack of correlation between rate processes and gene abundances often reported in the literature. It also raises the question as to what this seemingly inactive group is doing?

Microbial stratification and DOM removal in drinking water biofilters: Implications for enhanced performance.

Biofiltration is a low-cost, low-energy technology that employs a biologically activated bed of porous medium to reduce the biodegradable fraction of the dissolved organic matter (DOM) pool in source water, resulting in the production of drinking water. Microbial communities at different bed depths within the biofilter play crucial roles in the degradation and removal of dissolved organic carbon (DOC), ultimately impacting its performance. However, the relationships between the composition of microbial communities inhabiting different biofilter depths and their utilisation of various DOC fractions remain poorly understood. To address this knowledge gap, we conducted an experimental study where microbial communities from the upper (i.e., top 10 cm) and lower (i.e., bottom 10 cm) sections of a 30-cm long laboratory-scale biofilter were recovered. These communities were then individually incubated for 10 days using the same source water as the biofilter influent. Our study revealed that the bottom microbial community exhibited lower diversity yet had a co-occurrence network with a higher degree of interconnections among its members compared to the top microbial community. Moreover, we established a direct correlation between the composition and network structure of the microbial communities and their ability to utilise various DOM compounds within a DOM pool. Interestingly, although the bottom microbial community had only 20 % of the total cell abundance compared to the top community at the beginning of the incubation, it utilised and hence removed approximately 60 % more total DOC from the DOM pool than the top community. While both communities rapidly utilised labile carbon fractions, such as low-molecular-weight neutrals, the utilisation of more refractory carbon fractions, like high-molecular-weight humic substances with an average molecular weight of more than ca. 1451 g/mol, was exclusive to the bottom microbial community. By employing techniques that capture microbial diversity (i.e., flow cytometry and 16S rRNA amplicon sequencing) and considering the complexities of DOM (i.e., LCOCD), our study provides novel insights into how microbial community structure could influence the microbial-mediated processes of engineering significance in drinking water production. Finally, our findings could offer the opportunity to improve biofilter performances via engineering interventions that shape the compositions of biofilter microbial communities and enhance their utilisation and removal of DOM, most notably the more classically humified and refractory DOM compound groups.

Mechanisms of contextual risk for adolescent self-injury: invalidation and conflict escalation in mother-child interactions.

According to developmental theories of self-injury, both child characteristics and environmental contexts shape and maintain problematic behaviors. Although progress has been made toward identifying biological vulnerabilities to self-injury, mechanisms underlying psychosocial risk have received less attention. In the present study, we compared self-injuring adolescents (n = 17) with typical controls (n = 20) during a mother-child conflict discussion. Dyadic interactions were coded using both global and microanalytic systems, allowing for a highly detailed characterization of mother-child interactions. We also assessed resting state psychophysiological regulation, as indexed by respiratory sinus arrhythmia (RSA). Global coding revealed that maternal invalidation was associated with adolescent anger. Furthermore, maternal invalidation and coerciveness were both related to adolescent opposition/defiance. Results from the microanalytic system indicated that self-injuring dyads were more likely to escalate conflict, suggesting a potential mechanism through which emotion dysregulation is shaped and maintained over time. Finally, mother and teen aversiveness interacted to predict adolescent resting RSA. Low-aversive teens with highly aversive mothers had the highest RSA, whereas teens in high-high dyads showed the lowest RSA. These findings are consistent with theories that emotion invalidation and conflict escalation are possible contextual risk factors for self-injury.

Competitive and substrate limited environments drive metabolic heterogeneity for comammox Nitrospira.

Nitrospira has been revealed as a high versatile genus. Although previously considered only responsible for the conversion of nitrite to nitrate, now we know that Nitrospira can perform complete ammonia oxidation to nitrate too (comammox). Comammox activity was firstly reported as dominant in extremely limited oxygen environments, where anaerobic ammonia oxidation was also occurring (anammox). To explain the comammox selection, we developed an Individual-based Model able to describe Nitrospira and anammox growth in suspended flocs assembled in a dynamic nitrogen and oxygen-limiting environment. All known and hypothesized nitrogen transformations of Nitrospira were considered: ammonia and nitrite oxidation, comammox, nitrate-reducing ammonia oxidation, and anaerobic nitrite-reducing ammonia oxidation. Through bioenergetics analysis, the growth yield associated to each activity was estimated. The other kinetic parameters necessary to describe growth were calibrated according to the reported literature values. Our modeling results suggest that even extremely low oxygen concentrations (~1.0 µM) allow for a proportional growth of anammox versus Nitrospira similar to the one experimentally observed. The strong oxygen limitation was followed by a limitation of ammonia and nitrite, because anammox, without strong competitors, were able to grow faster than Nitrospira depleting the environment in nitrogen. These substrate limitations created an extremely competitive environment that proved to be decisive in the community assembly of Nitrospira and anammox. Additionally, a diversity of metabolic activities for Nitrospira was observed in all tested conditions, which in turn, explained the transient nitrite accumulation observed in aerobic environments with higher ammonia availability.

