Despite advancements in urban wastewater treatment, final disinfection remains the primary, yet often inefficient or costly, measure to control fecal bacteria and antibiotic resistance genes (ARGs). A solution to enhance performance while reducing the impact of chemical disinfection could be found in a different management of the often-overlooked tertiary clarifiers integrated into the coagulation-flocculation stage. By analyzing three different wastewater treatment plants (WWTPs) combining amplicon and shotgun metagenomics, flow cytometry, and water chemistry, we demonstrated that tertiary clarifiers shape microbial communities, reducing bacterial abundance while maintaining community richness. Whereas fecal/opportunistic pathogens decline, environmental bacteria either persist or increase, suggesting selective pressures at play. Furthermore, while ARG abundance decreased, their relative prevalence remained constant or even increased, raising concerns about potential horizontal gene transfer. Biofilms within clarifiers act as reservoirs, stabilizing diversity despite environmental fluctuations. These findings challenge the traditional perception of tertiary clarifiers as mere sedimentation tanks and highlight their potential role for microbial control. Through optimized retention times and ecological management, tertiary clarifiers could complement disinfection by reducing environmental footprints and operational costs, lowering the overall intensity required to mitigate bacterial and genetic pollution. This study provides a foundation for an additional use of tertiary clarifiers in WWTP design, integrating eco-engineered solutions to enhance treatment efficacy.

Abstract While Antarctic terrestrial ecosystems support low metazoan diversity, the surrounding marine macrobenthos is rich. However, marine meiofauna remains historically neglected, leaving its diversity patterns unclear. In this study, we used 18S rRNA gene metabarcoding alongside an enhanced taxonomic annotation pipeline to characterize marine meiofauna diversity in the Ross Sea, comparing it to global datasets. We evaluated how depth, habitat type, and mesh size influence community structures to test if habitat heterogeneity drives diversity despite the harsh Southern Ocean conditions. Our results revealed exceptionally high diversity, with metazoans richness comparable to or higher than temperate regions. Although environmental variables had limited effects on taxonomic richness, they significantly shaped community composition, with habitat type explaining the highest proportion of variance. Interestingly, we detected several ASVs 100% identical to North Sea and North Atlantic sequences, likely reflecting the limited taxonomic resolution of the 18S marker rather than global dispersal (the “meiofaunal paradox”). Overall, these findings demonstrate that Antarctic marine sediments host rich meiofaunal communities where ecological processes operate similarly to other global regions, contrasting sharply with depauperate continental Antarctic ecosystems.

Aquatic microbial community can be defined as an assemblage of co-occurring, and potentially interacting, microbes, present in a defined habitat in space and time. Despite the small size, microorganisms are key elements for the ecological dynamics of the biosphere. They are not only the most diffused life forms; they are also characterized by an incredible functional and genetic diversity, contributing fundamentally to the biogeochemical processes on Earth. In this article, we describe the main ecological characteristics of aquatic microbial communities. We begin from microbial lifestyles in freshwater and marine habitat, considering then the diversity and functions of prokaryotes, eukaryotes, and viruses, to end with an analysis of the intrinsic and extrinsic community drivers. Since planetary biogeochemical transformations are the emergent results of accumulated processes working at microscale, we conclude the article with an opening to novel molecular and single-cell approaches that promise to shed light on micro-scale interactions.

The endangered anchialine squat lobster Munidopsis polymorpha is restricted to northern Lanzarote. To understand genetic isolation and conservation risks, we genotyped 26 individuals at eight microsatellite loci from four sites in La Corona lava tube and Charcos de Luis. Lava tube sites shared most alleles and were effectively undifferentiated, whereas Charcos de Luis harbored nine private alleles, the highest allelic richness, and clear differentiation from the lava tube complex. We detected no Hardy-Weinberg deviations among testable locus x site combinations and no linkage disequilibrium. These results are consistent with two candidate management units: La Corona lava tube and Charcos de Luis. We outline genomic priorities to guide future conservation plans. To account for our reduced sampling size, we screened for null alleles and repeated the analyses on a reduced set of loci passing conservative quality filters, obtaining congruent patterns of differentiation.

