Roxarsone reduces earthworm-mediated nutrient cycling by suppressing aggregate formation and enzymic activity in soil with manure application.

The application of manure and earthworms are frequently used in fertilization practices to improve C, N, and P cycling in soil, which may be adversely affected by roxarsone (ROX), as an organoarsenical pollutant. To effectively address this issue, in this work, the interactive impacts of ROX and earthworm Eisenia foetida on the aggregate formation, input of organic carbon (OC), and changes in the available N and P following 56-day cultivation were systematically investigated. Compared to the control, earthworms increased the mean weight diameter (MWD) of the soil aggregates from 0.6 to 1.1 mm. Thereby, they activated soil enzymes including catalase (CAT), sucrase (SC), urease (UE), and neutral phosphatase (NP), with the soil's pH decreased to 7.1. Consequently, the values of OC, soluble nitrite (NO3-N), and Olsen-P content were respectively increased by 0.78-, 1.69-, and 0.87- folds in the E treatment (14.3 vs. 25.5 g/kg, 12.8 vs. 33.3 mg/kg, and 7.8 vs. 14.6 mg/kg). Although the changes in the R treatment were slight, ROX reduced the earthworm-mediated improvements of soil fertility during the application of the RE treatment compared to the E treatment, i.e., the values of MWD, OC, NO3-N, and Olsen-P were reduced to 0.9 mm, 20.4 g/kg, 25.4 mg/kg, and 11.6 mg/kg, respectively. From the well-fitted structural equation models, it was demonstrated that earthworms enhanced the aggregate formation and nutrient cycling of OC, NO3-N, and Olsen-P, which were inhibited by ROX. Overall, these adverse effects can be offset by earthworm addition, which can play the dual role of monitor and driver for the soil properties. Our work provides insightful strategies for ROX-bearing manure management.

Biotransformation of roxarsone by earthworms and subsequent risk of soil arsenic release: The role of gut bacteria.

The organoarsenical feed additive roxarsone (ROX) is a ubiquitous threat due to the unpredictable levels of arsenic (As) released by soil bacteria. The earthworms representing soil fauna communities provide hotspots for As biotransformation genes (ABGs). Nonetheless, the role of gut bacteria in this regard is unclear. In this study, the changes in As speciation, bacterial ABGs, and communities were analyzed in a ROX-contaminated soil (50 mg/kg As in ROX form) containing the earthworm Eisenia feotida. (RE vs. R treatment). After 56 d, earthworms reduced the levels of both ROX and total As by 59 % and 17 %, respectively. The available As content was 10 % lower in the RE than in R treatment. Under ROX stress, the total ABG abundance was upregulated in both earthworm gut and soil, with synergistic effects observed following RE treatment. Besides, the enrichment of arsM and arsB genes in earthworm gut suggested that gut bacteria may facilitate As removal by enhancing As methylation and transport function in soil. However, the bacteria carrying ABGs were not associated with the ABG abundance in earthworm gut indicating the unique strategies of earthworm gut bacteria compared with soil bacteria due to different microenvironments. Based on a well-fit structural equation model (P = 0.120), we concluded that gut bacteria indirectly contribute to ROX transformation and As detoxification by modifying soil ABGs. The positive findings of earthworm-induced ROX transformation shed light on the role of As biomonitoring and bioremediation in organoarsenical-contaminated environments.

Discovery and Mechanism of Action of a Novel Antimicrobial Peptide from an Earthworm.

The robust innate immune system of the earthworm provides a potential source of natural antimicrobial peptides (AMPs). However, the cost and high rediscovery rate of direct separation and purification limits their discovery. Genome sequencing of numerous earthworm species facilitates the discovery of new antimicrobial peptides. Through predicting potential antimicrobial peptides in the open reading frames of the Eisenia andrei genome and sequence optimization, a novel antimicrobial peptide, named EWAMP-R (RIWWSGGWRRWRW), was identified. EWAMP-R demonstrated good activity against various bacteria, including drug-resistant strains. The antibacterial mechanisms of EWAMP-R were explored through molecular simulation and wet-laboratory experiments. These experiments demonstrated that the bacterial membrane may be one of the targets of EWAMP-R but that there may be different interactions with Gram-negative and Gram-positive bacterial membranes. EWAMP-R can disrupt bacterial membrane integrity; however, at low concentrations, it appears that EWAMP-R may get through the membrane of Escherichia coli instead of damaging it directly, implying the existence of a secondary response. Gene expression studies identified that in E. coli, only the apoptosis-like cell death (ALD) pathway was activated, while in Staphylococcus aureus, the MazEF pathway was also upregulated, limiting the influence of the ALD pathway. The different antimicrobial actions against Gram-positive and -negative bacteria can provide important information on the structure-activity relationship of AMPs and facilitate AMP design with higher specificity. This study identified a new source of antibacterial agents that has the potential to address the increasingly serious issue of antibiotic resistance. IMPORTANCE Drug-resistant bacteria are a great threat to public health and drive the search for new antibacterial agents. The living environment of earthworms necessitates a strong immune system, and therefore, they are potentially a rich resource of novel antibiotics. A novel AMP, EWAMP-R, with high antibacterial activity was found through in silico analysis of the Eisenia andrei genome. Molecular analysis investigating the interactions between EWAMP-R and the cell membrane demonstrated the importance of tryptophan and arginine residues to EWAMP-R activity. Additionally, the different secondary responses found between E. coli and S. aureus were in accordance with a common phenomenon where some antibacterial agents only target specific species of bacteria. These results provided useful molecular information to support further AMP research and design. Our study expands the sources of antimicrobial peptides and also helps to explain the adaptability of earthworms to their environment.

