Environmental Microbiology Reports

Background: Cyanobacteria are the major phototrophs in many endolithic ecosystems, yet their genomic diversity and metabolic adaptations remain poorly characterized. Here, we investigated cyanobacterial diversity in endolithic dolomite rocks from the Piora Valley in the southern Swiss Alps using genome-resolved metagenomics. Results: We reconstructed 138 medium-quality metagenome-assembled genomes (MAGs) from four neighboring endolithic rock communities, including 15 cyanobacterial MAGs distributed across six order-level lineages. The cyanobacterial communities showed limited taxonomic overlap among rocks, and most recovered MAGs represented potentially novel species. Only one Gloeobacter lineage occurred in all sampled rocks, which we propose as Gloeobacter dolomitensis sp. nov., representing the first Gloeobacter genome recovered from an endolithic environment. Comparative genomic analyses identified a cynS gene encoding cyanase unique to G. dolomitensis among the Gloeobacter. Phylogenetic analyses support its horizontal acquisition from Pseudomonadota. Interestingly, the genome of G. dolomitensis lacks a canonical CynABD cyanate transporter and instead contains a distinct ATP-synthase binding cassette (ABC) transporter adjacent to cynS. Although the available genomic and structural evidence does not establish cyanate transport, its genomic association with cynS suggests a potentially non-canonical mechanism. We further show that horizontal gene transfer was unevenly distributed among cyanobacterial lineages, with Acaryochloridales acting predominantly as donors, Chroococcidiopsidales as recipients, and Gloeobacterales showing a comparatively high frequency of interphylum gene exchange. Conclusions: The Piora Valley dolomite environment represents an underexplored reservoir of cyanobacterial diversity, including basal and previously undescribed lineages. The discovery of G. dolomitensis and its unusual cyanate-associated genomic architecture expands our understanding of Gloeobacterales diversity and highlights the potential importance of horizontal gene acquisition and metabolic innovation in endolithic microbial communities.

The human gut is home to more than 100 trillion bacteria, where various environmental factors, such as nutrients and gases, exist and influence them. The levels of fermentation gases in the gut, including CO2, fluctuate depending on the host's diet. However, how CO2, an environmental factor, affects the physiological states of gut anaerobes remains poorly understood, but CO2 is often utilized in their cultivation. Akkermansia muciniphila (A. muciniphila) is widely studied as a beneficial human gut microbe. However, the effects of CO2 on the physiological state of A. muciniphila are not fully understood. In this study, we focused on CO2 as an environmental factor and aimed to clarify how CO2 affects the physiological state of A. muciniphila. Culture experiments showed that compared with the absence of CO2, the presence of CO2 significantly increased the growth of A. muciniphila. The results of the metabolomic analysis revealed distinct metabolic profiles in the presence or absence of CO2. The tracing analysis using stable 13C-labelled CO2 demonstrated that CO2 was incorporated into various metabolites in the TCA cycle, the de novo purine/pyrimidine synthesis pathways, and other pathways. In addition, the activity of pyruvate carboxylase, which catalyses an anaplerotic reaction involving CO2 fixation to supply oxaloacetate (OAA) to the TCA cycle, was significantly higher in A. muciniphila cells grown in the presence of CO2. Moreover, supplementation with OAA increased the growth of A. muciniphila in a dose-dependent manner, mirroring the effects of CO2 supplementation. These findings suggest that A. muciniphila utilizes CO2 for an anaplerotic reaction in the TCA cycle and other pathways involving CO2 incorporation, facilitating metabolic flux to promote its robust growth.

