Freshwater Biology

Passive sampling is the deployment of a collection material in the environment to continuously capture environmental DNA (eDNA) over time, offering the potential to integrate biodiversity signals while reducing the need for repeated active water collection. However, the mechanisms governing eDNA capture and retention on passive samplers remain poorly understood, limiting the interpretation of passive eDNA signals and their broader application. Here, we investigated the mechanistic performance of glass fibre passive samplers using controlled mesocosm experiments with three invasive freshwater bivalves: zebra mussels (Dreissena polymorpha), quagga mussels (Dreissena bugensis), and Asian clams (Corbicula fluminea). Specifically, we quantified eDNA accumulation dynamics, evaluated the contribution of different eDNA states, tested the persistence of captured eDNA, and compared passive sampler signals with conventional active sampling. Passive samplers rapidly accumulated target eDNA within hours of deployment, after which concentrations either plateaued or continued to increase depending on species. Sequential transfer of passive samplers between mesocosms containing different species showed that previously captured eDNA declined while new target eDNA accumulated to concentrations comparable to freshly deployed samplers, demonstrating continual turnover rather than permanent retention. Dissolved eDNA showed little evidence of accumulation beyond the concentration retained in the pore water within the membrane, suggesting that it is unlikely to be the dominant contributor to long-term passive sampler signals. Instead, the observed variability among replicate samplers, together with the physical properties of glass fibre membranes, suggests that membrane-bound and particulate eDNA are the primary contributors to passive eDNA capture. Collectively, these findings support a model in which glass fibre passive sampler signals reflect a dynamic equilibrium between ongoing eDNA capture and concurrent loss processes rather than cumulative accumulation over time. This mechanistic framework provides a foundation for interpreting passive eDNA data and informs the future development of passive sampling materials, deployment strategies, and biodiversity monitoring applications.

Understanding the evolutionary history of species within a geographic context is key to historical biogeography, as it reveals how geological and climatic changes shaped biodiversity. This is especially important in ecologically significant regions like the Amazon and Orinoco basins. Together, they host the worlds greatest freshwater fish diversity ([~]3,500 species), sharing a common but not yet fully understood evolutionary history. The gilded catfish (Brachyplatystoma rousseauxii), an ancient species widely distributed as a metapopulation in Neotropics, is an important model for studying past connectivity, divergence, and historical processes shaping fish diversity between these basins. This study analyzed the genetic structure, connectivity routes, and demographic history of B. rousseauxii using nuclear (microsatellite and ddRADseq) and mitochondrial DNA. Population structure analyses and coalescent models indicate that B. rousseauxii populations from the Orinoco and Amazon basins are genetically distinct, with no evidence of current gene flow. However, our results support the occurrence of a possible secondary contact event after the divergence, with the Boa Vista population retaining the genetic signal of this process. The ancestral population split occurred at the Rupununi Portal around 2.54 Ma (ddRAD) or 1.31 Ma (mtDNA). Then, the species colonized the Branco and Orinoco Rivers [~]1.90 Ma (ddRAD) or 0.6 Ma (mtDNA), rapidly expanding in the Orinoco (>1.3 or >0.29 Ma), while colonization of the Amazon from the Branco River was more recent ([≤]1.0 or [≤]0.15 Ma). Population expansion signal was detected in the Orinoco ([~]0.20 Ma), whereas the Amazon remained stable. Our findings suggest that the rise of the Vaupes Arch in the Late Miocene does not explain the observed genetic divergence. Likewise, the Casiquiare Canal and Japura-Guaviare headwaters are not connectivity routes between basins. Instead, the Rupununi Portal, including the recent capture of the Branco River by the Negro River, was the last point of connection and played a key role in shaping B. rousseauxiis distribution. These findings provide insights into Neotropical fish biogeography and the historical configuration of the Orinoco and Amazon basins.

