Enantiomerization and enantioselective bioaccumulation of diclofop-methyl in tubifex worms.

In this study, the hydrolysis of diclofop-methyl (DM) in aqueous system and the bioaccumulation of its main metabolite, Diclofop (DA), in the tubifex worms were investigated using enantioselective high-performance liquid chromatography. With the addition of tubifex, rapid hydrolysis was observed for DM. It is revealed that the hydrolysis of DM in the water and the accumulation of DA in the worms were both enantioselective. Meanwhile, either the hydrolysis amount or the levels of enantioselectivity were influenced by the tubifex. After incubated for 24 h, about 94.6% of the DM was hydrolyzed and the enantiomer fraction of metabolite (DA) deviated from 0.5 with R-DA significantly higher than S-DA. The enantiopure S-DM and R-DM and S-DA and R-DA were incubated, and enantiomerizations were detected between the two DM enantiomers with S-form inversing into R-form and vice versa. It was found that the S-DM exhibited a higher tendency to invert than the R-DM.

The influence of ambient temperature and polyphenols from plant leaves on growth and the response to oxidative and nitrosative stress in African nightcrawler earthworm, Eudrilus eugeniae (Kinberg, 1867).

Earthworms are one of the organisms that may be affected by climate change. Finding ways to help them deal with this problem is, therefore, important and necessary. The objective of this experiment was to understand the influence of ambient temperature and polyphenols from mulberry (Morus alba L.), almond (Terminalia catappa L.) and cassava (Manihot esculenta (L.) Crantz) leaves on growth, ferric reducing antioxidant power (FRAP), malondialdehyde (MDA), hydrogen peroxide (H2O2) and nitric oxide (NO) concentration of the African night crawler, Eudrilus eugeniae (Kinberg, 1867) earthworm. The earthworms were cultured in 2 different conditions of ambient temperature, and in 4 types of substrate i.e. dairy cow faeces (BS), dairy cow faeces + mulberry leaves (BS + MA), almond leaves (BS + TC), and cassava leaves (BS + ME), respectively. At week 2 of the experiment, body weight, FRAP, MDA, H2O2 and NO were measured in the earthworms. It was found that the body weight gain (BWG) of the earthworms cultured in BS at cyclical temperature (26 + 1oC - 34 + 1oC - 26 + 1oC, CyT) was higher than the constant temperature (26 ± 1 °C, CoT) (P < 0.05). FRAP of earthworms cultured in BS + TC was higher than in other groups (P < 0.05). MDA of earthworms cultured at CyT was higher than ambient temperature at CoT (P < 0.05). At CyT, the MDA of earthworms cultured in BS + MA was higher than that of those cultured in BS, BS + TC and BS + ME (P < 0.05). NO of earthworms at CoT was higher than at CyT(P < 0.05). At CoT, the NO of earthworms cultured in BS + TC was lower than that of those cultured in BS + MA and BS + ME (P < 0.05). H2O2 of earthworms at CoT was higher than those at CyT (P < 0.05). The level of H2O2 of the earthworms cultured in BS + ME at CoT was higher than at CyT (P < 0.05). In addition, the H2O2 of earthworms cultured in both ambient temperatures and cultured in BS + MA was higher than the other groups (P < 0.05). These phenomena indicated that low and high ambient temperatures induced nitrosative and oxidative stress in earthworms, respectively. Mulberry leaves are toxic to earthworms. On the other hand, almond leaves could reduce nitrosative stress in earthworms. While at the CoT, cassava leaves induced the production of H2O2 in the earthworms.

Biorecognition of hydrogen peroxide using a novel electrochemical platform designed with Glossoscolex paulistus giant hemoglobin.

