Biochemical and functional characterization of heat-inactivated coelomic fluid from earthworms as a potential alternative for fetal bovine serum in animal cell culture.

Fetal bovine serum (FBS) plays a pivotal role in animal cell culture. Due to ethical and scientific issues, searching for an alternative, comprising the three R's (Refinement, Reduction and Replacement) gained global attention. In this context, we have identified the heat inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a potential alternative for FBS. Briefly, we formulated HI-CF (f-HICF) containing serum free medium which can aid the growth, attachment, and proliferation of adherent cells, similar to FBS. In this study, we investigated the biochemical characterization, sterility, stability, formulation, and functional analysis of HI-CF as a supplement in culturing animal cells. Notably, vitamins, micronutrients, proteins, lipids, and trace elements are identified and compared with FBS for effective normalization of the serum free media. HI-CF is tested to be devoid of endotoxin and mycoplasma contamination thus can qualify the cell culture grade. The f-HICF serum free media was prepared, optimised, and tested with A549, HeLa, 3T3, Vero and C2C12 cell lines. Our results conclude that f-HICF is a potential alternative to FBS, in accordance with ethical concern; compliance with 3R's; lack of unintended antibody interactions; presence of macro and micronutrients; simple extraction; cost-effectiveness and availability.

Rainforest transformation reallocates energy from green to brown food webs.

Terrestrial animal biodiversity is increasingly being lost because of land-use change1,2. However, functional and energetic consequences aboveground and belowground and across trophic levels in megadiverse tropical ecosystems remain largely unknown. To fill this gap, we assessed changes in energy fluxes across 'green' aboveground (canopy arthropods and birds) and 'brown' belowground (soil arthropods and earthworms) animal food webs in tropical rainforests and plantations in Sumatra, Indonesia. Our results showed that most of the energy in rainforests is channelled to the belowground animal food web. Oil palm and rubber plantations had similar or, in the case of rubber agroforest, higher total animal energy fluxes compared to rainforest but the key energetic nodes were distinctly different: in rainforest more than 90% of the total animal energy flux was channelled by arthropods in soil and canopy, whereas in plantations more than 50% of the energy was allocated to annelids (earthworms). Land-use change led to a consistent decline in multitrophic energy flux aboveground, whereas belowground food webs responded with reduced energy flux to higher trophic levels, down to -90%, and with shifts from slow (fungal) to fast (bacterial) energy channels and from faeces production towards consumption of soil organic matter. This coincides with previously reported soil carbon stock depletion3. Here we show that well-documented animal biodiversity declines with tropical land-use change4-6 are associated with vast energetic and functional restructuring in food webs across aboveground and belowground ecosystem compartments.

Spent coffee grounds as a suitable alternative to standard soil in ecotoxicological tests.

Eisenia andrei is considered in OECD and ISO guidelines to be a suitable replacement for Eisenia fetida in ecotoxicological assays. This suggests that other alternative materials and methods could also be used in standard procedures for toxicity testing. The guidelines also favor using less time-consuming procedures and reducing costs and other limitations to ecotoxicological assessments. In recent years, spent coffee grounds (SCG) have been used to produce vermicompost and biochar and as an additive to organic fertilizers. In addition, the physicochemical characteristics of SCG indicate that the material is a suitable substrate for earthworms, with the organisms performing as well as in natural soil. In the present study, a battery of ecotoxicological tests was established with unwashed and washed SCG and a natural reference soil (LUFA 2.2). The test substrates were spiked with different concentrations of silver nitrate. Survival and reproduction of the earthworm E. andrei were assessed under different conditions, along with substrate basal respiration (SBR) as a proxy for microbial activity. Seedling emergence and the germination index of Lepidium sativum were also determined, following standard guidelines. Exposure to silver nitrate had similar effects on earthworm survival and reproduction, as the estimated effective concentrations (EC10 and EC50) in unwashed SCG and LUFA 2.2 overlapped. A hormetic effect was observed for SBR in LUFA 2.2 spiked with 12.8 mg/kg but not in unwashed SCG. Both SBR and root development were inhibited by similar concentrations of silver nitrate in washed SCG. The findings indicate that unwashed SCG could potentially be used as a substrate in E. andrei toxicity tests and support the eventual inclusion of this material in the standard guidelines.

