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Nitrous oxide (N 2 O) is a potent greenhouse gas with various production pathways. N 2 O reductase (N 2 OR) is the primary N 2 O sink, but the distribution of its gene clades, typically nosZ I and atypically nosZ II, along urbanization gradients remains poorly understood. Here we sampled soils from forests, parks, and farmland across eight provinces in eastern China, using high-throughput sequencing to distinguish between two N 2 O-reducing bacteria clades. A deterministic process mainly determined assemblies of the nosZ I communities. Homogeneous selection drove nosZ I deterministic processes, and both homogeneous and heterogeneous selection influenced nosZ II. This suggests nosZ II is more sensitive to environmental changes than nosZ I, with significant changes in community structure over time or space. Ecosystems with stronger anthropogenic disturbance, such as urban areas, provide diverse ecological niches for N 2 O-reducing bacteria (especially nosZ II) to adapt to environmental fluctuations. Structural equation modeling (SEM) and correlation analyses revealed that pH significantly influences the community composition of both N 2 O-reducing bacteria clades. This study underscores urbanization's impact on N 2 O-reducing bacteria in urban soils, highlighting the importance of nosZ II and survival strategies. It offers novel insights into the role of atypical denitrifiers among N 2 O-reducing bacteria, underscoring their potential ecological importance in mitigating N 2 O emissions from urban soils.

Antibiotic resistance genes (ARGs) are ancient but have become a modern critical threat to health. Gut microbiota, a dynamic reservoir for ARGs, transfer resistance between individuals. Surveillance of the antibiotic resistome in the gut during different host growth phases is critical to understanding the dynamics of the resistome in this ecosystem. Herein, we disentangled the ARG profiles and the dynamic mechanism of ARGs in the egg and adult phases of Tetramorium caespitum . Experimental results showed a remarkable difference in both gut microbiota and gut resistome with the development of T. caespitum . Meta-based metagenomic results of gut microbiota indicated the generalizability of gut antibiotic resistome dynamics during host development. By using Raman spectroscopy and metabolomics, the metabolic phenotype and metabolites indicated that the biotic phase significantly changed lipid metabolism as T. caespitum aged. Lipid metabolites were demonstrated as the main factor driving the enrichment of ARGs in T. caespitum . Cuminaldehyde, the antibacterial lipid metabolite that displayed a remarkable increase in the adult phase, was demonstrated to strongly induce ARG abundance. Our findings show that the gut resistome is host developmental stage-dependent and likely modulated by metabolites, offering novel insights into possible steps to reduce ARG dissemination in the soil food chain.

Resistomes are ubiquitous in natural environments. Previous studies have shown that both the plant phyllosphere and soil-borne nematodes were reservoirs of above- and below-ground resistomes, respectively. However, the influence of plant identity on soil, nematode, and phyllosphere resistomes remains unclear. Here, a microcosm experiment was used to explore the characteristics of bacterial communities and resistomes in soil, nematode, and phyllosphere associated with six different plant identities ( Lactuca sativa , Cichorium endivia , Allium fistulosum , Coriandrum sativum , Raphanus sativus , and Mesembryanthemum crystallinum ). A total of 222 antibiotic resistance genes (ARGs) and 7 mobile genetic elements (MGEs) were detected by high-throughput quantitative PCR from all samples. Plant identity not only significantly affected the diversity of resistomes in soil, nematode, and phyllosphere but also influenced the abundance of resistomes in nematodes. Shared bacteria and resistomes indicated a possible pathway of resistomes transfer through the soil-nematode-phyllosphere system. Structural equation models revealed that plant identity had no direct effect on phyllosphere ARGs, but altered indirectly through complex above- and below-ground interactions (soil-plant-nematode trophic transfer). Results also showed that bacteria and MGEs were key factors driving the above- and below-ground flow of resistomes. The study extends our knowledge about the top-down and bottom-up dispersal patterns of resistomes.