Island coastal aquifers, though spatially limited, sustain key ecosystem functions linked to locally critical provisioning, maintenance and cultural ecosystem services. These functions are largely dependent on the presence of highly adapted biological communities, whose microbial components remain poorly understood. Here, we describe bacterial communities across groundwater-dependent ecosystems on Lanzarote (Canary Islands, Spain), spanning habitats with contrasting environmental conditions and degrees of human influence, using 16 S rRNA gene amplicon sequencing. We then infer the processes shaping community variation by integrating diversity partitioning, indicator species analysis, and machine-learning classification. Bacterial taxonomic diversity varied significantly among habitats, with community composition primarily structured by turnover, consistent with environmental filtering. In contrast, predicted human-associated and potentially pathogenic taxa showed patterns dominated by nestedness, indicating localized enrichment linked to anthropogenic inputs. Caves, enclosed marine bays, and hypersaline systems hosted the most compositionally distinct microbial communities, whereas wells and anchialine pools showed greater overlap in community composition. Together, our results suggest that groundwater microbial communities are influenced by the interplay between environmental filtering and anthropogenic inputs, and that coastal aquifers can act simultaneously as reservoirs of natural biodiversity and sinks of human-associated bacteria. These findings highlight the need for integrative monitoring and conservation strategies that incorporate both hydrological and biological components to safeguard groundwater-dependent ecosystems on oceanic islands.

Abstract Picocyanobacteria, typically associated with oxygenated waters, thrive also in oxygen minimum zones (OMZs) and anoxic environments. The extent to which retention of diverse ancestral anaerobic traits facilitate their metabolic flexibility with regards to transitioning between oxic and anoxic habitats is poorly understood. Here, using Black Sea Cyanobium (BSA11S), we performed 13C-glucose and 15N-ammonium incubations coupled with nanoscale ion mass spectrometry (NanoSIMS) and transcriptomics to characterize the growth mode and gene expression during transition from light-to-dark and oxic-to-anoxic conditions. Incubated picocyanobacteria assimilate 13C-glucose and ammonium under anoxic-dark conditions, grow slowly without light, and retain the capability to synthesize photosynthetic pigments. They moderately upregulate genes responsible for chlorophyll-a/bilin biosynthesis, uptake of C sources, debranching of glycogen storage, and the pentose phosphate pathway for ATP, NADH, and NADPH production, as well as genes involved in NAD+ regeneration, including proton-reduction hydrogen-metabolism coupled with lactic acid fermentation. Our results support the remarkable versatility, plasticity, and potential for mixotrophy in photoautotrophs that allow them to dominate diverse aquatic systems worldwide.

The deep subsurface is a dynamic and biologically active environment that harbors a vast array of microbial communities, accounting for a substantial fraction of Earth’s biomass. Most of the available information about subsurface ecosystems in continental regions is derived from studies on sedimentary rock formations and the analysis of groundwater and deep fluids accessed through boreholes and mines. Research on microbial life within crystalline bedrock has historically been more limited, primarily focusing on rocks such as granites, schists, and serpentinized ophiolites. Nevertheless, several studies have demonstrated that fractured crystalline rocks can host unique and diverse microbial ecosystems. In this study, we present the microbiological characterization of a water overflow at the borehole 5071_1_B (IGSN: ICDP5071EH30001) in the context of the ICDP-sponsored DIVE (Drilling the Ivrea-Verbano zonE) project (expedition number 5071) aiming for a full geophysical and petrological characterization of the continental lower crust in the Ivrea-Verbano Zone and for the identification of microbial communities inhabiting the different lithologies encountered in borehole 5071_1_B. During the drilling operation, a water overflow was observed at a depth of 300-316 m below current surface, due to the presence of a deep aquifer. The fluids were analysed geochemically and through a combination of 16S rRNA gene amplicon sequencing, metagenomic analysis, and epifluorescence microscopy. By using the waters of the nearby Toce River and on-site contamination tracking procedures we provide hypotheses on the origins of the rising fluids, as well as insights into the microbial taxonomic and functional diversity within the deep aquifer fluids.