The residues and health risk assessment of polychlorinated biphenyls (PCBs) in Pheretima (an earthworm-derived traditional medicine) from southeastern China.

Minimal research exists on polychlorinated biphenyl (PCB) exposure from traditional Chinese medicines (TCMs), despite their significant contributions to domestic and international health protection. This study is the first to investigate the levels, profiles, and health risks of PCB residue in Pheretima, a typical TCM produced from earthworm. Seventy-seven Pheretima samples from different regions of China were analyzed for 45 PCB congeners. PCBs were found in all samples exhibiting species-dependent discrepancies. ∑45PCBs was ranging from 0.532 to 25.2 µg/kg (mean 4.46 µg/kg), with CB-11 being the most abundant congener contributing 71.8% ± 10.8% to ∑45PCBs, followed by CB-47, which were all non-Aroclor congeners called unintentionally produced PCBs (UP-PCBs). The average estimated daily intake of ∑45PCBs, ∑7ID-PCBs (indicative polychlorinated biphenyls), and CB-11 were 0.71, 0.04, and 0.51 ng/kg bw/d, respectively. The ∑HQ of PCBs in Pheretima samples was 2.97 × 10-4-2.46 × 10-2 (mean 2.77 × 10-3, 95th 4.21 × 10-3), while the ∑RQ ranged from 1.19 × 10-8 to 2.88 × 10-6 (mean 4.87 × 10-7, 95th 2.31 × 10-6). These findings indicate that Pheretima ingestion does not pose significant non-carcinogenic risks. However, certain individual samples exhibit an acceptable level of potential risks, particularly when considering that PCBs are recognized as endocrine disruptors and classified as probable carcinogens. These results contribute to the safety evaluation of traditional medicines and suggest the potential use of Pheretima as a bioindicator for PCB pollution. It is advisable to monitor UP-PCBs as indicator congeners and gather additional toxicological data.

Water extract of earthworms mitigates mouse liver fibrosis by potentiating hepatic LKB1/Nrf2 axis to inhibit HSC activation and hepatocyte death.

ETHNOPHARMACOLOGICAL RELEVANCE: When left untreated, liver fibrosis (LF) causes various chronic liver diseases. Earthworms (Pheretima aspergillum) are widely used in traditional medicine because of their capacity to relieve hepatic diseases. AIM OF THE STUDY: This study aimed to explore the anti-LF effects of water extract of earthworms (WEE) and the underlying molecular mechanisms. MATERIALS AND METHODS: A CCl4-induced mouse model of LF was used to study the impact of WEE on LF in vivo. The anti-LF activity of WEE in mice was compared with that of silybin, which can be clinically applied in LF intervention and was used as a positive control. Activation of LX-2 hepatic stellate cells (HSCs) and apoptosis and ferroptosis of AML-12 hepatocytes induced by TGFß1 were used as in vitro models. RESULTS: WEE drastically improved LF in mice. WEE reduced markers of activated HSCs in mice and inhibited TGFß1-induced activation of LX-2 HSCs in vitro. Additionally, WEE suppressed CCl4-induced apoptosis and ferroptosis in mouse hepatocytes. Mechanistically, WEE induced Nrf2 to enter the nuclei of the mouse liver cells, and the hepatic levels of Nrf2-downstream antioxidative factors increased. LKB1/AMPK/GSK3ß is an upstream regulatory cascade of Nrf2. In the LF mouse model, WEE increased hepatic phosphorylated LKB1, AMPK, and GSK3ß levels. Similar results were obtained for the LX-2 cells. In AML-12 hepatocytes and LX-2 HSCs, WEE elevated intracellular Nrf2 levels, promoted its nuclear translocation, and inhibited TGFß1-induced ROS accumulation. Knocking down LKB1 abolished the impact of WEE on the AMPK/GSK3ß/Nrf2 cascade and eliminated its protective effects against TGFß1. CONCLUSIONS: Our findings reveal that WEE improves mouse LF triggered by CCl4 and supports its application as a promising hepatoprotective agent against LF. The potentiation of the hepatic antioxidative AMPK/GSK3ß/Nrf2 cascade by activating LKB1 and the subsequent suppression of HSC activation and hepatocyte apoptosis and ferroptosis are implicated in WEE-mediated alleviation of LF.