Multitrophic communities inhabit in soil faunal gut, including bacteria, fungi, and protists, which have been considered a hidden reservoir for antibiotic resistance genes (ARGs). However, there is a dearth of research focusing on the relationships between ARGs and multitrophic communities in the gut of soil faunas. Here, we studied the contribution of multitrophic communities to variations of ARGs in the soil woodlouse gut. The results revealed diverse and abundant ARGs in the woodlouse gut. Network analysis further exhibited strong connections between key ecological module members and ARGs, suggesting that multitrophic communities in the keystone ecological cluster may play a pivotal role in the variation of ARGs in the woodlouse gut. Moreover, long-term application of sewage sludge significantly altered the woodlice gut resistome and interkingdom communities. The variation portioning analysis indicated that the fungal community has a greater contribution to variations of ARGs than bacterial and protistan communities in the woodlice gut after long-term application of sewage sludge. Together, our results showed that changes in gut microbiota associated with agricultural practices (e.g., sewage sludge application) can largely alter the gut interkingdom network in ecologically relevant soil animals, with implications for antibiotic resistance, which advances our understanding of the microecological drivers of ARGs in terrestrial ecosystem.

Applying organic fertilizers has been well documented to facilitate the dissemination of antibiotic resistance genes (ARGs) in soil ecosystems. However, the role of soil fauna in this process has been seldom addressed, which hampers our ability to predict the fate of and to manage the spread of ARGs. Here, using high-throughput quantitative polymerase chain reaction (HT-qPCR), we examined the effect of long-term (5-, 8-, and 10-year) fertilization treatments (control, inorganic fertilizers, and mixed fertilizers) on the transfer of ARGs between soil, nematodes, and earthworms. We found distinct fates for ARGs in the nematodes and earthworms, with the former having higher enriched levels of ARGs than the latter. Fertilization impacted the number and abundance of ARGs in soil, and fertilization duration altered the composition of ARGs. Shared ARGs among soil, nematodes, and earthworm guts supported by a fast expectation-maximization microbial source tracking analysis demonstrated the trophic transfer potential of ARGs through this short soil food chain. The transfer of ARGs was reduced by fertilization duration, which was mainly ascribed to the reduction of ARGs in the earthworm gut microbiota. This study identified the transfer of ARGs in the soil-nematode-earthworm food chain as a potential mechanism for a wider dissemination of ARGs in the soil ecosystem.

Microplastics pollution in the environment is now receiving worldwide attention; however, the effects of copollution of antibiotics and microplastics on the gut microbiome of globally distributed and functionally important nontarget soil animals remain poorly understood. We studied a model collembolan ( Folsomia candida ) and found that the ingestion of microplastics (polystyrene, 2-2.9 μm) substantially altered the gut microbiome, antibiotic resistance gene (ARG) profile, and the isotopic fractionation in the soil collembolan tissue. Importantly, collembolans exposed to polystyrene microplastics loaded with sulfamethoxazole (MA) presented a distinctive gut microbiome, ARG profile, and isotopic fractionation compared to those exposed to polystyrene alone (MH). We observed that the abundance of ARGs and mobile genetic elements (MGEs) in the MA-treated collembolan guts was significantly higher than in the MH and the control treatments. There were also strong interactions between the gut microbiome and ARGs in the collembolan guts. We further found that bacterial β-diversity correlated significantly with the δ 13 C and δ 15 N values in collembolan body tissues. Together, our results indicate that changes in isotopic fractionation and ARG profiles in the collembolan were induced by the changes in gut microbiota and suggest that microplastics from diverse sources may have profound influences on soil fauna and soil food webs.

The presence and spread of antibiotic resistance genes (ARGs) are causing substantial global public concern; however, the dispersal of ARGs in the food chain is poorly understood. Here, we experimented with a soil collembolan ( Folsomia candida)-predatory mite ( Hypoaspis aculeifer) model food chain to study trophic transfer of ARGs in a manure-contaminated soil ecosystem. Our results showed that manure amendment of soil could significantly increase ARGs in the soil collembolan microbiome. With the ARGs in the prey collembolan microbiome increasing, an increase in ARGs in the predatory mite microbiome was also observed, especially for three high abundant ARGs ( blaSHV, fosX and aph6ia). Three unique ARGs were transferred into the microbiome of the predatory mite from manure amended soil via the prey collembolan ( aac(6' )-lb(akaaacA4), yidY_mdtL and tolC). Manure amendment altered the composition and structure and reduced the diversity of the microbiomes of the prey collembolan and the predatory mite. We further reveal that bacterial communities and mobile genetic elements were two important drivers for the trophic transfer of ARGs, not just for ARGs distribution in the samples. These findings suggest that the importance of food chain transmission of ARGs for the dispersal of resistance genes in soil ecosystems may be underestimated.