Context Pumping stations pose a threat to fish globally through land use change, habitat fragmentation and entrainment risk, with the catadromous and critically endangered European eel particularly impacted. Objectives/methods Establish, model, assess and understand the present-day distribution of European eel and resident fishes in 152 pumping station catchments in a once extensive wetland (The Fens) using eDNA metabarcoding (855 samples over two and half years), with specific focus on anthropogenic influences on hydrological connectivity and habitat quality. A removal survey design maximised confidence in negative results while minimising time and consumable costs. Results Eel occurrence upstream of pumping stations was low (occupancy = 28.3%) and positively associated with catchment area, fish species richness and natural hydrological connectivity (gravity drainage or flooding) and negatively associated with distance from the tidal limit. Fish species richness replaced catchment area and improved model performance, potentially acting as a biotic indicator of habitat quality and connectivity. Pumped catchments with manually operated upstream water transfers had reduced eel presence, potentially linked to the direction of water flow or the timing of operation. By contrast, fish species richness increased in these catchments during summer, suggesting displacement into unsuitable long-term habitats. Physical habitat maintenance had no detectable effect on eel occurrence or fish species richness. Conclusions This study provides the first landscape-scale assessment of European eel distribution and drivers of occurrence in pumped river catchments. The highly novel and comprehensive insights have implications for European eel conservation as well as infrastructure and catchment management, including compliance with legislation (EC Regulation No. 1100/2007).

Climate driven aridification and intensified human activity are placing increasing pressure on Mediterranean freshwater ecosystems. These impacts propagate from land to water, altering nutrient regimes and reshaping aquatic microbial communities. We analysed Arcellinida diversity across 363 lentic inland saline and freshwater sediment samples spanning broad gradients of land use, water chemistry, soil properties, and climate in southern Spain. Random forest models identified terrestrial land use intensity followed by water chemistry as main predictors of community diversity. Diversity declined sharply in sites with population densities above [~]33 inhabitants/km{superscript 2} and under eutrophic conditions, but peaked in oligotrophic systems with stable, carbon rich soils. These threshold responses demonstrate that aquatic protist assemblages integrate both long term terrestrial pressures and current water conditions. Overall, our findings show that landscape transformation and its cascading effects on water quality dominate community assembly, and that the combination of community level diversity metrics with selected taxon-level indicators capture ecosystem degradation more consistently than relying on a single metric.

Intertidal mudflats are low hydrodynamic energy environments hosting microphytobenthic communities that experience strong spatiotemporal variability in light regimes, including changes in spectral quality and light intensity that can lead to cellular photooxidative stress. To cope with these fluctuations, autotrophs exhibit diverse and highly plastic adaptations that are often species-dependent and shaped by their ecological niches. This study investigates photophysiological responses and metabolic remodeling in a diatom assemblage originating from a natural winter microphytobenthic biofilm under contrasting red and blue light intensities. To this end, photosynthetic parameters were monitored alongside changes in lipophilic metabolites, including untargeted lipids and lipophilic pigments. While few metabolites showed temporal remodeling, rapid and contrasting changes were observed within 30 minutes in response to both spectral quality and light intensity. Red light treatments induced broader remodeling of lipophilic metabolites than blue light, whereas blue light appeared to have a greater impact on photosynthetic parameters. Moreover, red light induced xanthophyll-cycle responses comparable to those observed under blue light at equivalent incident intensity. We discuss these metabolic responses in relation to diatom photoadaptive strategies, placing these findings within the intertidal environmental framework. This work further underlines the importance of understanding rapid metabolic plasticity in coping with light fluctuations, providing new insights into the photoregulatory strategies of natural microphytobenthic communities.