Island coastal aquifers, though physically small compared to continental groundwater systems, are of huge ecological and societal importance, sustaining functions that connect to locally crucial provision, maintenance and culture ecosystem services. Those functions are largely dependent on the presence of highly adapted biological communities, for which, their microbial communities remain understudied. Our goal is to describe the bacterial communities across the groundwater-dependent ecosystems on Lanzarote, spanning a gradient of anthropogenic pollution using 16SrRNA amplicon sequencing. We sampled coastal caves and pools, wells and water galleries, springs, saltworks and marine bays affected by submarine groundwater discharge. Ecological analyses highlight that richness and composition of bacterial communities strongly depend on the type of habitats. Pathogens and human-derived species were ubiquitous in our samples, but, strikingly, caves and wells were strongly enriched with them compared to other habitats. We propose that our results highlight the susceptibility of groundwater environments to pollution and indicate that aquifers act as reservoirs of biological contamination in addition to natural diversity—regardless of their salinity. Since this enrichment might compromise some of the functions and services that groundwater-dependent ecosystems provide in oceanic islands, we call for integrative conservation strategies that include hydrological and biological perspectives into the decision making.

By hosting significant amounts of extra-atmospheric dissolved gases, including geogenic CO2 and CH4, volcanic lakes provide relevant ecosystem services through the key role the aquatic microbial community in mediating freshwater carbon fluxes. In view of elucidating the mechanisms governing the microbial spatial distribution and the possible implications for ecosystem functioning, we compared the hydrogeochemical features and the microbial community structure of two adjacent stratified volcanic lakes (Lake Grande - LG and Lake Piccolo - LP). Water chemistry, gases and their isotopic composition were coupled with microbial pigment profiling, cell counting, and phylogenetic analyses. LP showed transparent waters with low concentrations of chlorophyll-a and the occurrence of phycoerytrin-rich cyanobacteria. LG was relatively more eutrophic with a higher occurrence of diatoms and phycocyanine-rich cyanobacteria. Considering the higher concentrations of CO2 and CH4 in bottom waters, the oligotrophic LP was likely a more efficient sink of geogenic CO2 in comparison to the adjacent eutrophic LG. The prokaryotic community was dominated by the mixothrophic hgcI clade (family Sporichthyaceae) in the LG surface waters, while in LP this taxon was dominant down to -15 m. Moreover, in LP, the bottom dark waters harbored a unique strictly anaerobic bacterial assemblage associated with methanogenic Archaea (i.e. Methanomicrobiales), resulting in a high biogenic methane concentration. Water layering and light penetration were confirmed as major factors affecting the microbial distribution patterns. The observed differences in the geochemical and trophic conditions reflected the structure of the aquatic microbial community, with direct consequences on the dynamics of dissolved greenhouse gases.

IntroductionHorizontal gene transfer (HGT) of antibiotic resistance genes (ARGs) is one of the primary routes of antimicrobial resistance (AMR) dissemination. In the One Health context, tracking the spread of mobile genetic elements (MGEs) carrying ARGs in agri-food ecosystems is pivotal in understanding AMR diffusion and estimating potential risks for human health. So far, little attention has been devoted to plant niches; hence, this study aimed to evaluate the conjugal transfer of ARGs to the bacterial community associated with the plant rhizosphere, a hotspot for microbial abundance and activity in the soil. We simulated a source of AMR determinants that could enter the food chain via plants through irrigation.MethodsAmong the bacterial strains isolated from treated wastewater, the strain Klebsiella variicola EEF15 was selected as an ARG donor because of the relevance of Enterobacteriaceae in the AMR context and the One Health framework. The strain ability to recolonize lettuce, chosen as a model for vegetables that were consumed raw, was assessed by a rifampicin resistant mutant. K. variicola EEF15 was genetically manipulated to track the conjugal transfer of the broad host range plasmid pKJK5 containing a fluorescent marker gene to the natural rhizosphere microbiome obtained from lettuce plants. Transconjugants were sorted by fluorescent protein expression and identified through 16S rRNA gene amplicon sequencing.Results and discussionK. variicola EEF15 was able to colonize the lettuce rhizosphere and inhabit its leaf endosphere 7 days past bacterial administration. Fluorescence stereomicroscopy revealed plasmid transfer at a frequency of 10−3; cell sorting allowed the selection of the transconjugants. The conjugation rates and the strain’s ability to colonize the plant rhizosphere and leaf endosphere make strain EEF15::lacIq-pLpp-mCherry-gmR with pKJK5::Plac::gfp an interesting candidate to study ARG spread in the agri-food ecosystem. Future studies taking advantage of additional environmental donor strains could provide a comprehensive snapshot of AMR spread in the One Health context.