Eco-risk management of tylosin fermentation residues using vermicomposting.

Tylosin fermentation residues (TFR) pose an ecotoxicological risk through antibiotic resistant bacteria (ARBs) and their corresponding genes (ARGs). This study evaluated the ecotoxicity of TFR to soil biological activity, and further explored the mechanisms of vermicomposting to reduce the toxicological risk. The results showed that tylosin (TYL) was moderately degradable with a half-life (t1/2) of 37.5 d, inducing 28-44% inhibition rate of nitrogen transformation in soil, and the EC50 of earthworm avoidance was 880 mg/kg. The 30-d vermicomposting reduced the pH and OM content, while increased the EC and TN content, accelerated compost maturation (C/N ratio up to 20), and enriched the microbial community. ARGs were reduced by earthworm through removal of TYL (>70% degradation, t1/2 of <20 d), inhibiting abundance of intI1 and ARBs. We conclude that vermicomposting is an efficient method for TFR treatment and its eco-risk management.

Mitochondrial bioenergetic, oxidative stress and burrowing responses in earthworm exposed to roxarsone in soil.

The eco-risk of roxarsone (ROX) was evaluated using multiple responses of earthworm biomarkers under different ROX concentrations for 28 d. With the increasing total arsenic accumulation (TAs-E), biological responses in earthworm generally showed a two-stage changes of homeostasis dysregulation and dose-dependent alterations. At the early periods, ROX stress increased the reactive oxygen species (ROS) and lipid peroxidation (LPO) in a similar manner, and apparently disrupted mitochondrial calcium ([Ca2+]m). But earthworms regulated their mitochondrial and redox homeostasis through stable mitochondrial membrane potential (MMP) and increase of ATP level, superoxide dismutase (SOD) and catalase (CAT). After 14 d, the positively correlated mitochondrial effects of ROS, [Ca2+]m, MMP and ATP were related to the behavioral inhibition of burrow length, depth and reuse rate as well as antioxidant up-regulation of Nrf2, HO-1, sod1 and cat. These results contributed possible biomarkers from the dose-dependent relationship between mitochondrial, antioxidant and behavioral responses. Multiple biological detection in earthworms can better reflect the sub-chronic ecotoxicity of phenylarsenic pollutants in soil.

Toxicity assessment of earthworm exposed to arsenate using oxidative stress and burrowing behavior responses and an integrated biomarker index.

Arsenate (As-V) is a ubiquitous contaminant in soil as a result of excessive use of veterinary drugs and pesticides, causing enormous environmental risks. Multiple biomarkers have been used to assess the ecotoxicity of arsenic, however, the mechanisms of toxicity remain unclear. This paper describes the exposure of the earthworm (Eisenia fetida) to natural soil with different As-V concentrations for 28 days, then biomarkers from oxidative stress and burrowing behavior were quantified to evaluate As-V stress. Dynamic changes in reactive oxygen species (ROS), lipid peroxidation (MDA), adenosine triphosphate (ATP) content and antioxidant enzymes activity (Gpx, SOD, CAT) implied two stages of intensified stress responses and physiological adaptability. The transcriptional expression and regulation of antioxidant enzymes showed different responses. The mRNA expression of sod1 was up-regulated, while that of cat showed no significant change. The related regulators, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), showed dose-dependent activation, suggesting antioxidant defense induced by Nrf2 signaling. The burrowing behavior after 14-day exposure indicated that As-V inhibited burrowing activity, especially the burrow length and maximum burrow depth. These multiple biomarkers were integrated using a biomarker response index (BRI) model, which showed significant dose-effect relationship especially on day 28, and suggested that ATP was a sensitive and representative biomarker. This study provided evidence that burrowing activity, Nrf2 and HO-1 were useful biomarkers warranting inclusion into the BRI model. Arsenic toxicity was comprehensively understood through redox homeostasis regulation, biochemical and behavioral changes, and these results suggested new strategies for soil pollutants diagnosis.