Sinorhizobium meliloti is a nitrogen-fixing soil bacterium that forms a facultative mutualism with its host alfalfa. S. meliloti strains exhibit diverse colony morphologies on solid culture media that span a continuum from dry to mucoid. Studies on how this trait affects symbiosis rely on mutations in exopolysaccharide II production genes; however, whether natural variation in morphology confers a selective advantage on rhizobia in their natural habitats remains unknown. Here, we used 90 mucoid and 27 dry natural isolates to test whether colony morphology is associated with rhizobial fitness in the soil and in the host, or with the nitrogen-fixation benefits they provide to the plant. Colony morphologies were widely distributed across a phylogenetic tree, suggesting that morphological shifts occurred repeatedly and no single genetic variant was strongly associated with this trait. We inoculated soil mesocosms and plants with a mixture of dry and mucoid bacteria. We observed that dry rhizobia have higher fitness, particularly at 22{degrees}C and 32{degrees}C, but not under cold or drought conditions, independent of the initial rhizobial load. Conversely, mucoid bacteria have a modestly higher relative fitness in host nodules, and single inoculations showed that they tend to confer greater benefits to all three alfalfa varieties. Over time, we also observed rare, repeated conversion from mucoid to dry morphologies in soil and host plants. Taken together, our study reveals that morphological variation within a species in a single agricultural field is important for bacterial fitness in the soil and can have cascading effects on the rhizobia's plant benefits.

Hydrostatic pressure is a defining feature of the deep ocean, yet how it structures microbial activity across the subseafloor biosphere is poorly understood. Here, we assess pressure effects on anabolic activity in seafloor and subseafloor communities across a margin-to-hadal-to-abyssal transect of the Puerto Rico Trench region (493-8,385 m water depth). Community-level responses to pressure reverse with burial, revealing a previously unrecognized shift toward piezophily in resident subseafloor communities down to 20 meters below seafloor (mbsf). In pressure-incubation experiments, atmospheric pressure yielded higher proportions of active cells in surficial sediments, whereas in situ pressure yielded higher proportions in the subseafloor. Pressure also structured active-community composition; in trench-axis sediments extending to 8.35 mbsf, JS1/Atribacterota populations belonging to geographically widespread hadal lineages exhibited a physiological response to in situ pressure, indicative of subseafloor piezophily. These findings suggest that hydrostatic pressure acts as an ecological filter after burial, favoring piezophilic traits that may support microbial persistence in the deep subseafloor biosphere.

Dark waters harbour two-thirds of the ocean's bacterial and archaeal cells, yet the energy sources sustaining microbial life at these depths remain poorly understood. Here we integrate hydrographic profiles with metagenomic analyses and ex situ activity measurements to investigate whether carbon monoxide (CO) and molecular hydrogen (H2) are viable energy sources for epipelagic and mesopelagic microbial populations in the Mariana Trench region. Both gases were detected from 5 to 9,371 m, with concentrations generally higher in the photic zone but anomalously high in several deeper waters. Metagenomic analyses indicate a shift from phototrophic towards lithotrophic energy acquisition with depth. Microbial populations encoding CO dehydrogenases increased from 6.1% at 5 m to 45% at 1,000 m, while those encoding uptake hydrogenases were much less abundant, but increased from 0.34% to 0.63%. Ex situ incubations revealed that in situ cell-specific CO oxidation rates declined with depth, whereas corresponding H2 oxidation rates were significantly higher at 1,000 m than at the surface. At 1,000 m, cell-specific H2 oxidation rates were approximately 4,500-fold higher than those of CO. These contrasting trends suggest that CO oxidation is a widespread auxiliary energy-acquisition strategy, whereas H2 oxidation may sustain hydrogenotrophic growth in dark waters. Together, our results suggest H2 and CO are readily available to pelagic microbial populations and are biologically oxidised in the dark zone, supporting microbial energy acquisition and oceanic biogeochemical cycling.