A review on the fate of micro and nano plastics (MNPs) and their implication in regulating nutrient cycling in constructed wetland systems.

This review discusses the micro-nano plastics (MNPs) and their interaction with physical, chemical and biological processes in a constructed wetland (CW) system that is typically used as a nature-based tertiary wastewater treatment for municipal as well as industrial applications. Individual components of the CW system such as substrate, microorganisms and plants were considered to assess how MNPs influence the CW processes. One of the main functions of a CW system is removal of nutrients like nitrogen (N) and phosphorus (P) and here we highlight the pathways through which the MNPs influence CW's efficacy of nutrient removal. The presence of morphologically (size and shape) and chemically different MNPs influence the growth rate of microorganisms important in N and P cycling, invertebrates, decomposers, and the plants which affect the overall efficiency of a CW treatment system. Certain plant species take up the MNPs, and some toxicity has been observed. This review focuses on two significant aspects: (1) the presence of MNPs in a significant concentration affects the efficiency of N and P removal, and (2) the removal of MNPs. Because MNPs reduce the enzyme activities in abundance and overproduction of ROS oxidizes the enzyme active sites, resulting in the depletion of proteins, ultimately inhibiting nitrogen and phosphorus removal within the substrate layer. The review found that the majority of the studies used sand-activated carbon (SAC), granular-activated carbon (GAC), rice straw, granular limestone, and calcium carbonate, as a substrate for CW treatment systems. Common plant species used in the CW include Phragmites, Arabidopsis thaliana, Lepidium sativum, Thalia dealbata, and Canna indica, which were also found to be dominant in the uptake of the MNPs in the CWs. The MNPs were found to affect earthworms such as Eisenia fetida, Caenorhabditis elegans, and, Enchytraeus crypticus, whereas Metaphire vulgaris were found unaffected. Though various mechanisms take place during the removal process, adsorption and uptake mechanism effectively emphasize the removal of MNPs and nitrogen and phosphorus in CW. The MNPs characteristics (type, size, and concentration) play a crucial role in the removal efficiency of nano-plastics (NPs) and micro-plastics (MPs). The enhanced removal efficiency of NPs compared to MPs can be attributed to their smaller size, resulting in a faster reaction rate. However, NPs dose variation showed fluctuating removal efficiency, whereas MPs dose increment reduces removal efficiency. MP and NPs dose variation also affected toxicity to plants and earthworms as observed from data. Understanding the fate and removal of microplastics in wetland systems will help determine the reuse potential of wastewater and restrict the release of microplastics. This study provides information on various aspects and highlights future gaps and needs for MNP fate study in CW systems.

Alleviation of endothelial dysfunction of Pheretima guillemi (Michaelsen)-derived protein DPf3 in ponatinib-induced thrombotic zebrafish and mechanisms explored through ox-LDL-induced HUVECs and TMT-based proteomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Thrombus generation is one of the leading causes of death in human, and vascular endothelial dysfunction is a major contributor to thrombosis. Pheretima guillemi (Michaelsen), a traditional medicinal animal known as "Dilong", has been utilized to cure thrombotic disorders for many years. DPf3, a group of functional proteins extracted from P. guillemi, has been characterized and identified to possess antithrombotic bioactivity via in vitro and ex vivo experiments. AIM OF THE STUDY: This study is aimed to investigate the vascular-protection activity and related mechanism of antithrombotic protein DPf3 purified from Pheretima guillelmi systematically. MATERIALS AND METHODS: The antithrombotic activity and vascular endothelium protection effect of DPf3 was explored in vivo using ponatinib-induced vascular endothelial injury zebrafish thrombus model. Then, (hi) ox-LDL-induced HUVECs was applied to investigate the protection mechanism of DPf3 against the injury of vascular endothelium. In addition, TMT-based proteomics analysis was used to study the biomarkers, biological processes and signal pathways involved in the antithrombotic and vascular protective effects of DPf3 holistically. RESULTS: DPf3 exerted robust in vivo antithrombosis and vascular endothelial protection ability. DPf3 was identified to prevent HUVECs from damage by reducing ROS production, and to reduce monocyte adhesion by decreasing the protein content of adhesion factor VCAM 1. DPf3 was also observed to weaken the migration ability of injured cells and inhibit abnormal angiogenesis. The mechanism of DPf3's antithrombotic and vascular protective activity was mainly related to the regulation of lipid metabolism, energy metabolism, complement and coagulation system, ECM receptor interaction, MAPK signal pathway, etc. CONCLUSIONS: This study demonstrates that DPf3 has strong antithrombotic and endothelial protective effects. The endothelial protective ability and related mechanisms of DPf3 provide a scientific reference for the traditional use of earthworms in the treatment of thrombosis.