Antibiotic resistance genes (ARGs) are abundant in aquatic ecosystems affected by human activities. Understanding the fate of ARGs across different ecosystems is essential because of the significant role aquatic environments play in the cycle of antibiotic resistance. We quantified selected ARGs in Lake Maggiore, its main tributaries, and the effluent of the main wastewater treatment plant (WWTP) discharging directly into the lake. We linked their dynamics to the different anthropogenic impacts in each tributary's watershed. The dynamics of tetA in the lake were influenced by those of the rivers and the WWTP effluent, and by the concentration of N-NH4, related to anthropogenic pollution, while sul2 abundance in the lake was not influenced by any water inflow. The dynamics of the different ARGs varied across the different rivers. Rivers with watersheds characterized by high population density, touristic activities, and secondary industries released more ARGs, while ermB correlated with higher numbers of primary industries. This study suggests a limited contribution of treated wastewater in the spread of ARGs, indicating as prevalent origin other sources of pollution, calling for a reconsideration on what are considered the major sources of ARGs into the environment.

Aquatic ecosystems are crucial in the antimicrobial resistance cycle. While intracellular DNA has been extensively studied to understand human activity's impact on antimicrobial resistance gene (ARG) dissemination, extracellular DNA is frequently overlooked. This study examines the effect of anthropogenic water pollution on microbial community diversity, the resistome, and ARG dissemination. We analyzed intracellular and extracellular DNA from wastewater treatment plant effluents and lake surface water by shotgun sequencing. We also conducted experiments to evaluate anthropogenic pollution's effect on transforming extracellular DNA (using Gfp-plasmids carrying ARGs) within a natural microbial community. Chemical analysis showed treated wastewater had higher anthropogenic pollution-related parameters than lake water. The richness of microbial community, antimicrobial resistome, and high-risk ARGs was greater in treated wastewaters than in lake waters both for intracellular and extracellular DNA. Except for the high-risk ARGs, richness was significantly higher in intracellular than in extracellular DNA. Several ARGs were associated with mobile genetic elements and located on plasmids. Furthermore, Gfp-plasmid transformation within a natural microbial community was enhanced by anthropogenic pollution levels. Our findings underscore anthropogenic pollution's pivotal role in shaping microbial communities and their antimicrobial resistome. Additionally, it may facilitate ARG dissemination through extracellular DNA plasmid uptake.

Phylosymbiosis, the association between the phylogenetic relatedness of hosts and the composition of their microbial communities, is a widespread phenomenon in diverse animal taxa. However, the generality of the existence of such a pattern has been questioned in many animals across the tree of life, including small-sized aquatic invertebrates. This study aims to investigate the microbial communities associated with poorly known marine interstitial nemerteans to uncover their microbiota diversity and assess the occurrence of phylosymbiosis. Specimens from various Central American sites were analyzed using morphology-based taxonomy and molecular techniques targeting the host 18S rRNA gene whereas their microbial association was analyzed by targeting the prokaryotic 16S rRNA gene. Phylogenetic and statistical analyses were conducted to examine the potential effects of host nemertean taxa and sampling locations on the host-associated microbial communities. The results provide compelling evidence of phylosymbiosis in meiofaunal nemertean species, highlighting the significant impact of host genetic relatedness on microbiome diversity in small-sized animals. This finding supports previous studies that demonstrate how certain nemertean species harbor distinct microbial communities with functional and ecological implications. Given the remarkable diversity of meiofaunal animals-spanning numerous phyla with varying lifestyles and co-existing in the same habitat-combined with advancements in multi-omics approaches, there is a promising opportunity to deepen our understanding of the evolutionary and ecological interactions between hosts and their microbiota throughout the animal tree of life.