The giant extracellular hemoglobin of the annelid Glossoscolex paulistus (HbGp; 3.6 MDa) is a valuable and underexplored supramolecular hemoprotein system for the biorecognition of reactive oxygen species. In this work, an efficient and simple electrochemical platform was designed for analyzing H2O2, using HbGp covalently immobilized on Nafion®-modified glassy carbon electrode, named as HbGp/Nafion/GCE. Voltammetric and spectroscopic studies revealed the importance of prior modification of the electrodic support with the conducting polymer to obtain satisfactory hemoglobin electroactivity, as well as a biocompatible microenvironment for its immobilization. In terms of biological activity, it was observed a greater reactivity of the biomolecule in acidic medium, enabling the detection of the analyte by a quasi-reversible mechanism, whose kinetics was limited by analyte diffusion. In the presence of H2O2, the native structure of hemoglobin (oxy-HbGp (Fe2+)) oxidizes to ferryl-HbGp (Fe4+) and this redox reaction can be monitored on HbGp/Nafion/GCE with a detection limit of 8.5 × 10‒7  mol L-1. In addition to high sensitivity, the electrochemical biosensor also provided reproducible, consistent, and accurate measurements. The electroanalytical method showed an appropriate performance to quantify different levels of H2O2 in milk samples, proving the potential of HbGp/Nafion/GCE for this purpose.

Authentication of three main commercial Pheretima based on amino acids analysis.

Pheretima has been used as an animal-derived traditional Chinese medicine for thousands of years in Asian countries due to its multi-activities. However, more than half of the commercial Pheretima are adulterants according to the previous research. Besides, the standards of Pheretima are still inadequate in the identification of Pheretima species. In this study, an amino acids (AAs) analytical method established based on the ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-QqQ-MS) in multiple reaction monitoring (MRM) mode through derivatization with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) was used for qualitative and quantitative analysis of the total AAs of three main commercial Pheretima (two major Pheretima species, Amynthas aspergillum, Metaphire vulgaris, and one main counterfeit, M. magna). As a result, 16 AAs were detected and quantitated in their hydrolyzed samples. Then, multivariate statistical analysis was applied to distinguish the three commercial Pheretima based on their AAs level. Finally, four AAs (Thr, Glu, Asp, and Arg) were screened as species-differential AAs, which may be used as chemical markers to distinguish the three commercial Pheretima. This study deeply described the outline of AAs in Pheretima and offered a good reference for its species authentication.

Annelid Coelomic Fluid Proteins.

The coelomic cavity is part of the main body plan of annelids. This fluid filled space takes up a considerable volume of the body and serves as an important site of exchange of both metabolites and proteins. In addition to low molecular substances such as amino acids and glucose and lactate, the coelomic fluid contains different proteins that can arise through release from adjacent tissues (intestine) or from secretion by coelomic cells. In this chapter, we will review the current knowledge about the proteins in the annelid coelomic fluid. Given the number of more than 20,000 extant annelid species, existing studies are confined to a relatively few species. Most studies on the oligochaetes are confined to the earthworms-clearly because of their important role in soil biology. In the polychaetes (which might represent a paraphyletic group) on the other hand, studies have focused on a few species of the Nereidid family. The proteins present in the coelomic fluid serve different functions and these have been studied in different taxonomic groups. In oligochaetes, proteins involved antibacterial defense such as lysenin and fetidin have received much attention in past and ongoing studies. In polychaetes, in contrast, proteins involved in vitellogenesis and reproduction, and the vitellogenic function of coelomic cells have been investigated in more detail. The metal binding metallothioneins as well as antimicrobial peptides, have been investigated in both oligochaetes and polychaetes. In the light of the literature available, this review will focus on lipoproteins, especially vitellogenin, and proteins involved in defense reactions. Other annelid groups such as the Pogonophora, Echiura, and Sipuncula (now considered polychaetes), have not received much attention and therefore, this overview is far from being complete.

Earthworms and their cutaneous excreta can modify the virulence and reproductive capability of entomopathogenic nematodes and fungi.