Anaerobic gut fungi (AGF) are an integral part of the herbivore gut microbiome, primary decomposers of lignocellulose, and play a crucial role in establishing foundational links of syntrophic associations. The syntrophy with methanogens appears beneficial for mitigating feedback inhibition and results in the augmentation of AGF activity. However, the thermodynamic advantages of the presence of methanogens to AGF remain poorly understood. This study explores the preferred pathways adopted by Cyllamyces sp. and Methanobrevibacter olleyae, using 13C-glucose as a substrate and spiked with malate and pyruvate in different experimental set-ups. The results indicated reduced biomass formation by Cyllamyces sp. in monoxenic culture with Methanobrevibacter olleyae compared to axenic AGF culture supplemented with glucose. Additionally, metabolic analyses revealed the pyruvate:ferredoxin oxidoreductase (PFO) as a key contributor to pyruvate metabolism. Malate supplementation notably enhanced the growth of Cyllamyces sp. and M. olleyae, whereas pyruvate supplementation enhanced the growth of Cyllamyces sp. only in axenic culture. This investigation provides insights into the preferred metabolic pathways operating in the hydrogenosomes of axenic anaerobic gut fungi (AGF) cultures and monoxenic AGF-methanogen co-cultures.

Unlike most other heterotrophic protist lineages which synthesize glycogen, alveolates accumulate -glucan storage polysaccharides in the form of starch. Within Alveolata, ciliates do so by using a simple pathway analogous to that used for glycogen metabolism in eukaryotes. However, Myzozoa have evolved five additional enzymatic steps which are common to Dinoflagellata, Chromerids and Apicomplexa. Moreover, dinoflagellates have acquired seven additional enzymes that distinguish them from the Chromerids-Apicompexa lineages, while chromerids possess only a single enzyme not found in dinoflagellates. Such modifications are not found in other eukaryotic lineages save for those resulting directly from primary or secondary plastid endosymbiosis. The many differences affecting the starch pathway of Myzozoa can be explained by a single secondary or tertiary plastid endosymbiosis of a starch-containing alga followed by distinct compartmentalisation outcomes of starch metabolism in Dinoflagellata relative to chromerids and Apicomplexa.

Due to their highly polyphagous capacities, black soldier fly (Hermetia illucens) larvae (BSFL) are increasingly valued for their ability to convert organic waste into valuable biomass that can be used for a variety of purposes. These remarkable digestive capabilities are highly dependent on an extremely plastic gut microbiota. However, the distribution and functioning of bacterial communities in the various gut compartments - particularly in the hindgut - remain little understood. In this study, we used a metabarcoding approach based on 16S gene sequencing to investigate the effect of three carbohydrate-rich diets with distinct molecular compositions on the functional diversity of the BSFL gut microbiota. Our results showed that the midgut harbors a highly substrate-sensitive microbiota, with a high abundance of Actinomyces spp., regardless of the substrate. A bacterial diversity oriented toward fatty acid biosynthesis pathways is promoted by starch-rich environment, whereas a lignocellulosic substrate fosters a midgut microbiota dominated by Paenibacillus spp. In contrast, the hindgut exhibits a distinctly stable and homogeneous bacterial composition dominated by Dysgonomonas spp. Overall, our results provide clear evidence of a two-compartment microbial system, in which the midgut primarily serves as a substrate-adaptive primary degradation chamber, while the hindgut functions as a stable terminal compartment for the final processing of residual substrates and the recycling of nutrients. These findings contribute to our understanding of the functional diversity of the bacterial microbiota along the BSFL digestive tract, which is a key factor in explaining this insect's remarkable polyphagous behavior and optimizing its use for industrial purposes.

Prevotella melaninogenica is a Gram-negative bacterium that has been implicated in numerous disorders such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis, and despite its abundance in the human microbiome, there is still limited knowledge on the fundamental physiology describing Prevotella melaninogenica. To properly advance Prevotella biology, it is critical to report and document such characterizations of Prevotella; therefore, the purpose of this study is to examine and define the phenotypic and growth characteristics of P. melaninogenica ATCC 25845. In this study, we found that Prevotella melaninogenica ATCC 25845 is a Gram-negative bacterium that is predominantly short and medium rod-shaped, with occasional filamentous rods. SEM imaging revealed that this filamentation may serve as division centers, points from which multiple daughter cells form. We also describe the antibiotic profile, which showed susceptibility to four classes and resistance to six classes of antibiotics. Carbon source growth kinetic characterization revealed a preference for carbohydrate sources.