Vermicomposting as an effective approach to municipal sewage sludge management through optimization of the selected process variables.

In most developing countries, municipal sewage sludge end-use practices appear unsustainable; rather, it poses environmental concerns. This study examined the potential of vermicomposting of municipal sewage sludge and its blend with other biowaste for agricultural application. Using a response surface methodology and the Box-Behnken design in Design Expert Software (Version 10.0.7), the current study optimized the moisture content (60-90%), turning frequency (1-3 turnings/week), and substrate mixing ratios (50:50 to 80:20 wt.%) to maximize the content of nitrogen, phosphorus, and potassium. As a result, an optimal moisture content (72%), substrate mixing ratio (72.34:27.6 wt.%), and turning frequency (2 per week), producing a promising-quality vermicompost with a maximum yield of nitrogen (2.76%), phosphorus (1.80%), and potassium (1.88%) is achieved. Thus, vermicomposting can effectively turn the concerning municipal sewage sludge into useful agricultural input for its sustainable management.

Vermiremediation of heavy metals (HMs)-contaminated agricultural land: synergistic changes in soil enzyme activities and earthworm's growth parameters.

In this investigation the heavy metals (Cu, Zn, Mn, Cr and Ni) remediation potential of Eisenia fetida was studied in the crude oil polluted soil. The potential of E. fetida was evaluated based on the decrease in concentrations of Cu, Zn, Mn, Cr and Ni, and improvement in the soil enzyme activities at the end of 90 days of experimental trials. Moreover, soil health quality, inter-relationship between the enzyme activities and the growth parameters of E. fetida and synergistic relation among the enzyme activities were also evaluated through G-Mean and T-QSI indices, chord plot analysis and principal component analysis (PCA) to confirm the performance of E. fetida during vermiremediation. The results revealed that the soil treated with E. fetida showed a reduction in the concentration of Cu, Zn, Mn, Cr and Ni by 17.4% 19.45%, 9.44%, 23.8% and 9.6% respectively by end of the experimental trials. The cellulase, amylase, polyphenol oxidase, peroxidase, urease, dehydrogenase and catalase activities in the E. fetida-treated soil were enhanced by 89.83%, 99.17%, 142%, 109.9%, 92.9%, 694.3% and 274.5% respectively. The results of SEM-EDS revealed enhancement in the O, K, Na, Mg and P content by 62.36%, 96.2%, 97.9%, 93.7% and 98.2% respectively by the end of the experimental trial. The G-Mean and T-QSI indices also confirmed the improvement in soil enzyme activities thereby indicating the positive influence of E. fetida on soil decontamination process. The chord plot indicated the interrelationship between the earthworm's growth parameters and enzyme activities of the soil as indicated by the high linkage between the nodes. Finally, the PCA confirmed the negative effect of the heavy metals on the soil enzyme activities and synergistic interrelationship between the enzyme activities during the vermiremediation process. Thus, this study demonstrated the changes in the soil enzyme activities and their interconnected influences during vermiremediation of crude oil sourced heavy metals from polluted soil.

Elucidating response mechanisms at the metabolic scale of Eisenia fetida in typical oil pollution sites: A native driver in influencing carbon flow.