Freshwater bioassessment relies on assessing aquatic assemblages to infer ecological conditions, yet conventional surveys require extensive field sampling, specimen processing, and specialized taxonomic expertise. Existing environmental DNA (eDNA) methods have not yet provided a practical alternative to conventional macroinvertebrate assays in part because current approaches cannot feasibly recover broad taxonomic diversity at sufficient taxonomic resolution. Here, we evaluated targeted hybridization capture of mitochondrial cytochrome oxidase I (COI) target sequences as a unified molecular approach for cross-phylum freshwater bioassessment. Environmental DNA was collected at 18 sites along 63 km of Boulder Creek spanning nearly 1,500 m of elevation from forested headwaters to agricultural plains. COI targets were enriched using custom RNA bait panels designed to target regional freshwater arthropods, annelids, and molluscs. Hybridization capture increased recovery of COI sequences [~]1,760-fold relative to unenriched shotgun libraries, generating Folmer-region COI contigs that averaged [~]400 bp. Across the watershed, we recovered sequences for approximately 450 macroinvertebrate genera across 8 phyla. Detected macroinvertebrate richness averaged 56 genera per site and increased down Boulder Canyon before declining downstream of the city. Macroinvertebrate assemblage composition from hybridization capture paralleled patterns observed with past conventional bioassessment. These results demonstrate that targeted hybridization capture enables robust, cross-phylum detection of species used for freshwater bioassessment from environmental DNA.

While the suppression of toxic cyanobacteria by aquatic plants has long been recognized, few studies have clearly differentiated between the allelopathic effects of the plant itself and the inhibitory influence of its associated microbiome. This study aimed to clarify the primary inhibitory agent by pre-culturing Myriophyllum spicatum (Eurasian watermilfoil) under three conditions: (1) BG11 medium, (2) live Microcystis aeruginosa KW culture, and (3) a Microcystis-symbiotic microbiome (excluding Microcystis cells). After a 7-day pre-culture, Myriophyllum shoots were transferred to fresh Microcystis culture. The Myriophyllum pre-cultured in Microcystis culture exhibited rapid inhibition against Microcystis (84% within day 1), whereas the Myriophyllum pre-cultured in BG11 medium showed delayed responses (89% by day 7). In contrast, inhibition remained below 50% in the Myriophyllum pre-cultured with the Microcystis-symbiotic microbiome. Notably, plant-derived soluble compounds exhibited weak inhibitory effects, whereas the microbiome showed stronger inhibitory activity, indicating that the plant-associated microbiome plays a more dominant role than the plant itself. Exposure to Microcystis triggered significant shifts in plant-symbiotic microbial community composition, leading to rapid enhancement of inhibitory activity in the Myriophyllum microbiome. Microbial community analysis identified 28 bacterial taxa closely associated with the inhibitory response, including strains involved in organic matter degradation, adhesion, biofilm formation, and predatory behavior. Meta-transcriptomic analysis further confirmed increased expression of genes related to bacterial adhesion, biofilm formation, and carbohydrate metabolism following Microcystis exposure, highlighting functional adaptations linked to cyanobacterial suppression. These findings underline the role of microbiome-mediated cyanobactericidal mechanisms, providing new insights into a nature-based solution for mitigating Microcystis-dominated harmful algal blooms.

Replication is central to most experimental and sampling designs, increasing inferential power and capturing fine-scale data heterogeneity. However, its importance remains poorly evaluated in some ecological and evolutionary settings. This is the case of metabarcoding studies using DNA recovered from sedimentary archives, in which biological signals may integrate ecological information through depositional and burial processes, and are often inferred from a single sediment core per site. Here, we evaluated the effect of different types of replication using sedimentary DNA (sedaDNA) metabarcoding data from two genetic markers (mitochondrial COI and nuclear 18S), under a nested sampling design. The design included three intertidal sites, three spatially separated sediment cores per site (biological replicates), two sediment depth horizons per core, and eight PCR (technical) replicates per sediment sample. Variance partitioning showed that site identity and sediment age group together explained >70% of the variation in beta diversity, indicating that among-site spatial variation and stratigraphic variation were the dominant drivers of community composition. In contrast, variation among different cores within sites was small and non-significant (<5%). Among PCR replicates from the same sediment sample, richness varied substantially, whereas Shannon diversity was more consistent. Despite this variability, differences in community composition among technical replicates remained smaller than among biological replicates and site identity, indicating limited influence on broader ecological patterns. Community composition was highly similar among replicate cores within sites, consistent with stratigraphic coherence. These results indicate limited within-site heterogeneity and suggest that, under stratigraphically coherent conditions, increasing biological replication may yield limited additional information, whereas enhancing technical replication and stratigraphic resolution can improve ecological inference from sedaDNA metabarcoding datasets.