Wastewater treatment plants (WWTP) effluents can release microbiological pollutants, including the int I1 gene (integrases of class 1 integrons), which has been proposed as a target for monitoring anthropogenic pollution in surface waters. This gene has also a strong correlation with antibiotic resistance, making of it an important proxy to evaluate the level of genetic contamination in aquatic environments. he ecological factors that influence the abundance and dynamics of intI 1 within natural water bodies are largely unknown. To better understand the fate of class 1 integrons in aquatic systems, we resorted to classical limnological monitoring of intI 1 over multiple years. We also conducted experiments to elucidate the impact of Daphnia grazing on its abundance. The monitoring of different size fractions of the Lake Maggiore microbial community has shown a particle-bound life-style for intI 1-harbouring bacteria. Most of the bacteria hosting intI 1, originating from both a wastewater effluent that discharges intro Lake Maggiore and lake water itself, grow on particulate substrates in open waters, making them particularly vulnerable to grazing by large filter feeders such as Daphnia . Daphnia grazing is independent from the origin (lake water or wastewater) of the bacterial genera; it selectively removes bacteria that are present in aggregates or even filamentous forms from both origins. To understand if intI 1 is related to viable bacteria or just DNA residues, it is important to study the persistence of class 1 integrons with their gene cassettes, which often contain antibiotic resistance genes in freshwater ecosystems.Significance Statement While faecal pollution of freshwaters is commonly monitored, genetic pollution through wastewater treatment plant outflows, such as antibiotic resistance genes, is difficult to monitor due to the diverse nature of genes present. The intI 1 gene is proposed as a proxy for anthropogenic pollution; however, there is a major lack of understanding regarding the persistence of this gene in freshwaters. In this study, we demonstrate that intI 1 in freshwaters is associated with both the natural microbial community and allochthonous microbes arriving from wastewater. Furthermore, we show that intI 1 harbouring bacteria preferentially reside in the aggregated microbial fraction and are easily removed by zooplankton grazing. This study is the first limnological investigation of this gene and highlights a significant gap in our knowledge regarding the ecology of class 1 integrons.Genetic pollution of surface waters is however a global problem and of very broad interest on the one hand, on the other hand, the question of the establishment of an allochthonous gene into a natural microbial community is also an interesting fundamental question in ecology, thus this study has both more applied and more fundamental aspects. Therefore, we consider it perfect for the readership of L&O.### Competing Interest StatementThe authors have declared no competing interest.

Wastewater treatment plant effluents release microbiological pollutants, including the intI1 gene (integrases of class 1 integron), which has been proposed as a target for monitoring anthropogenic pollution in surface waters. This gene correlates with antibiotic resistance genes, making it an important proxy for genetic contamination in aquatic environments. It is currently unclear whether intI1 found in lake water is mainly present due to continuous seeding or if autochthonous bacteria harbor this gene. To better understand the fate and dynamics of class 1 integrons in aquatic systems, we resorted to classical limnological monitoring of intI1 (qPCR) over multiple years in three different size fractions: free-living bacteria, particle-attached bacteria, and zooplankton-attached bacteria. We also conducted experiments to elucidate the impact of grazers on the abundance of intI1. The monitoring of different size fractions of the Lake Maggiore microbial community showed a particle-bound lifestyle for intI1-hosting bacteria. Most of these bacteria originated from both a wastewater effluent that discharges into Lake Maggiore and the lake water itself (amplicon sequencing). We hypothesize that these bacteria grow on particles in open waters, making them particularly vulnerable to grazing by large filter feeders such as Daphnia. Therefore, the presence of Daphnia reduced the abundance of intI1 in lake water, whereas this was not true for other grazers such as Rotaria macrura or Poterioochromonas sp. Our study shows that the food web structure and temporal changes in the lake influence the abundance of intI1 and consequently the assessment of anthropogenic pollution.