Previous investigations on the stress response patterns of earthworms (Eisenia fetida) in practical petroleum hydrocarbon (PH) contamination systems were less focused. Therefore, this study investigated the ecotoxicological effect of PH contamination on earthworms based on metabonomics and histological observation, followed by correlation analysis between the earthworm metabolism, PH types and concentrations, soil physicochemical characteristics, and the microbial community structures (i.e., diversity and abundance) and functions. The results showed that due to the abundant PH organics, the cell metabolism of earthworms shifts under PH contamination conditions, leading them to use organic acids as alternative energy sources (i.e., gluconeogenesis pathway). Simultaneously, biomarker metabolites related to cellular uptake, stress response, and membrane disturbance were identified. In addition, when compared to the controls, considerable epicuticle and cuticle layer disruption was observed, along with PH internalization. It was demonstrated that PH pollution preferentially influences the physiological homeostasis of earthworms through indirect (i.e., microbial metabolism regulation) than direct (i.e., direct interaction with earthworms) mechanisms. Moreover, the varied CO2 releasement was verified, which highlights the potential role of earthworms in influencing carbon transformation and corresponds with the considerably enriched energy metabolism-related pathway. This study indicated that PH contamination can induce a strong stress response in earthworms through both direct and indirect mechanisms, which in turn, substantially influences carbon transformation in PH contamination sites.

The Protective Effect of Lithium Against Rotenone may be Evolutionarily Conserved: Evidence from Eisenia fetida, a Primitive Animal with a Ganglionic Brain.

Chronic exposure to stress is a non-adaptive situation that is associated with mitochondrial dysfunction and the accumulation of reactive oxygen species (ROS), especially superoxide anion (SA). This accumulation of ROS produces damage-associated molecular patterns (DAMPs), which activate chronic inflammatory states and behavioral changes found in several mood disorders. In a previous study, we observed that an imbalance of SA triggered by rotenone (Ro) exposure caused evolutionarily conserved oxi-inflammatory disturbances and behavioral changes in Eisenia fetida earthworms. These results supported our hypothesis that SA imbalance triggered by Ro exposure could be attenuated by lithium carbonate (LC), which has anti-inflammatory properties. The initial protocol exposed earthworms to Ro (30 nM) and four different LC concentrations. LC at a concentration of 12.85 mg/L decreased SA and nitric oxide (NO) levels and was chosen to perform complementary assays: (1) neuromuscular damage evaluated by optical and scanning electron microscopy (SEM), (2) innate immune inefficiency by analysis of Eisenia spp. extracellular neutrophil traps (eNETs), and (3) behavioral changes. Gene expression was also evaluated involving mitochondrial (COII, ND1), inflammatory (EaTLR, AMP), and neuronal transmission (nAchR α5). LC attenuated the high melanized deposits in the circular musculature, fiber disarrangement, destruction of secretory glands, immune inefficiency, and impulsive behavior pattern triggered by Ro exposure. However, the effects of LC and Ro on gene expression were more heterogeneous. In summary, SA imbalance, potentially associated with mitochondrial dysfunction, appears to be an evolutionary component triggering oxidative, inflammatory, and behavioral changes observed in psychiatric disorders that are inhibited by LC exposure.

A nano-Liposomal formulation potentiates antioxidant, anti-inflammatory, and fibrinolytic activities of Allolobophora caliginosa coelomic fluid: formulation and characterization.