This research addresses a central question in Palaeolithic research: how hunter-gatherer mobility was structured across space and time in the Cantabrian Region (northern Iberia), which has human occupation evidence spanning from the Middle Pleistocene through the Holocene. A multidisciplinary framework integrating primarily {delta}3S isotope values, combined with {delta}{superscript 1}3C and {delta}{superscript 1}N, palaeoproteomics, Bayesian age modelling, palaeoclimatic reconstruction, isoscape mapping, and ecological diversity was developed. A total of 905 animal bone collagen samples, with evidence of anthropogenic modifications, from 16 key archaeological sites from the Mousterian to Mesolithic (Marine Isotopic Stage 5 to 1, between 100-7 ka BP) were analysed, permitting the reconstruction of spatial patterns of resource exploitation and human mobility. The {delta}3S isotope values show weak, inconsistent relationships with climatic proxies, suggesting that sulfur signatures are primarily driven by geographic and ecological factors rather than climate. Strong spatial trends are observed, with higher {delta}3S values in coastal zones and lower values inland. Diachronic trends reveal marked shifts in human mobility: smaller ranges during the Mousterian, increasing mobility through the Chatelperronian and especially the Aurignacian, followed by reduced mobility in the Gravettian and Solutrean, and renewed territorial expansion during the Magdalenian and, likely, the Azilian. In contrast, the Mesolithic is characterised by decreased mobility and thus increased territoriality in both coastal and inland contexts. Faunal isotope values and isoscape predictions reveal that some animals were acquired beyond local foraging ranges during the Palaeolithic, particularly in inland regions with lower {delta}3S values. Isotopic niche analyses indicate partial interspecific overlap consistent with ecological flexibility. Macromammal and micromammal diversity exhibit contrasting patterns, with a significant negative correlation in Simpson and Shannon indices. Macromammal diversity correlates negatively with {delta}3S values, linking increased hunting diversity to expanded catchment areas and longer-distance foraging, whereas micromammal diversity shows positive correlations with {delta}3S, {delta}{superscript 1}3C and {delta}{superscript 1}N reflecting stronger climatic influence. Overall, these results demonstrate that hunter-gatherer behaviour in northern Iberia during the Middle and Late Palaeolithic was highly dynamic, combining logistical and residential strategies that shifted in response to changing environmental conditions, resource distributions and cultural adaptations.

Phenotypic differences among populations can arise through heritable genetic divergence, phenotypic plasticity, or both, making it difficult to determine whether trait-environment correlations observed in nature reflect adaptive evolution. Within threespine stickleback (Gasterosteus aculeatus) studies, numerous document morphological differences among allopatric-, parapatric-, and even sympatric populations. These phenotypic differences among populations are often correlated with diet and lake habitat (e.g., lake size), suggesting an adaptive value to the population differences. However, many studies of ecomorphological divergence in stickleback use wild-caught stickleback, which may differ due to evolution or plasticity. Although common garden experiments have confirmed that population differences can be heritable, such experiments typically entail small numbers of populations. Consequently, we still do not know to what extent well-known trait-environment correlations in stickleback are a result of evolution. To address this gap, we reared stickleback embryos from 27 lake populations on Vancouver Island, in a laboratory environment. Morphological differences among populations persist in common-garden fish, confirming a large role for divergent evolution. These heritable differences were associated with environmental variation among lakes, implying an adaptive value. However, some well-known trait-environment relationships in stickleback did not persist in common-garden fish and may be primarily plastic.