Anaerobic ammonium oxidation coupled to iron reduction (Feammox) is a recently discovered pathway contributing to nitrogen loss in various ecosystems such as paddy soils and sediments. However, little is known about the microbes driving Feammox in an agricultural ecosystem. Here, we demonstrated the occurrence of Feammox in paddy soils of Southern China using a 15 N isotopic tracing technique, and examined the microbial communities associated with Feammox using RNA based stable isotope probing (RNA-SIP) combined with Illumina sequencing. Feammox was detected in all collected soils with direct N 2 production as the dominant Feammox pathway. It was estimated that approximately 6.91% of the applied nitrogen fertilizers were lost through Feammox in the paddy soils. RNA-SIP results showed that the composition of enriched active microbial communities were dependent on soil properties, especially the soil pH and grain size. Geobacter were enriched in most soils across various properties. The abundance of enriched GOUTA19 were significantly higher in soils with low pH than those in soils with medium pH and high pH, and the relative abundance of active Nitrososphaeraceae and Pseudomonas only increased in soils with medium and high pH during 4-day of incubation. These results suggested Feammox is a ubiquitous and important process for N loss. Geobacter, GOUTA19, Nitrososphaeraceae and Pseudomonas were active during the incubation that favored Feammox and the growth of Feammox microbes, suggesting these microbes were potentially associated with Feammox in natural agricultural soils.

Diets of soil-feeding earthworms contain abundant nitrate and iron(III) oxides, which are potential electron acceptors for mineralization of organic compounds. The earthworm gut provides an ideal habitat for ingested iron(III)-reducing microorganisms. However, little is known about iron(III) reduction and its interaction with other processes in the guts of earthworms. Here, we determined the dynamics of iron(III) and revealed its interaction with the turnover of organic acids and nitrate in the gut of the earthworm Pheretima guillelmi. Samples from gut contents combined with anoxic incubation were used for chemical analysis and 16S rRNA based Illumina sequencing. Chemical analysis showed that higher ratios of iron(II)/iron(III), nitrite/nitrate, and more abundant organic acids were contained in the in vivo gut of the earthworm P. guillelmi than those in the in situ soil. A higher rate of iron(III) reduction was detected in treatments of microcosmic incubation with gut contents (IG gut) than that with soil (IG soil), and nitrate reduction occurred earlier than iron(III) reduction in both treatments. Potential iron(III) reducers were dominated by fermentative genera Clostridium, Bacillus, and Desulfotomaculum in the treatment of IG gut, while they were dominated by dissimilatory iron(III)-reducing genera Geobacter in the treatment of IG soil. The iron(III)-reducing microbial community shared several genera with denitrifers in the treatment of IG gut, revealing a close link between iron(III) reduction and denitrification in the gut of earthworms. Collectively, our findings demonstrated that iron(III) reduction occurred along the gut and provided novel insights into the great contribution of earthworm gut microbiota on Fe and the associated C and N cycling in soil environments.

Numerous studies have investigated the composition and diversity of antibiotic resistance genes (ARGs) in multiple environments but the pattern of ARGs in field-collected soil fauna remains poorly understood. In the present study soil collembolans were collected from six sites with three different land use types (parkway land, park land, and arable land) and 285 ARGs and 10 mobile genetic elements (MGEs) in the microbiome of these "wild" collembolans were quantified by high-throughput quantitative PCR. A total of 76 unique ARGs and 5 MGEs were detected. There were significant differences between collection sites in the antibiotic resistome in the collembolans. Land use significantly altered the distribution patterns of collembolan ARGs. Thirty shared ARGs and three shared MGEs were identified. The co-occurrences of shared resistomes were largely random, and more positive relationships were found in the coassociation network. Partial redundancy analysis confirms that the changes in bacterial communities explained 27.77% of the variation in ARGs. These findings suggest that resistance genes are pervasive in the microbiome associated with the field collembolan and the activity of the collembolans may contribute to the spread and dissemination of resistance genes in the environment, an aspect of ARGs that has until now been largely overlooked.