Extremophilic fungi are a relatively unexplored source of stress-resilient microbes for sustainable agriculture. Here, we describe Talaromyces pseudofuniculosus FAR, isolated from the highly acidic site of Piccolo Inferno in southern Italy and identified by ITS and {beta}-tubulin (BenA) sequencing. Despite its origin from an extreme environment, T. pseudofuniculosus FRA remained fully functional under non-stress conditions and displayed tolerance to salinity and heavy metals while maintaining key plant growth-promoting traits, including phosphate solubilization, auxin-related compound production and hydrolytic activities. In soybean (Glycine max L.), the live fungus promoted plant growth under both non-stress conditions and agriculturally relevant salinity and copper stress, and successfully colonized the roots. A cell-free extracellular extract (exCFS) promoted early seedling development in vitro, but its effects were not sustained in pot experiments. GC-MS analysis provided a qualitative characterization of the extracellular metabolite profile and revealed condition-dependent differences between control, NaCl and CuSO4; growth conditions, indicating environment-dependent modulation of fungal metabolism. Overall, these findings highlight the physiological and metabolic plasticity of T. pseudofuniculosus FRA and support its potential as a fungal biostimulant for saline- and metal-impacted agricultural soils.

Eutrophication-driven hypoxia and harmful algal blooms are an expanding threat to water quality in estuaries. Engineered aeration is a technology used in freshwater and, to a lesser extent, estuarine systems to mitigate both hypoxia and algal blooms. However, the efficacy of engineered aeration has not been well studied within estuarine ecosystems and very little is known about the impact of aeration on microbial communities in either estuarine or freshwater environments. Here we present a time series investigation of microbial community response to aeration and cessation of aeration in a eutrophic estuary. Samples for 16S rRNA gene analysis were collected over about a month-long period during which time the aerators transitioned from being "on" to "off" to "on" again. Cessation of aeration selected for the dominance of a eukaryotic algae similar to Heterosigma akashiwo. After the resumption of aeration, H. akashiwo dominance decreased rapidly. Cessation of aeration also shifted the structure of the entire microbial community, where diversity decreased and the structure of microbial communities at the surface diverged from those from the bottom of the water column. These changes did not occur at non-aerated control sites. Distinct groups of taxa responded to the disruption in a successional pattern through time, with most taxa never returning to their pre-aeration-cessation abundance during the observational period. These findings demonstrate the complex community response to anthropogenic interventions and provide support for bloom suppression through aeration.

The high stability of enteric viruses in freshwater increases their risk of waterborne transmission. In aquatic environments, bacteria are known to enhance viral removal and inactivation. However, the differences in viral inactivation across freshwater ecosystems and the influence of community diversity on this process remains poorly understood. Here, we used dilution-to-extinction to generate 64 distinct freshwater communities from three lakes and three aquifers. After establishing freshwater communities in chemostat cultures, each community was challenged with two viruses, coxsackievirus B5 and human adenovirus 2. Both viruses were inactivated more rapidly in lake-derived than in groundwater-derived communities, and adenovirus was inactivated faster than coxsackievirus in both freshwater types. Community-level parameters (cell numbers, richness, evenness) were not significant predictors of viral inactivation. In contrast, differential abundance analyses identified bacterial OTUs associated with inactivation: Acidovorax sp. were associated across sources and Brevundimonas sp., Sphingopyxis sp., and Hydrogenophaga sp. showed source-specific associations. Together, these findings provide new insights into the ecological drivers of viral stability and suggest that viral inactivation may depend more strongly on specific microbial taxa and their ecological context than on overall community diversity. Identifying these associations improves our understanding of the factors governing viral persistence in freshwater environments.