BACKGROUND: Coelomic fluid, a pharmacologically active compound in earthworms, exhibits a range of biological activities, including antioxidant, anti-inflammatory, and anticancer. However, the biological activities exerted by the coelomic fluid can be restrained by its low bioavailability and stability. Liposomes are progressively utilized as an entrapment system for natural bioactive compounds with poor bioavailability and stability, which could be appropriate for coelomic fluid. Thus, the present study was designed to fabricate, characterize, and evaluate the stability of liposomal formulation for Allolobophora caliginosa coelomic fluid (ACCF) as a natural antioxidant compound. METHODS: The ACCF-liposomes were developed with a subsequent characterization of their physicochemical attributes. The physical stability, ACCF release behavior, and gastrointestinal stability were evaluated in vitro. The biological activities of ACCF and its liposomal formulation were also determined. RESULTS: The liposomal formulation of ACCF had a steady characteristic absorption band at 201 nm and a transmittance of 99.20 ± 0.10%. Its average hydrodynamic particle size was 98 nm, with a PDI of 0.29 ± 0.04 and a negative zeta potential (-38.66 ± 0.33mV). TEM further confirmed the formation of vesicular, spherical nano-liposomes with unilamellar configuration. Additionally, a remarkable entrapment efficiency percent (77.58 ± 0.82%) with a permeability rate equal to 3.20 ± 0.31% and a high retention rate (54.16 ± 2.20%) for ACCF-liposomes were observed. The Fourier transform infrared spectroscopy (FTIR) result demonstrated that ACCF successfully entrapped inside liposomes. The ACCF-liposomes exhibited a slow and controlled ACCF release in vitro. Regarding stability studies, the liposomal formulation enhanced the stability of ACCF during storage and at different pH. Furthermore, ACCF-liposomes are highly stable in intestinal digestion conditions comparable to gastric digestion. The current study disclosed that liposomal formulation potentiates the biological activities of ACCF, especially antioxidant, anti-inflammatory, and thrombolytic activities. CONCLUSION: These promising results offer a novel approach to increasing the bioaccessibility of ACCF, which may be crucial for the development of pharmaceuticals and nutraceutical-enriched functional foods.

Effects of soybean oil exposure on the survival, reproduction, biochemical responses, and gut microbiome of the earthworm Eisenia fetida.

With increasing production of kitchen waste, cooking oil gradually enters the soil, where it can negatively affect soil fauna. In this study, we explored the effects of soybean oil on the survival, growth, reproduction, tissue structure, biochemical responses, mRNA expression, and gut microbiome of earthworms (Eisenia fetida). The median lethal concentration of soybean oil was found to be 15.59%. Earthworm growth and reproduction were significantly inhibited following exposure to a sublethal concentration of soybean oil (1/3 LC50, 5.2%). The activity of the antioxidant enzymes total superoxide dismutase (T-SOD), peroxidase (POD), and catalase (CAT) were affected under soybean oil exposure. The glutathione (GSH) content decreased significantly, whereas that of the lipid peroxide malondialdehyde (MDA) increased significantly after soybean oil exposure. mRNA expression levels of the SOD, metallothionein (MT), lysenin and lysozyme were significantly upregulated. The abundance of Bacteroides species, which are related to mineral oil repair, and Muribaculaceae species, which are related to immune regulation, increased within the earthworm intestine. These results indicate that soybean oil waste is toxic to earthworms. Thus, earthworms deployed defense mechanisms involving antioxidant system and gut microbiota for protection against soybean oil exposure-induced stress.

Bioremediation of polycyclic aromatic hydrocarbons in crude oil by bacterial consortium in soil amended with Eisenia fetida and rhamnolipid.

The present study investigated the concerted effort of Eisenia fetida and rhamnolipid JBR-425 in combination with a five-member bacterial consortium exhibiting elevated degradation levels of low and high molecular weight polycyclic aromatic hydrocarbons (PAH) from soil contaminated with Digboi crude oil. Application of bacterial consortium (G2) degraded 30-89% of selected PAH from the artificial soil after a 45-day post-exposure, in which chrysene showed the highest level of degradation with 89% and benzo(a)pyrene is the lowest with 30%, respectively. Moreover, an acute exposure study observed that earthworm biomass decreased, and mortality rates increased with increasing crude oil concentrations (0.25 to 2%). Earthworms with a 100% survival rate at 1% crude oil exposure suggest the tolerance potential and its mutual involvement in the bioremediation of crude oil with selected bacterial consortia. Bacterial consortium assisted with E. fetida (G3) showed 98% chrysene degradation with a slight change in benzo(a)pyrene degradation (35%) in crude oil spiked soil. Besides, the most dominant PAH in crude oil found in the current work, fluoranthene, undergoes 93% and 70% degradation in G3 and G5 groups, respectively. However, rhamnolipid JBR-425 coupled with the bacterial consortium (G5) has resulted in 97% degradation of chrysene and 33% for benzo(a)pyrene. Overall, bacterial consortium assisted with earthworm group has shown better degradation of selected PAH than bacterial consortium with biosurfactant. Catalase (CAT), glutathione reductase (GST) activity and MDA content was found to be reduced in earthworms after sub-lethal exposure, suggesting oxidative stress prevalence via reactive oxygen species (ROS). Hence, the findings of the present work suggest that the application of a bacterial consortium, along with earthworm E. fetida, has huge potential for field restoration of contaminated soil with PAH and ecosystem sustainability.