Ecological Observations Based on Functional Gene Sequencing Are Sensitive to the Amplicon Processing Method.

Until recently, the de facto method for short-read-based amplicon reconstruction was a sequence similarity threshold approach (operational taxonomic units [OTUs]). This has changed with the amplicon sequence variant (ASV) method where distributions are fitted to abundance profiles of individual genes using a noise-error model. While OTU-based approaches are still useful for 16S rRNA/18S rRNA genes, where thresholds of 97% to 99% are used, their use for functional genes is still debatable as there is no consensus on clustering thresholds. Here, we compare OTU- and ASV-based reconstruction approaches and taxonomy assignment methods, the naive Bayesian classifier (NBC) and Bayesian lowest common ancestor (BLCA) algorithm, using a functional gene data set from the microbial nitrogen-cycling community in the Brouage mudflat (France). A range of OTU similarity thresholds and ASVs were used to compare amoA (ammonia-oxidizing archaea [AOA] and ammonia-oxidizing bacteria [AOB]), nxrB, nirS, nirK, and nrfA communities between differing sedimentary structures. Significant effects of the sedimentary structure on weighted UniFrac (WUniFrac) distances were observed for AOA amoA when using ASVs, an OTU at a threshold of 97% sequence identity (OTU-97%), and OTU-85%; AOB amoA when using OTU-85%; and nirS when using ASV, OTU-90%, and OTU-85%. For AOB amoA, significant effects of the sedimentary structures on UniFrac distances were observed when using OTU-97% but not ASVs, and the inverse was found for nrfA. Interestingly, conclusions drawn for nirK and nxrB were consistent between amplicon reconstruction methods. We also show that when the sequences in the reference database are related to the environment in question, the BLCA algorithm leads to more phylogenetically relevant classifications. However, when the reference database contains sequences more dissimilar to the ones retrieved, the NBC obtains more information. IMPORTANCE Several analysis pipelines are available to microbial ecologists to process amplicon sequencing data, yet to date, there is no consensus as to the most appropriate method, and it becomes more difficult for genes that encode a specific function (functional genes). Standardized approaches need to be adopted to increase the reliability and reproducibility of environmental amplicon-sequencing-based data sets. In this paper, we argue that the recently developed ASV approach offers a better opportunity to achieve such standardization than OTUs for functional genes. We also propose a comprehensive framework for quality filtering of the sequencing reads based on protein sequence verification.

Microbiomes in drinking water treatment and distribution: A meta-analysis from source to tap.

A meta-analysis of existing and available Illumina 16S rRNA datasets from drinking water source, treatment and drinking water distribution systems (DWDS) were collated to compare changes in abundance and diversity throughout. Samples from bulk water and biofilm were used to assess principles governing microbial community assembly and the value of amplicon sequencing to water utilities. Individual phyla relationships were explored to identify competitive or synergistic factors governing DWDS microbiomes. The relative importance of stochasticity in the assembly of the DWDS microbiome was considered to identify the significance of source and treatment in determining communities in DWDS. Treatment of water significantly reduces overall species abundance and richness, with chlorination of water providing the most impact to individual taxa relationships. The assembly of microbial communities in the bulk water of the source, primary treatment process and DWDS is governed by more stochastic processes, as is the DWDS biofilm. DWDS biofilm is significantly different from bulk water in terms of local contribution to beta diversity, type and abundance of taxa present. Water immediately post chlorination has a more deterministic microbial assembly, highlighting the significance of this process in changing the microbiome, although elevated levels of stochasticity in DWDS samples suggest that this may not be the case at customer taps. 16S rRNA sequencing is becoming more routine, and may have several uses for water utilities, including: detection and risk assessment of potential pathogens such as those within the genera of Legionella and Mycobacterium; assessing the risk of nitrification in DWDS; providing improved indicators of process performance and monitoring for significant changes in the microbial community to detect contamination. Combining this with quantitative methods like flow cytometry will allow a greater depth of understanding of the DWDS microbiome.