Background Symbiosis with microbes is a key strategy that has enabled plants to colonize extreme environments. Since the benefits conferred by root-associated microbes depend on both environmental conditions and host-microbe combinations, plant adaptation to harsh environments is closely linked to the assembly of root microbial communities. Understanding how environmental and host filtering jointly shape these communities is therefore fundamental to elucidating the mechanisms underlying plant adaptation to extreme environments. Results In this study, we investigated the differentiation of root-associated prokaryotic and fungal communities and individual operational taxonomic units (OTUs) across two contrasting habitats surrounding fumaroles, solfatara-field and forest-edge habitats, and six dominant Ericaceae and ectomycorrhizal plant taxa. Prokaryotic and fungal OTUs rarely exhibited strong preferences for both habitat and host identity. Instead, many of prokaryotic and fungal OTUs specialized to one of these niches, collectively generating root microbial communities differentiated by both factors. Nonetheless, striking specializations in habitat and host niches were observed in the fungal family Hyaloscyphaceae (Helotiales). To gain insight into the evolutionary basis of microbial specialization, we examined phylogenetic signals in preference phenotypes. The resulting weak phylogenetic signals in these preference phenotypes further suggest that this fungal clade has undergone substantial ecological divergence. Conclusion Overall, our findings indicate that root-associated microbial communities in extreme environments are assembled through the accumulation of microbial taxa specialized to either habitat or host, and that strong ecological specialization in fungi can arise with little phylogenetic constraint.

Oral multispecies biofilms developing on dental implants drive peri-implant mucositis and peri-implantitis. They are shaped by the physicochemical conditions of their surrounding environment, among which oxygen availability is an important influential factor. However, while oxygen tension fluctuates significantly within the peri-implant pocket, whether these environmental shifts are sufficient to influence the structure and composition of an established microbial community remains unclear. For this purpose, we evaluated the structure and composition of a four-species oral biofilm model comprising the species Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis, which was cultivated under normoxic (21% O2), hypoxic (1% O2) and anoxic (0% O2;) conditions for a period of 21 days. Biofilm 3D architecture, volume and membrane integrity was analyzed by LIVE/DEAD staining combined with confocal laser scanning microscopy. Community composition was assessed by quantitative real-time PCR and complemented by Fluorescence In Situ Hybridization (FISH) to provide structural context. While normoxia transiently promoted higher early biofilm volume and membrane integrity, the community exhibited remarkable long-term resilience, consistently maintaining a V. dispar-dominated structure across all oxygen gradients. Crucially, the obligate anaerobic pathogen P. gingivalis persisted as a viable minority (~1%) even under 21-day normoxic cultivation, sheltered within the deeper layers of the biofilm. These results suggest that clinical oxygen levels alone are not able to drastically modulate established oral biofilm communities and that oxygen-sensitive pathogens persist in a subclinical sanctuary protected by the surrounding non-pathogenic species.

Marine microbial communities vary seasonally and spatially, shaped by physiochemical gradients and biotic interactions. Spatiotemporal variability of seawater microbiomes in tropical coastal regions is comparatively understudied, especially in Southeast Asia with high pressures from development and anthropogenic activities. Here, we examined the microspatial and temporal variability of bacterioplankton in the Singapore Strait, which is a tropical system in Southeast Asia influenced by monsoons. By applying total community amplicon sequencing to three particle size fractions reflecting microspatial habitats of seawater (0.22-1 {micro}m ''free-living'', 1-10 {micro}m ''large cells and small aggregates'' and 10-150 {micro}m ''particle-associated''), we show that the seawater microbial community was structured by their microspatial habitats, monsoon seasons and physiochemical variables. Monsoon-driven alternation of water masses originating from the South China Sea to the East and from off the Sumatran coast to the West resulted in a distinct community during the Southwest monsoon across all three size fractions. Total DNA (biomass) was lowest in the 10-150 {micro}m fraction, but these particle-associated sequences had the highest diversity and were surprisingly enriched with picocyanobacterial and some pathogenic taxa, potentially reflecting aggregate formation and/or host-derived particle association. This fraction also exhibited greater temporal variability than the two smaller fractions, which could be related to a more heterogenous environment in particle microhabitats. Overall, this study demonstrated compositional variability in the bacterioplankton across space and time in an equatorial region influenced by monsoons.