Earthworms are ecological engineers that can contribute to the displacement of biological control agents such as the entomopathogenic nematodes (EPNs) and fungi (EPF). However, a previous study showed that the presence of cutaneous excreta (CEx) and feeding behavior of the earthworm species Eisenia fetida (Haplotaxida: Lumbricidae) compromise the biocontrol efficacy of certain EPN species by reducing, for example, their reproductive capability. Whether this phenomenon is a general pattern for the interaction of earthworms-entomopathogens is still unknown. We hypothesized that diverse earthworm species might differentially affect EPN and EPF infectivity and reproductive capability. Here we investigated the interaction of different earthworm species (Eisenia fetida, Lumbricus terrestris, and Perionyx excavatus) (Haplotaxida) and EPN species (Steinernema feltiae, S. riojaense, and Heterorhabditis bacteriophora) (Rhabditida) or EPF species (Beauveria bassiana and Metarhizium anisopliae) (Hypocreales), in two independent experiments. First, we evaluated the application of each entomopathogen combined with earthworms or their CEx in autoclaved soil. Hereafter, we studied the impact of the earthworms' CEx on entomopathogens applied at two different concentrations in autoclaved sand. Overall, we found that the effect of earthworms on entomopathogens was species-specific. For example, E. fetida reduced the virulence of S. feltiae, resulted in neutral effects for S. riojaense, and increased H. bacteriophora virulence. However, the earthworm P. excavates increased the virulence of S. feltiae, reduced the activity of H. bacteriophora, at least at specific timings, while S. riojaense remained unaffected. Finally, none of the EPN species were affected by the presence of L. terrestris. Also, the exposure to earthworm CEx resulted in a positive, negative or neutral effect on the virulence and reproduction capability depending on the earthworm-EPN species interaction. Concerning EPF, the impact of earthworms was also differential among species. Thus, E. fetida was detrimental to M. anisopliae and B. bassiana after eight days post-exposure, whereas Lumbricus terrestris resulted only detrimental to B. bassiana. In addition, most of the CEx treatments of both earthworm species decreased B. bassiana virulence and growth. However, the EPF M. anisopliae was unaffected when exposed to L. terrestris CEx, while the exposure to E. fetida CEx produced contrasting results. We conclude that earthworms and their CEx can have positive, deleterious, or neutral impacts on entomopathogens that often coinhabit soils, and that we must consider the species specificity of these interactions for mutual uses in biological control programs. Additional studies are needed to verify these interactions under natural conditions.

Bioevaluation of Pheretima vulgaris Antithrombotic Extract, PvQ, and Isolation, Identification of Six Novel PvQ-Derived Fibrinolytic Proteases.

Thrombosis is a disease that seriously endangers human health, with a high rate of mortality and disability. However, current treatments with thrombolytic drugs (such as recombinant tissue-plasminogen activator) and the oral anticoagulants (such as dabigatran and rivaroxaban) are reported to have a tendency of major or life-threatening bleeding, such as intracranial hemorrhage or massive gastrointestinal bleed with non-specific antidotes. In contrast, lumbrokinase is very specific to fibrin as a substrate and does not cause excessive bleeding. It can dissolve the fibrin by itself or convert plasminogen to plasmin by inducing endogenous t-PA activity to dissolve fibrin clots. Therefore, searching for potentially new therapeutic molecules from earthworms is significant. In this study, we first collected a strong fibrinolytic extract (PvQ) from the total protein of the Pheretima vulgaris with AKTA pure protein purification systems; its fibrinolytic bioactivity was verified by the fibrin plate assay and zebrafish thrombotic model of vascular damage. Furthermore, according to the cell culture model of human umbilical vein endothelial cells (HUVECs), the PvQ was proven to exhibit the ability to promote the secretion of tissue-type plasminogen activator (t-PA), which further illustrated that it has an indirect thrombolytic effect. Subsequently, extensive chromatographic techniques were applied to reveal the material basis of the extract. Fortunately, six novel earthworm fibrinolytic enzymes were obtained from the PvQ, and the primary sequences of those functional proteins were determined by LC-MS/MStranscriptome cross-identification and the Edman degradation assay. The secondary structures of these six fibrinolytic enzymes were determined by circular dichroism spectroscopy and the three-dimensional structures of these proteases were predicted by MODELLER 9.23 based on multi-template modelling. In addition, those six genes encoding blood clot-dissolving proteins were cloned from P. vulgaris by RT-PCR amplification, which further determined the accuracy of proteins primary sequences identifications and laid the foundation for subsequent heterologous expression.