A framework based on fundamental biochemical principles to engineer microbial community dynamics.

Microbial communities are complex but there are basic principles we can apply to constrain the assumed stochasticity of their activity. By understanding the trade-offs behind the kinetic parameters that define microbial growth, we can explain how local interspecies dependencies arise and shape the emerging properties of a community. If we integrate these theoretical descriptions with experimental 'omics' data and bioenergetics analysis of specific environmental conditions, predictions on activity, assembly and spatial structure can be obtained reducing the a priori unpredictable complexity of microbial communities. This information can be used to define the appropriate selective pressures to engineer bioprocesses and propose new hypotheses which can drive experimental research to accelerate innovation in biotechnology.

Changes in benthic denitrification, nitrate ammonification, and anammox process rates and nitrate and nitrite reductase gene abundances along an estuarine nutrient gradient (the Colne estuary, United Kingdom).

Estuarine sediments are the location for significant bacterial removal of anthropogenically derived inorganic nitrogen, in particular nitrate, from the aquatic environment. In this study, rates of benthic denitrification (DN), dissimilatory nitrate reduction to ammonium (DNRA), and anammox (AN) at three sites along a nitrate concentration gradient in the Colne estuary, United Kingdom, were determined, and the numbers of functional genes (narG, napA, nirS, and nrfA) and corresponding transcripts encoding enzymes mediating nitrate reduction were determined by reverse transcription-quantitative PCR. In situ rates of DN and DNRA decreased toward the estuary mouth, with the findings from slurry experiments suggesting that the potential for DNRA increased while the DN potential decreased as nitrate concentrations declined. AN was detected only at the estuary head, accounting for approximately 30% of N2 formation, with 16S rRNA genes from anammox-related bacteria also detected only at this site. Numbers of narG genes declined along the estuary, while napA gene numbers were stable, suggesting that NAP-mediated nitrate reduction remained important at low nitrate concentrations. nirS gene numbers (as indicators of DN) also decreased along the estuary, whereas nrfA (an indicator for DNRA) was detected only at the two uppermost sites. Similarly, nitrate and nitrite reductase gene transcripts were detected only at the top two sites. A regression analysis of log(n + 1) process rate data and log(n + 1) mean gene abundances showed significant relationships between DN and nirS and between DNRA and nrfA. Although these log-log relationships indicate an underlying relationship between the genetic potential for nitrate reduction and the corresponding process activity, fine-scale environmentally induced changes in rates of nitrate reduction are likely to be controlled at cellular and protein levels.

Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology.

Quantitative PCR (Q-PCR or real-time PCR) approaches are now widely applied in microbial ecology to quantify the abundance and expression of taxonomic and functional gene markers within the environment. Q-PCR-based analyses combine 'traditional' end-point detection PCR with fluorescent detection technologies to record the accumulation of amplicons in 'real time' during each cycle of the PCR amplification. By detection of amplicons during the early exponential phase of the PCR, this enables the quantification of gene (or transcript) numbers when these are proportional to the starting template concentration. When Q-PCR is coupled with a preceding reverse transcription reaction, it can be used to quantify gene expression (RT-Q-PCR). This review firstly addresses the theoretical and practical implementation of Q-PCR and RT-Q-PCR protocols in microbial ecology, highlighting key experimental considerations. Secondly, we review the applications of (RT)-Q-PCR analyses in environmental microbiology and evaluate the contribution and advances gained from such approaches. Finally, we conclude by offering future perspectives on the application of (RT)-Q-PCR in furthering understanding in microbial ecology, in particular, when coupled with other molecular approaches and more traditional investigations of environmental systems.

Parent-child interactions, peripheral serotonin, and self-inflicted injury in adolescents.