Fucoidan is the second most abundant polysaccharide in brown macroalgae, contributing up to 50% of the dissolved organic matter released daily from these algae into the ocean, substantially influencing the marine carbon cycle. Due to its complex chemical structure, fucoidan is highly resistant to microbial degradation and accumulates on the surface of macroalgae, where it contributes to regulating their microbiome. Here, we used a range of microfluidic assays to quantify the chemotactic and physiological responses of marine bacteria to fucoidan. Our results reveal that all the strains tested (14 out of 14) are attracted to fucoidan produced by two different species of brown macroalgae. Attraction occurred only at high fucoidan concentrations (0.1 to 1 mg/mL), such as those expected in the immediate vicinity of algal surfaces, whereas low concentrations elicited no response or modest chemorepulsion. Although fucoidan did not support bacterial growth under our experimental conditions, it did not impair the utilization of other carbon sources. Together, these findings suggest that fucoidan gradients can act as chemical cues guiding marine bacteria toward the nutrient-rich microenvironment of macroalgal surfaces, pointing to a potential ecological role of fucoidan in shaping macroalgal microbiota, bacterial behavior, and ultimately carbon cycling in the ocean.

Coinfections are common in a nature and often involve competition among coinfecting parasites to extract resources from the host. As a consequence of this competition, parasites can face trade-offs between investing in competition with other parasites and exploiting the host for their own growth and transmission. Such within-host interactions among parasites can either increase or decrease virulence depending on how they alter parasite density, resource use, and host damage. We tested the inhibitory effect of different species of the entomopathogenic fungus Metarhizium on one another in vitro and the impact of coinfection on virulence towards Tribolium castaneum. We found that Metarhizium pemphigi is able to inhibit all other isolates of Metarhizium tested, whereas Metarhizium pingshaense was relatively resistant to inhibition by other Metarhizium strains. Virulence towards the host, Tribolium castaneum, differed very little among isolates tested. We did, however, observe differences between virulence in single and coinfections in two cases. Where there were virulence differences, these were both consistent with in vitro inhibition patterns and interestingly resulted in reduced virulence in coinfection. This is inconsistent with broader patterns of virulence in coinfection, where virulence seems to be driven by the more virulent parasite. It is, however, consistent with data from other systems where there is evidence of interference competition. Our data thus support the idea that, in contrast to other forms of between parasite interaction, interference competition results in reduced virulence in coinfection.

Anoxygenic phototrophic green sulfur bacteria (GSB; Chlorobiia) are important primary producers in anoxic and sulfidic environments, whose significance in aquatic ecosystems will expand with coastal deoxygenation. Here, we employed a uniquely comprehensive analytical approach to investigate the formation, maintenance, and collapse of a GSB bloom. We combined multi-omics -- V4V5 and synthetic long read 16S rRNA amplicon sequencing, metagenomics, and metaproteomics -- with total and GSB-specific cell counts, biogeochemical measurements, and isotopic analysis. The GSB bloom exceeded 109 cells ml-1, among the highest environmental cell densities reported to date, with up to 96 % of the bloom consisting of a single strain-level Prosthecochloris lineage (GSB-TRL01). Extreme sulfide concentrations (> 17 mM) coincided with peaking cell density and a shift in isotopic composition. Bloom persistence is supported by tightly coupled sulfur cycling, high rates of nitrogen fixation, mechanisms to tolerate oxidative stress and maintain redox balance, and nutrient acquisition through outer membrane transport systems. A shift in sulfur oxidizing enzymes towards enzymes with higher sulfide affinity proceeded bloom demise. Prosthecochloris GSB-TRL01 differs from closely related lineages in several outer membrane transport systems, including porins to transport phosphate and tonB-dependent transporters. Collectively, these findings identify physiological and metabolic strategies that may enable near-clonal Prosthecochloris populations to attain extraordinary biomass while coupling the sulfur, carbon, and nitrogen cycles in coastal euxinic environments.