Combination of c oxidase subunit I based deoxyribonucleic acid barcoding and HPLC techniques for the identification and quality evaluation of Pheretima aspergillum.

This study aimed to identify Pheretima aspergillum (Guang-Pheretima) and its adulterants using the cytochrome c oxidase subunit I based deoxyribonucleic acid barcoding technology, and further to evaluate their quality using an optimized high-performance liquid chromatography method. For deoxyribonucleic acid barcoding identification, the Kimura-2-Parameter model was used to analyze genetic distance, and phylogenetic neighbor-joining tree was constructed for species identification of 20 labeled Guang-Pheretima samples. A high-performance liquid chromatography method was developed for the simultaneous determination of seven nucleoside components for quality evaluation. Compared with the GenBank database, 10 samples were identified as real Guang-Pheretima (P. aspergillum), and the others as the adulterants-Metaphire magna. The maximum intraspecific genetic distances of c oxidase subunit I sequence for P. aspergillum were smaller than the minimum interspecific genetic distances between P. aspergillum and M. magna. Ten P. aspergillum and 10 M. magna samples were clearly clustered in the neighbor-joining tree. The contents of seven nucleosides components in P. aspergillum were significantly higher than that in its adulterant-M. magna. The incidence of adulterants for Guang-Pheretima was high (up to 50%) with an alarming quality. This study provided a powerful idea for the quality evaluation of other highly valuable plant- or animal-derived products for safety concerns to avoid misidentification.

The impact of Last Glacial climate variability in west-European loess revealed by radiocarbon dating of fossil earthworm granules.

The characterization of Last Glacial millennial-timescale warming phases, known as interstadials or Dansgaard-Oeschger events, requires precise chronologies for the study of paleoclimate records. On the European continent, such chronologies are only available for several Last Glacial pollen and rare speleothem archives principally located in the Mediterranean domain. Farther north, in continental lowlands, numerous high-resolution records of loess and paleosols sequences show a consistent environmental response to stadial-interstadial cycles. However, the limited precision and accuracy of luminescence dating methods commonly used in loess deposits preclude exact correlations of paleosol horizons with Greenland interstadials. To overcome this problem, a radiocarbon dating protocol has been developed to date earthworm calcite granules from the reference loess sequence of Nussloch (Germany). Its application yields a consistent radiocarbon chronology of all soil horizons formed between 47 and 20 ka and unambiguously shows the correlation of every Greenland interstadial identified in isotope records with specific soil horizons. Furthermore, eight additional minor soil horizons dated between 27.5 and 21 ka only correlate with minor decreases in Greenland dust records. This dating strategy reveals the high sensitivity of loess paleoenvironments to Northern Hemisphere climate changes. A connection between loess sedimentation rate, Fennoscandian ice sheet dynamics, and sea level changes is proposed. The chronological improvements enabled by the radiocarbon "earthworm clock" thus strongly enhance our understanding of loess records to a better perception of the impact of Last Glacial climate changes on European paleoenvironments.

Assessment of the immobilization effectiveness of several amendments on a cadmium-contaminated soil using Eisenia fetida.