Self-inflicted injury in adolescence indicates significant emotional and psychological suffering. Although data on the etiology of self-injury are limited, current theories suggest that the emotional lability observed among self-injuring adolescents results from complex interactions between individual biological vulnerabilities and environmental risk. For example, deficiencies in serotonergic functioning, in conjunction with certain family interaction patterns, may contribute to the development of emotional lability and risk for self-injury. The authors explored the relation between peripheral serotonin levels and mother-child interaction patterns among typical (n = 21) and self-injuring (n = 20) adolescents. Findings revealed higher levels of negative affect and lower levels of both positive affect and cohesiveness among families of self-injuring participants. Peripheral serotonin was also correlated with the expression of positive affect within dyads. Furthermore, adolescents' serotonin levels interacted with negativity and conflict within dyads to explain 64% of the variance in self-injury. These findings underscore the importance of considering both biological and environmental risk factors in understanding and treating self-injuring adolescents.

16S rRNA sequencing reveals likely beneficial core microbes within faecal samples of the EU protected slug Geomalacus maculosus.

The EU-protected slug Geomalacus maculosus Allman occurs only in the West of Ireland and in northern Spain and Portugal. We explored the microbial community found within the faeces of Irish specimens with a view to determining whether a core microbiome existed among geographically isolated slugs which could give insight into the adaptations of G. maculosus to the available food resources within its habitat. Faecal samples of 30 wild specimens were collected throughout its Irish range and the V3 region of the bacterial 16S rRNA gene was sequenced using Illumina MiSeq. To investigate the influence of diet on the microbial composition, faecal samples were taken and sequenced from six laboratory reared slugs which were raised on two different foods. We found a widely diverse microbiome dominated by Enterobacteriales with three core OTUs shared between all specimens. While the reared specimens appeared clearly separated by diet in NMDS plots, no significant difference between the slugs fed on the two different diets was found. Our results indicate that while the majority of the faecal microbiome of G. maculosus is probably dependent on the microhabitat of the individual slugs, parts of it are likely selected for by the host.

Characterization of the bacterial community associated with the surface and mucus layer of whiting (Merlangius merlangus).

The bacterial community inhabiting the mucus layer and surface of whiting was examined to determine whether the bacteria present are a reflection of the surrounding water or an indigenous bacterial flora is present. The outer mucus, mouth mucus and gut of four whiting harvested from a site in the Irish Sea and the surrounding water were examined by terminal restriction fragment length polymorphism (tRFLP) analysis of the 16S rRNA gene and clone library construction. The water community was the most diverse, with only a small number of shared water-mucus phylotypes present. The bacterial flora associated with the outer mucus layer were more diverse than that of the mouth mucus and gut. All three mucus layers were characterized by the presence of a dominant phylotype, identified as clone wom-1, highly similar to Photobacterium iliopiscarium. In addition to other Photobacterium phylotypes, members of the CFB and Clostridia groups were also detected. Subsequently, whiting from 11 different sites along the east and south coast of Ireland were compared by tRFLP analysis. Strikingly, the mucus layer of whiting at all sites was characterized by the presence and dominance of a TRF corresponding to the clone wom-1 which was virtually absent from the water column.

Small-scale variation of ammonia oxidisers within intertidal sediments dominated by ammonia-oxidising bacteria Nitrosomonas sp. amoA genes and transcripts.

While numerous studies have investigated the abundance of ammonia oxidising bacteria and archaea (AOB/AOA) via the ammonia monooxygenase gene amoA, less is known about their small-scale variation and if amoA gene abundance equates to activity. Here we present a spatial and temporal study of ammonia oxidation in two small intertidal bays, Rusheen and Clew bay, Ireland. Potential Nitrification Rate (PNR) was ten-fold higher in Rusheen bay (Clew: 0.27 ± SD 0.55; Rusheen: 2.46 ± SD 3.4 NO2- µg-1 g-1 day-1, P < 0.001) than in Clew bay but amoA gene abundances were similar between bays, and comparable to those in other coastal ecosystems. Within bays AOB genes increased towards the muddy sediments and were positively correlated with PNR and pH. Less spatial variation was observed in AOA abundances which nevertheless positively correlated with pH and temperature and negatively with salinity and ammonia. Transcriptionally active AOB and AOA were quantified from all sites in Rusheen bay, February 2014, following the general trends observed at DNA level. AOB phylotypes predominantly from the known Nitrosomonas group were distributed across the bay, while Nitrosomonas group B phylotypes were absent from low salinity sites. AOA genes and transcripts were primarily affiliated with Thaumarchaeota group I.1a.