Gut microbiota contribute to host health. Numerous recent studies have focused on the survival and reproduction of nontarget soil animals exposed to the toxicity of silver nanoparticles (AgNPs) but changes in the gut microbiota due to nanoparticle toxicity are largely unknown. Here, we examine some effects of AgNPs and silver nitrate (ionic Ag) on the gut microbiota of the common soil collembolan Folsomia candida using Illumina sequencing and concomitant changes in antibiotic resistance genes (ARGs) of the gut microbiota using high-throughput quantitative PCR. A large number of Ag accumulated in Ag-exposed individuals after 28 days and ionic Ag significantly inhibited the reproduction of the collembolan (by 19.3%). Exposure to AgNPs disturbed the composition of the collembolan gut bacterial community, resulting in dysbiosis of the gut microbiota. However, the dominant microbiota was shared among different treatments. In addition, AgNPs exposure did indeed reduce the incidence of ARGs in the collembolan gut microbiota. A weak relationship was identified between gut bacterial communities and ARG profiles. These results extend our knowledge regarding the role of the gut microbiota in assessing the soil ecotoxicology of AgNPs.

Gut microbiota make an important contribution to host health but the effects of environmental pressures on the gut microbiota of soil fauna are largely uncharacterized. Here, we examine the effects of norfloxacin and oxytetracycline on the gut microbiome of the common soil collembolan Folsomia candida and concomitant changes in the incidence of antibiotic resistance genes (ARGs) in the gut and in growth of the collembolan. Exposure to 10 mg antibiotics kg -1 for 2 weeks significantly inhibited the growth of the collembolan with roughly a 10-fold decrease in 16S rRNA gene abundance. Antibiotics did alter the composition and structure of the collembolan gut microbiome and decreased the diversity of the gut bacteria. A decline in the firmicutes/bacteroidetes ratio in the antibiotic-treated collembolans may be responsible for the decrease in body weight. Exposure to antibiotics significantly increased the diversity and abundance of ARGs in the collembolan gut. The Mantel test and Procrustes analysis both reveal that ARGs and gut microbiota were significantly correlated with one another ( P < 0.05). These results indicate that antibiotics may induce a shift in the gut microbiota of nontarget organisms such as soil collembolans and thereby affect their growth and enrichment of ARGs.

Anaerobic ammonium oxidation coupled to iron(III) reduction, termed Feammox, is a newly discovered nitrogen cycling process. However, little is known about the roles of electron shuttles in the Feammox reactions. In this study, two forms of Fe(III) (oxyhydr)oxide ferrihydrite (ex situ ferrihydrite and in situ ferrihydrite) were used in dissimilatory Fe(III) reduction (DIR) enrichments from paddy soil. Evidence for Feammox in DIR enrichments was demonstrated using the (15)N-isotope tracing technique. The extent and rate of both the (30)N2-(29)N2 and Fe(II) formation were enhanced when amended with electron shuttles (either 9,10-anthraquinone-2,6-disulfonate (AQDS) or biochar) and further simulated when these two shuttling compounds were combined. Although the Feammox-associated Fe(III) reduction accounted for only a minor proportion of total Fe(II) formation compared to DIR, it was estimated that the potentially Feammox-mediated N loss (0.13-0.48 mg N L(-1) day(-1)) was increased by 17-340% in the enrichments by the addition of electron shuttles. The addition of electron shuttles led to an increase in the abundance of unclassified Pelobacteraceae, Desulfovibrio, and denitrifiers but a decrease in Geobacter. Overall, we demonstrated a stimulatory effect of electron shuttles on Feammox that led to higher N loss, suggesting that electron shuttles might play a crucial role in Feammox-mediated N loss from soils.

Microbe-mediated arsenic (As) biotransformation in paddy soils determines the fate of As in soils and its availability to rice plants, yet little is known about the microbial communities involved in As biotransformation. Here, we revealed wide distribution, high diversity, and abundance of arsenite (As(III)) oxidase genes (aioA), respiratory arsenate (As(V)) reductase genes (arrA), As(V) reductase genes (arsC), and As(III) S-adenosylmethionine methyltransferase genes (arsM) in 13 paddy soils collected across Southern China. Sequences grouped with As biotransformation genes are mainly from rice rhizosphere bacteria, such as some Proteobacteria, Gemmatimonadales, and Firmicutes. A significant correlation of gene abundance between arsC and arsM suggests that the two genes coexist well in the microbial As resistance system. Redundancy analysis (RDA) indicated that soil pH, EC, total C, N, As, and Fe, C/N ratio, SO4(2-)-S, NO3(-)-N, and NH4(+)-N were the key factors driving diverse microbial community compositions. This study for the first time provides an overall picture of microbial communities involved in As biotransformation in paddy soils, and considering the wide distribution of paddy fields in the world, it also provides insights into the critical role of paddy fields in the As biogeochemical cycle.