Giant clams are some of the largest bivalve molluscs. They form a vital partnership with Symbiodiniaceae dinoflagellates that supply most of their energetic requirements. However, the factors that shape giant clam-associated photosymbiont communities remain unknown. Here, we profiled Symbiodiniaceae communities using ITS2 metabarcoding in eight giant clam species (Hippopus hippopus, H. porcellanus, Tridacna crocea, T. derasa, T. gigas, T. maxima, T. noae and T. squamosa) from 11 sites across the Philippine archipelago. Symbiodiniaceae community structure was shaped by an interplay between giant clam host and environment. Most giant clams were dominated by members of a single symbiont genus, with Cladocopium as the most prevalent, followed by Durusdinium and Symbiodinium. However, giant clam hosts also exhibited flexibility in their symbiotic partners that was evident across sites. Differences in giant clam-associated symbiont communities may contribute to differences in holobiont function and adaptability to variable environments. These findings deepen our understanding of giant clam-Symbiodiniaceae associations, offering a framework for predicting how giant clams may be affected by increasingly stressful reef conditions and, more importantly, informing strategies to improve mariculture and conservation practices.

Urbanization is reshaping freshwater ecosystems, with well-documented effects across gradients of land-use change, hydrologic alteration, and habitat degradation. However, how biodiversity is organized among neighboring urban aquatic habitats that differ in hydrologic connectivity, disturbance transmission, residence time, management history, and opportunities for species movement is often less clear. This creates a challenge for interpreting urban fish communities at local scales as species occurrence may reflect both contemporary habitat filtering and historical contingencies including native persistence, interbasin transfer, stocking, and nonindigenous introductions. Here we use eDNA detections, historical records, phylogenetic information, and species trait data to investigate the fish assemblages of the Charlotte metropolitan region. We detect a highly mixed fauna that also depicts a strong signature of structured biodiversity profiles across taxonomic, phylogenetic, functional, and life-history dimensions between habitat types. In particular, bounded habitats contained assemblages with larger-bodied species that are fecund and faster to reproduce relative to free-flowing habitats. Species-level occurrence models did not support a simple trait-by-habitat rule. Instead our results demonstrate that urban aquatic habitats can sort historically mixed regional species pools into predictable assemblage-level life-history profiles while simultaneously retaining signatures of evolutionary and historical biogeographic contingency.

Flow regulation for hydropower production affects stream ecosystems through decreased connectivity and changed timing and magnitude of flows. Hydropeaking, a form of regulation in which infrequent large peak flows are replaced with frequent small peaks, increases riparian erosion and causes water and drought stress for riparian vegetation. Hydropeaking is likely to affect soil seed bank (SSB) formation and composition, while SSBs are important sources of self-restoration should a systems flow regulation be relaxed. We tested how hydropeaking intensity affects the size and composition of SSBs, including the functionally important group of large graminoids, and by calculating Ellenberg values for Moisture, Light and Soil disturbance. SSB samples were taken at 15 riparian zones across central and northern Sweden. Each site was regulated, but sites differed in their hydropeaking intensities. SSBs were subjected to a seedling emergence experiment, from which over 700 seedlings from 53 taxa emerged. We found that hydropeaking intensity affects the composition of soil seed banks on multiple levels. Seedling density was negatively correlated with hydropeaking intensity at the sites where samples were taken. SSB richness varied (two to eighteen species per site) and was not affected by hydropeaking intensity. The proportion of large graminoids in the seed bank showed a near-significant decrease with increasing hydropeaking intensity, and community-weighted means for Moisture, Light and Soil disturbance increased (non-significantly) with increasing intensity. Our results suggest that riparian SSBs, should flow regulation be relaxed or ceased, are not sufficient for self-restoration of functional riparian vegetation. Seeds of large graminoids and species that are tolerant to drought in the germination stage are less present in riparian SSBs of heavily-regulated streams. Supply of seeds of these groups, or even planting, may need to be considered when changes in flow management are implemented. HighlightsO_LIHydropeaking negatively affects riparian soil seed banks (SSBs) in Sweden C_LIO_LISSB size slightly decreases with hydropeaking intensity, but richness does not change C_LIO_LIThe proportion of large graminoid seeds in SSBs decreases with hydropeaking intensity C_LIO_LIRiparian SSBs from less-impacted sites have most potential for self-restoration C_LI