Organic waste recycling by vermicomposting amended with rock phosphate impacts the stability and maturity indices of vermicompost.

Recycling organic waste can help the land be nourished, properly disposed of, and protected from the negative impacts of chemical fertilizers. Organic additions like vermicompost can help restore and preserve the quality of the soil, however, producing vermicompost of a high enough standard is difficult. This study was planned to prepare vermicompost by utilizing two different organic wastes viz. household waste and organic residue amended with rock phosphate and further evaluate their stability and maturity indices during vermicomposting for quality of produce. For this study, the organic wastes were collected and vermicompost was prepared by using earthworm (Eisenia fetida) and with or without enriching with rock phosphate. Results showed that pH, bulk density, and biodegradability index were decreased and water holding capacity and cation exchange capacity was increased with the gradual progress of composting starting from 30 to 120 days of sampling/composting (DAS). Initially (upto 30 DAS) water-soluble carbon and water-soluble carbohydrate increased with rock phosphate enrichment. The population of the earthworms and enzymatic activities (CO2 evolution, dehydrogenase, and alkaline phosphatase) were also increased on enriching with rock phosphate and with the progression of the composting period. Rock phosphate addition (enrichment) also reflected the higher content of phosphorus (106% and 120% for household waste and organic residue, respectively) in the final product of vermicompost. The vermicompost prepared from household waste and enriched with rock phosphate showed greater maturity and stability indices. Overall, this can be concluded that the maturity and stability of vermicompost depend on the substrate used and improves on enriching with rock phosphate.Implications: Our study concludes that the quality of vermicompost depends on different substrates, composting period, and enrichment with rock phosphate. The qualities of vermicompost were best found under household waste-based vermicompost enriched with rock phosphate. The efficiency of vermicomposting process using earthworms was found maximum for enriched and without enriched household-based vermicompost. The study also indicated that several stability and maturity indices are influenced by different parameters and hence cannot be determined by a single parameter. The addition of rock phosphate increased the cation exchange capacity, phosphorus content, and alkaline phosphatase. Nitrogen, zinc, manganese, dehydrogenase, and alkaline phosphatase were found higher under household waste-based vermicompost relative to organic residue-based vermicompost. All four substrates promoted earthworm growth and reproduction in vermicompost.

A novel glycoprotein from earthworm extract PvE-3: Insights of their characteristics for promoting diabetic wound healing and attenuating methylglyoxal-induced cell damage.

Diabetic chronic wound is a worldwide medical burden related to overdosed methylglyoxal (MGO) synthesis, which is the major precursor of glycation of proteins and DNA and is related to the dysfunction of dermal cells thus leading to chronic refractory wounds. Previous studies proved that earthworm extract accelerates diabetic wound healing and possesses cell proliferation and antioxidative effects. However, the effects of earthworm extract on MGO-damaged fibroblasts, the inner mechanisms of MGO-induced cell damage and the functional components in earthworm extract are still poorly understood. Firstly, we evaluated the bioactivities of the earthworm extract PvE-3 on the diabetic wound model and the diabetic related cell damage model. Then the mechanisms were investigated through transcriptomics, flow cytometry and fluorescence probe. The results revealed that PvE-3 promoted diabetic wound healing and protected fibroblast function in cell-damaged conditions. Meanwhile, the high-throughput screening implied the inner mechanisms of diabetic wound healing and PvE-3 cytoprotection effect were involved in the muscle cell function, the cell cycle regulation and the mitochondrial transmembrane potential depolarization. The functional glycoprotein isolated from PvE-3 possessed EGF-like domain which had a strong binding affinity with EGFR. The findings provided references to explore the potential treatments of diabetic wound healing.

A new species of Enchytraeus (Enchytraeidae, Clitellata) from sewage sludge of a plum processing plant in Japan.