Soil Behaviour of the Veterinary Drugs Lincomycin, Monensin, and Roxarsone and Their Toxicity on Environmental Organisms.

Lincomycin, monensin, and roxarsone are commonly used veterinary drugs. This study investigated their behaviours in different soils and their toxic effects on environmental organisms. Sorption and mobility analyses were performed to detect the migration capacity of drugs in soils. Toxic effects were evaluated by inhibition or acute toxicity tests on six organism species: algae, plants, daphnia, fish, earthworms and quails. The log Kd values (Freundlich model) of drugs were: lincomycin in laterite soil was 1.82; monensin in laterite soil was 2.76; and roxarsone in black soil was 1.29. The Rf value of lincomycin, roxarsone, monensin were 0.4995, 0.4493 and 0.8348 in laterite soil, and 0.5258, 0.5835 and 0.8033 in black soil, respectively. The EC50 for Scenedesmus obliquus, Arabidopsis thaliana, Daphnia magna and LC50/LD50 for Eisenia fetida, Danio rerio, and Coturnix coturnix were: 13.15 mg/L,32.18 mg/kg dry soil,292.6 mg/L,452.7 mg/L,5.74 g/kg dry soil and 103.9 mg/kg (roxarsone); 1.085 mg/L, 25 mg/kg dry soil, 21.1 mg/L, 4.76 mg/L, 0.346 g/kg dry soil and 672.8 mg/kg (monensin); 0.813 mg/L, 35.40 mg/kg dry soil, >400 mg/L, >2800 mg/L, >15 g/kg dry soil, >2000 mg/kg (lincomycin). These results showed that the environmental effects of veterinary drug residues should not be neglected, due to their mobility in environmental media and potential toxic effects on environmental organisms.

Conjugation-broken thiophene-based electropolymerized polymers with well-defined structures: effect of conjugation lengths on electrochromic properties.

A series of monomers containing tetraphenylsilane connected to different thiophenes such as thiophene, bithiophene and terthiophene were designed and synthesized and were further used to prepare the corresponding polymers via electrochemical polymerization (pSiTPTP, pSiTPBTP and pSiTPTTP). From the polymers, the effective conjugate elements were well defined as bithiophene, quaterthiophene and sexithiophene because the sp3 Si atom can block the conjugation between the thiophene units in the polymer backbone. The spectroelectrochemical results indicated that pSiTPTP is incapable of electrochromism, which may be attributed to the insufficient conjugation length of the independent bithiophene. In contrast, both pSiTPBTP and pSiTPTTP exhibited obvious electrochromic properties and furthermore, pSiTPTTP displayed a shorter switching time and better stability. Such different electrochemical behaviors can be ascribed to the looser stacking structure and lower oxidation potential of pSiTPTTP with the independent sexithiophene unit. The EIS measurements also confirmed the lower charge-transfer resistance and higher ion-diffusion rate of pSiTPTTP with the independent sexithiophene unit. Hence, we can conclude that the effects of the electrochromic behavior of the conjugation-broken polythiophene derivatives depend on the increased conjugation length of the thiophene repeating unit, in which the inadequate electrochromism with bithiophene units can change to superior electrochromic properties with increased sexithiophene units.

Physicochemical properties, in vitro cytotoxic and genotoxic effects of PM1.0 and PM2.5 from Shanghai, China.

Exposure to ambient particulate matter (PM) links with a variety of respiratory diseases. However, compared with coarse particles (PM10) and fine particles (PM2.5), submicrometer particles (PM1.0) may be a more important indicator of human health risks. In this study, the cytotoxic and genotoxic effects of PM1.0 samples from Shanghai were examined using A549 cells, and compared with the effects of PM2.5, to better understand the health effects of PM1.0 in this area. The PM1.0 and PM2.5 samples were characterized for morphology, water-soluble inorganic ions, organic and elemental carbon, and metal elements. The cytotoxicity of PMs was measured using cell viability and cell membrane damage assays. The genotoxic effects of PMs were determined using the comet assay, and DNA damage was quantified using olive tail moment (OTM) values. The physicochemical characterization indicated that PM1.0 was enriched in carbonaceous elements and hazardous metals (Al, Zn, Pb, Mn, Cu, and V), whereas PM2.5 was more abundant in large, irregular mineral particles. The biological results revealed that both PM1.0 and PM2.5 could induce significant cytotoxicity and genotoxicity in A549 cells, and that exposure to PM1.0 caused more extensive toxic effects than exposure to PM2.5. The greater cytotoxic effects of PM1.0 can be attributed to the combined effects of size and chemical composition, whereas the genotoxic effects of PM1.0 may be mainly associated with chemical species.