Global change is driving rapid ocean warming, exposing organisms to both chronic temperature increases and acute marine heatwaves. Understanding how species cope with thermal stress is critical for predicting ecosystem resilience. Echinoderms are globally distributed and often function as foundational species, yet comparative assessments of upper thermal tolerance among species occupying contrasting thermal environments remain limited. Here, we address this gap by comparing upper thermal tolerance across three sea urchins with distinct biogeographic distributions: the latitudinally broad purple sea urchin (Strongylocentrotus purpuratus), the circumpolar green sea urchin (S. droebachiensis), and the tropical variegated sea urchin (Lytechinus variegatus). We quantified thermal limits after two acclimation treatments: ambient temperatures approximating native habitat conditions for each species and an elevated temperature (+6 C). We developed a novel assay to measure critical thermal maximum (CTmax), comparing variability and inconsistencies associated among multiple assays. Upper thermal tolerance increased with acclimation to elevated temperatures in all three species, but acclimatory capacity differed markedly, with S. droebachiensis showing the strongest response and S. purpuratus the weakest. Conversely, S. purpuratus had the highest thermal safety margin and thus the lowest proximity to its thermal ceiling. Our adhesion based CTmax method was more reproducible and the most precise compared to other metrics tested, providing an improved framework for quantifying physiological thermal limits of sea urchins. Together, these findings reveal substantial but unevenly distributed thermal resilience in ecologically diverse sea urchins, advancing our understanding of how foundational marine species may respond to future global change.

Ranges of small benthic fauna are notoriously difficult to assess. In some of these cases, modern eDNA methods can shed light on species occurrence. Here we conduct an exploratory study on the fish eDNA recoverable from the gastrovascular cavities of the easy-to-sample pore water siphoning benthic invertebrate, Cassiopea, across six sites within the Florida Keys. Twenty-seven fish 12S identities were recovered from water samples, two from sediment samples, and seventeen from Cassiopea gut swabs. In total, thirty-two different species were identified from nineteen families, including one shark species (Ginglymostoma cirratum), and five species of cryptobenthic reef fishes (f: Gobiidae, Labrisomidae). Additionally, five species were identified from medusae samples that were not recovered in water or sediment samples. The species identities recovered may provide insight into the fish in direct proximity to Cassiopea assemblages, as well as indicate that Cassiopea may accrue disproportionate eDNA from cryptobenthic reef fish compared to surrounding environmental samples. The unorthodox sampling technique of using eDNA recovered from jellyfish stomachs yields another avenue for epibenthic community data acquisition.

Previously, we have demonstrated that certain ferric salts common in Archean waters, such as iron trichloride and ferric ammonium citrate, can protect liposomes from destruction by short-wavelength UVC light. In this study, we investigate the propagation of 254 nm UV radiation through aqueous FeCl3 solutions and its interactions with liposomes. We then consider these findings in the context of early Earth UV environment, discuss their implications for our hypothesis of the Darwinian evolution of liposomes, and integrate them with our previous experimental results.

Arcellidae is a family of testate amoebae within Arcellinida (Amoebozoa), comprising three recognized genera: Arcella, Galeripora, and Antarcella. Although species in the family have been studied for nearly two centuries, many historically described taxa and major morphological groups remain unsampled at the molecular level. Here, we provide a comprehensive review of Arcellidae and generate new cytochrome c oxidase subunit I (COI) sequences for arcellid species from Canadian peatlands, focusing on tall-shelled Arcella historically classified in section Altae sensu Deflandre. COI phylogenetic analyses recover a strongly supported monophyletic clade corresponding to North American representatives of Altae, providing the first molecular corroboration of this morphologically defined group. Within this clade, we redescribe Arcella leidyana based on modern material from Eeyou Istchee (Quebec). We further describe Galeripora purdoni sp. nov. from a calcareous fen in eastern Ontario, representing a novel terrestrial lineage within the genus, and redescribe Galeripora artocrea, which we transfer to Arcella based on congruent molecular and morphological evidence. Phylogenomic analyses of Arcellidae isolates from the Protist 10,000 Genomes Project reveal an additional deep lineage basal to Arcella and Galeripora. Together, these results highlight hidden diversity and demonstrate the importance of integrative approaches for resolving arcellid systematics and refining its classification.