Enchytraeus ohtakai sp. nov. (Enchytraeidae, Clitellata, Oligochaeta) was discovered in the organic matter of a wastewater treatment facility of a plums processing plant in Honshu, Japan. The wastewater is characterized by high organic matter content and low salt concentration. Morphological analysis and DNA-sequencing of a fragment of the COI barcoding gene show that the new species belongs to the E. albidus species group. Within this group it differs in: vasa deferentia restricted to XII, preclitellar bundles with mostly three chaetae, postclitellar bundles with two or three, dorsal blood vessel from XII or XIII, spermathecal ectal duct completely glandular. spermatheca with a large diverticulum, accessory sexual glands present in XII, clitellum ventrally almost absent. The individual gene trees of COI analysis recovered this new species as a monophyletic group within the genus Enchytraeus, closely related to E. albidus species group.

[Effects of Earthworm, Straw, and Citric Acid on the Remediation of Zn, Pb, and Cd Contaminated Soil by Solanum photeinocarpum and Pterocypsela indica].

Regulation of exogenous substances and intercropping are effective methods to improve the efficiency of phytoremediation of heavy metal contaminated soil. A pot experiment was used to study the effects of earthworms, straw, and citric acid on the remediation of Zn, Pb, and Cd contaminated soil by monocropping and intercropping of Solanum photeinocarpum and Pterocypsela indica. The results showed that the bioaccumulation factors (BCF) of earthworms for Zn, Pb, and Cd were 0.07-0.13, 0.10-0.26, and 5.64-15.52, respectively. The addition of straw in the soil increased the biomass of earthworms by 22.29%-223.87% but reduced the heavy metal concentrations by 8.15%-62.58%. Straw and citric acid showed passivation and activation effects, respectively, but earthworms had no significant effect on the available concentrations of heavy metals in the soil. Earthworms had no significant effect on the heavy metal concentrations of P. indica but reduced the heavy metal concentrations of S. photeinocarpum. Straw showed an inhibitory effect on the concentrations of heavy metals in P. indica but promoted the concentrations of Cd in S. photeinocarpum. Citric acid had no significant effect on the heavy metal concentrations in S. photeinocarpum but significantly increased the Pb concentrations in P. indica. Intercropping significantly reduced the soil available heavy metal concentrations and increased the heavy metal concentrations in plant roots; however, it had no significant effect on heavy metal concentrations in plant shoots. The total extraction amounts of Zn, Pb, and Cd by plants were mainly manifested as P. indica>intercropping>S. photeinocarpum. The addition of earthworms increased the total extraction amounts of Zn, Pb, and Cd by 12.49%, 35.89%, and 29.01%, respectively, and the addition of straw+earthworms increased the total extraction amounts of Pb by 87.21%. The results indicated that straw significantly promoted the growth of earthworms and reduced their accumulation of heavy metals, and the addition of earthworms alone or in combination with straw can effectively improve the remediation potential of P. indica of Zn, Pb, and Cd contaminated soil.

Toxicity of methylparaben and its chlorinated derivatives to Allium cepa L. and Eisenia fetida Sav.

Methylparaben, chloro-methylparaben, and dichloro-methylparaben were evaluated in Allium cepa at 5, 10, 50, and 100 µg/L and in Eisenia fetida at 10 and 100 µg/L. In A. cepa roots, 100 µg/L methylparaben and 50 and 100 µg/L chlorinated methylparabens reduced cell proliferation, caused cellular changes, and reduced cell viability in meristems, which caused a reduction in root growth. Furthermore, they caused drastic inhibition of catalase, ascorbate peroxidase, and superoxide dismutase; activated guaiacol peroxidase and promoted lipid peroxidation in meristematic root cells. In earthworms, after 14 days exposure to the three compounds, there were no deaths, and catalase, ascorbate peroxidase, and superoxide dismutase were not inhibited. However, guaiacol peroxidase activity and lipid peroxidation were observed in animals exposed to dichloro-methylparaben. Soils with dichloro-methylparaben also caused the escape of earthworms. It is inferred that the recurrent contamination of soils with these methylparabens, with emphasis on chlorinated derivatives, can negatively impact different species that depend directly or indirectly on soil to survive.