Proper protocols for assessing the remediation effectiveness of contaminated soils are an important part of remediation projects. In the present study, the residual immobilization effectiveness of hydrated lime (L), hydroxyapatite (H), biochar (B) and organic fertilizer (F) alone and in combination was assessed by Eisenia fetida. The results showed that the application of amendments had no significant effect on the death rate and average fresh weight loss of earthworms. The earthworm Cd concentration increased with prolonged exposure time, however, the significant immobilization efficacy of amendments observed on the 7th day nearly disappeared after 28 days of exposure. The immobilization efficiencies, estimated by the earthworms internal Cd concentration, of L, H and B on the 7th day were 38.6%, 37.8% and 20.7%, respectively. These values decreased to 4.9%, 19.8% and 15.1%, respectively, on the 28th day. The detoxification effect of amendments was confirmed by the Cd subcellular fractionation in earthworms with lower proportions of Cd distributed in the metal-sensitive fractions in L, H and B treatments. The level of oxidative stress response of earthworms increased with exposure duration and amendments alleviated the oxidative damage induced by Cd to the earthworms. In addition, the pH and CaCl2-Cd in soils were both increased due to earthworm life activities and gut-related ingestion. In summary, the assessment of immobilization effectiveness of heavy metal-contaminated soils using Eisenia fetida was time-dependent. The immobilization efficacy of L and H performed better than B and F on the 7th day, while H and B performed better than L and F on the 28th day. Accordingly, the short-term earthworm exposure experiment (7 days) was recommended to be an alternative approach to time-consuming plant bioassays in assessment of reduced phytoavailability in chemical immobilization remediation. But the impact of earthworms on the immobilization effect of amendments needs to be considered in practical remediation.

Assessment of earthworm activity on Cu, Cd, Pb and Zn bioavailability in contaminated soils using biota to soil accumulation factor and DTPA extraction.

The study aims to investigate effect of earthworm activity on metal bioavailability in soils using their BSAF-metals. Based on a microcosmic laboratory experiment, epigeic species Amynthas corticis (A. corticis) and endogeic species Amynthas robustus (A. robustus) were cultured in two types of soils contaminated by Cd, Zn, Pb and Cu for 120 days. Earthworm characteristics (i.e. numbers, biomass and BSAF), soil properties (i.e. pH, organic C and N contents along with their components such as mineralization and microbial masses) and DTPA extracted metals in soil were determined. After the incubation, the biomass and survival numbers of both earthworm species decreased significantly (P < 0.05). The accumulation of Cd, Zn and Pb in earthworm tissues and BSAF-metals were earthworm species dependent. According to two-way ANOVA, BSAF-Pb clearly showed the effect of different species of earthworms while BSAF-Cu indicated an interactive effect of earthworms and soil type. Earthworms changed soil properties significantly, especially for mineralized C (Cmin), dissolved N (Ndis) and pH (P < 0.05). Earthworm activity increase DTPA extracted Zn and Cu, and the effect of A. robustus were stronger than for A. corticis. Redundancy analysis (RDA) showed that BSAF-Cu and BSAF-Pb contributed for respectively 51.9% and 51.7% of soil properties and DTPA metal changes, indicating that the effects of BSAF-Cu and BSAF-Pb on soil properties and on metal bioavailability in soil were similar. BSAF-Cu, indicating the interactive effect of earthworms and soil, accounted for 38.5% and 45.1% of soil properties and soil metal bioavailability changes. BSAF-Pb, representing the effect of earthworm species, accounted for 13.3% and 6.6% of soil property and soil metal bioavailability variations. Stepwise regression indicated that earthworm might change soil properties through their activities and interactions with soil, and hence increase heavy metal bioavailability. It suggested that BSAF is an important indicator for evaluating the effect of earthworm activity on soil metal bioavailability and designing remediation strategies.

Alternative Evaluation to Earthworm Toxicity Test in Polychlorinated Biphenyls Spiked and Remediated Soils.

Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that pose a threat to environment and human health. Aiming at predicting PCBs risk in actual soil ecosystem, this study was conducted by chemical and biological methods to assess the bioavailability of PCBs in spiked soil, and in field-contaminated soils before or after remediation. The three chemical methods were Soxhlet, n-butanol and hydroxypropyl-ß-cyclodextrin (HPCD). Results were compared to actual PCB bioaccumulation in earthworms (Eisenia fetida). HPCD extraction was the best to predict the actual PCB bioaccumulation in all soils. The results suggest that HPCD could be an effective alternative method to earthworm toxicity test. This study provides strategy to understand the toxicity assessment in contaminated soil and soil after remediation.

Assessment of trace metal contamination and ecological risk in the forest ecosystem of dexing mining area in northeast Jiangxi Province, China.