A native entomopathogenic nematode isolate, Heterorhabditis indica Che4.1, was recovered from soils in Valencia (Spain), and its symbiotic bacterium, Photorhabdus akhurstii Che4.1, was isolated and characterized. Nematode identification was confirmed by ITS and 28S rDNA sequencing, while genome-based analyses supported the taxonomic assignment of the bacterial symbiont. The nematode-bacterium complex exhibited insecticidal activity against Spodoptera exigua and Spodoptera littoralis. H. indica Che4.1 showed an LC of 53.7 infective juveniles (IJs)/mL against S. exigua, comparable to that of the commercial reference Larvanem (54.3 IJs/mL). Against S. littoralis, Che4.1 exhibited an approximately twofold lower LC than Larvanem (77.3 versus 155.9 IJs/mL), with non-overlapping 95% confidence intervals. The complete genome of P. akhurstii Che4.1 consists of a single circular chromosome of 5.66 Mb encoding a diverse repertoire of putative virulence factors, including toxin complex (Tc) components, YenTc-like proteins, Mcf toxins, App-family proteins, Pra/Prb proteins, and chitinases, together with several predicted secondary metabolite biosynthetic gene clusters. LC-MS/MS analyses of bacterial culture supernatants and infected insect cadavers detected multiple predicted virulence-associated proteins, including Tc-related, YenTc-like, App-family, and Pra/Prb proteins. In addition, bacterial cell-free supernatants caused 68.8-81.3% mortality in S. exigua neonates under laboratory conditions. Together, these findings reveal a diverse repertoire of potential virulence determinants in P. akhurstii Che4.1 and demonstrate the insecticidal potential of its native nematodebacterium complex. The performance of H. indica Che4.1 relative to a commercial reference supports further evaluation of this isolate for biological control of Spodoptera spp., particularly under greenhouse and field conditions.

Biological filtration in newly established aquaria depends on the establishment of nitrifying microorganisms that prevent accumulation of toxic ammonia and nitrite. Many aquarists use commercial nitrifying supplements to accelerate biofilter establishment and reduce ammonia and nitrite accumulation. However, the microbial composition and in situ efficacy of these products remain poorly characterized. We evaluated the ability of enrichment cultures containing comammox Nitrospira (CX) and the ammonia-oxidizing archaeon Ca. Nitrosotenuis aquarius (AQ) to serve as aquarium nitrifying supplements, tested alongside a conventional supplement (CS) containing ammonia-oxidizing bacteria. Inoculation of new aquaria containing zebrafish (Danio rerio) with nitrifying supplements resulted in distinct nitrifier profiles within biofilters of each treatment group corresponding with significant reductions in peak ammonia and nitrite concentrations, which were most evident when applied at the maximum tested supplement dose. Treating aquaria with CX, AQ, or CS maintained ammonia and nitrite at low concentrations, and CX effectively prevented ammonia and nitrite accumulation through all stages of aquarium operation and biofilter establishment. After removing all fish from the aquaria, ammonia "spike-in" experiments demonstrated biofilters treated with CX and AQ conferred long-term protection against nitrite accumulation following acute ammonia loading events. Whereas 16S rRNA gene sequences associated with CS were rarely detected within biofilter profiles, those associated with CX and AQ established early and persisted temporally. Overall, results demonstrate how nitrifiers might be differentially adapted to home aquarium conditions and the potential of AOA and comammox Nitrospira to improve biofilter establishment and maintaining safe water quality in newly established freshwater aquaria.