Terrestrial input to marine and freshwater ecosystems colors the water yellow-brown, causing a phenomenon called “brownification”. The effect of brownification on the marine pelagic microbial food web was studied in the oligotrophic eastern Mediterranean in June 2021 by adding HuminFeed in a 15-day mesocosm experiment with 2 treatments: Control (C, no addition) and HuminFeed (HF, single dose of HuminFeed, 2 mg L-1); and 3 replicates per treatment. HuminFeed caused shading, leading to a decrease in the abundance of photo-autotrophic organisms (cyanobacteria Synechococcus and diatoms). Bacteria were positively affected by the HF addition (mainly in terms of production rather than abundance), benefiting either directly from the dissolved organic carbon (DOC) contained in HuminFeed or indirectly from the trophic cascade through the food web. Despite the decrease in HF bacterial abundance during the experiment, an increase in both the high nucleic acid containing bacteria% and heterotrophic bacterial production were observed, suggesting higher activity at the single cell level. In the HF treatment, the increased abundance of dinoflagellates observed could be due to either a dominance of mixotrophic species or a release from predation by copepods. Both ciliates and copepods were severely impacted by HuminFeed, showing lower abundance and distorted forms (ciliates) and reduced reproductive potential (copepods). In conclusion, in the ultraoligotrophic eastern Mediterranean, the simulated brownification negatively affected autotrophs and top predators while benefiting bacteria, thus indicating a shift in the structure of the plankton food web.

A non-genetic transgenerational inhibitory effect on sexual reproduction has been demonstrated in Brachionus plicatilis in relation to environmental predictability. Indeed, clones of this species from more predictable environments do not respond to sex-inducing cues during several generations after leaving diapause. Notwithstanding, the molecular basis of this effect is still unknown. In this work, the expression level of genes related to the synthesis of sex hormones and to a potential epigenetic signalling mechanism were tracked along successive generations from diapausing eggs in clones of B. plicatilis populations inhabiting ponds with different level of environmental predictability. The selected genes were (1) the 17- β -dehydrogenase gene ( edh ), involved in the synthesis of 17- β- estradiol hormone in rotifers, and (2) the DNMT2 gene ( meth ), as a candidate epigenetic mechanism of control. According to expectations, results showed an increasing expression of edh across generations in clones from those the more predictable ponds. This finding provides a putative role of estradiol in the transgenerational effect. However, no differences were found in the meth gene neither across generations nor regarding the environmental predictability. Despite this, we point out alternatives for future research on the inherited gene regulation mechanism behind the transgenerational effect.

The management of Invasive Alien Species (IAS) is often hindered by ecological, social and economic factors, resulting in inadequate biodiversity protection and inefficient use of public money. A clear example of such inefficient management in aquatic ecosystems is the European catfish Silurus glanis L. in southern Europe. Native to central Eurasia, S. glanis is an emblematic and controversial freshwater fish, being the subject of extensive and profitable trophy angling in central Europe and of commercial fishing in eastern Europe. Concurrently, in western and southern Europe where it was introduced in the XIX century, S. glanis is considered a problematic invader. The lack of comprehensive information on S. glanis invasive populations has limited effective management, which is critical to successfully control the spread and minimize negative impacts on native ecosystems and species. LIFE PREDATOR, started in September 2022 with a budget of € 2.85 million and a consortium of six partners from three countries, aims at developing a multidisciplinary and transnational approach to control established populations of S. glanis, and prevent further spreading and future introductions in southern European lakes and reservoirs. The project will develop and test an early warning system based on eDNA and citizen science and identify the most effective and selective capture techniques to reduce the abundance of catfish, particularly in Natura 2000 lakes, actively involving anglers and professional fishermen on this. Massive raising awareness campaigns will be conducted targeting anglers but also the general public, and protocols and best practices will be transferred to management authorities. For the long-term sustainability of the project, a South European Management Group will be created. Additionally, in northern Italy, where the catfish invasion is more advanced, a local circular economy will be implemented, involving the increase in fishing pressure by encouraging catfish consumption as food.