Assessing intraspecific variation in thermal stress tolerance is key to predicting plant responses and long-term persistence under climate change, yet its underlying sources and temporal dynamics remain poorly understood. Using a common garden experiment with four ecologically-relevant temperatures, we evaluated the sources and temporal dynamics of variation in temperature stress tolerance of 18 Lemna minor clonal lines from contrasting climates. Our results showed that past adaptation, physiological acclimation, and within-line variation jointly contributed to variation in performance. The study provides the first evidence of adaptive genetic differentiation in heat stress tolerance in this ecologically-widespread freshwater species, with lines from warmer regions showing higher growth under heat stress. However, these differences were transient and diminished under prolonged exposure. Experimental lines also showed acclimation over time, but these responses were strongly temperature-dependent and occurred only under sub-optimal conditions. Additionally, replicates from some lines exhibited divergent performance trajectories under sustained heat stress, suggesting the emergence of novel phenotypic variation, potentially mediated by epigenetic mechanisms. These results show that heat stress tolerance in L. minor arises from multiple interacting sources and is dynamically shaped by both selective history and immediate exposure time, suggesting a more nuanced, multi-layer understanding of variation in heat stress tolerance.

Newly established freshwater aquaria rely on development of biofilter nitrifying populations to prevent ammonia and nitrite accumulation that can negatively impact fish health. Although initial fish loads impact water chemistry of new aquaria, little is known about the corresponding impact on microbial community succession within freshwater aquarium biofilters. To address this gap, fourteen home aquarium systems were established, stocked with a range of fish loads, and maintained for eight months. Aquaria were sampled regularly to monitor nitrogen species, microbial community composition (16S rRNA gene sequencing), and the abundance of nitrifiers (qPCR). Aquaria with higher fish loads developed microbial communities that were compositionally distinct from those with lower fish loads, and were dominated by Pseudomonas, Rhodobacter, and Planctomycetes. These patterns are consistent with increased nutrient availability supporting biofilm development, whereas lower fish loads may delay biofilm maturation. Increasing the number of fish in an aquarium significantly increased maximum ammonia and nitrite concentrations, although both were ultimately depleted within similar timeframes across treatments. Comammox Nitrospira were among the most abundant biofilter nitrifiers and were present in all biofilter samples regardless of fish load. Ammonia-oxidizing bacteria were detected at relatively low abundance but showed increases in relative abundance within high fish load aquarium filters. Ammonia-oxidizing archaea were below sequencing detection limits and detected only at low levels by qPCR, suggesting that their establishment in aquarium biofilters may require higher initial inoculation or longer timeframes. Overall, these results demonstrate that fish load shapes microbial community development in newly established aquarium biofilters, and that comammox Nitrospira dominate among nitrifiers during early biofilter establishment.

Karst groundwater supplies in Virginia are relied on to varying degrees for domestic, agricultural, and municipal water supplies. Further, Virginian caves harbor an estimated 200 endemic invertebrate species. The microbial occupants of Virginias karst aquifers are largely undescribed; characterizing them promises to inform both the scientific description of these systems and the management of a critical water resource. Karst aquifers are heterogeneous, and much of the water moving through them cannot be reached directly; we profiled cave waters both because cave passages offer direct access to active groundwater and because cave water specifically is relied upon by endemic invertebrate species living in caves. Using 16S rRNA sequencing, we characterized aquatic microbial communities in eight Virginia caves, across Virginias four major karst regions. We identified 3,899 unique amplicon sequence variants (ASVs) and found that caves hosted diverse microbial assemblages that differed markedly among sampled sites. These baseline data provide a starting point for future work to understand how seasonal cycles, weather events, and surface disturbances affect the microbial communities present in cave waters and the cave-endemic invertebrates that depend on these waters.