Samples of soil, earthworms, and tree roots from the forest ecosystem in the Dexing Pb/Zn mining area of Jiangxi Province were collected and the status of trace metal pollution analyzed to assess potential ecological risks. Chemometric and geographic information system methods were used to identify and describe the spatial distributions and the main contamination sources of trace metals. The order of potential ecological risks of trace metals in this area are as follows: cadmium (Cd) > arsenic (As) > copper (Cu) > nickel (Ni) > lead (Pb) > chromium (Cr) > zinc (Zn). Elemental spatial distribution maps showed the existence of zones heavily polluted by trace metals around the mining area. Earthworms and roots of three tree species were also heavily contaminated, with concentrations of trace metals in earthworms much higher than in previous studies. The potential ecological risk index and other soil quality indices indicated that soil had moderate to severe contamination and there were high ecological risks, with Cd making the greatest contribution. Multivariate statistical analyses showed that Cd, As, Cu, Pb, and Zn in soil came from a mining activity source, whereas Ni and Cr primarily originated from a natural source.

Coelomic Fluid of Lumbricus rubellus Synergistically Enhances Cytotoxic Effect of 5-Fluorouracil through Modulation of Focal Adhesion Kinase and p21 in HT-29 Cancer Cell Line.

Coelomic fluid of Lumbricus rubellus (CFL) has attracted interest due to its pharmacological properties, including antitumor effect. Furthermore, it is necessary to evaluate the response to treatment with new cancer therapeutic agents. This study aims to investigate whether the combination of CFL and 5-fluorouracil could reduce FAK protein level and iCa2+ and enhance p21 level. Furthermore, it is necessary to evaluate the response to treatment with new cancer therapeutic agents. After 24 hours of treatment, it was necessary to assess the percentage of apoptosis, FAK, and p21 protein expression by flow cytometry. iCa2+ concentration was measured using immunofluorescence. The combination therapy of CFL with 5-fluorouracil potently suppressed six treatment groups were included in this study. HT-29 cell lines were cultured and divided into six groups: group 1 was treated with vehicle (negative control), groups 2-5 were treated with 5-fluorouracil, groups 3-5 were treated with either CFL 5, 10, or 20 µg/ml immediately after 5-fluorouracil, and group 6 was treated with CFL 20 µg/ml, the progression of colorectal cancer. Combination of CFL and 5-fluorouracil significantly decreased FAK expression (p<0.05), iCa2+ (p<0.05), and increased p21 expression (p<0.05) in HT-29 cells. Our results suggest that CFL has an anticancer potential in colorectal cancer when combined with 5-fluorouracil.

Effects of the Chiral Fungicides Metalaxyl and Metalaxyl-M on the Earthworm Eisenia fetida as Determined by ¹H-NMR-Based Untargeted Metabolomics.

Although metalaxyl and metalaxyl-M are widely used fungicides, very little is known about their subacute and enantiospecific effects on the earthworm metabolome. In this study, Eisenia fetida were exposed to metalaxyl and metalaxyl-M at three concentrations (0.5, 5 and 50 mg/kg) for seven days. ¹H nuclear magnetic resonance (¹H-NMR)-based untargeted metabolomics showed that metalaxyl and metalaxyl-M exposure disturbed earthworms' metabolism at all three concentrations. Endogenous metabolites, such as succinate, arginine, aspartate, urea, asparagine, alanine, trimethylamine, taurine, cysteine, serine, threonine, histidine, lysine, glucose, choline, carnitine, citric acid, alpha-ketoisovaleric acid, fumaric acid and so on, were significantly changed. These results indicate that metalaxyl and metalaxyl-M produce different, enantiospecific disturbances in the earthworm metabolism, particularly in the tricarboxylic acid (TCA) and urea cycles. The application of untargeted metabolomics thus provides more information for evaluating the toxic risks of metalaxyl and metalaxyl-M.

Trace Elements in Surface Soils and Megascolecidae Earthworms in Urban Forests Within Four Land-Uses Around Poughkeepsie, New York, USA.

Amynthas agrestis and Metaphire hilgendorfi are being distributed across North America with unknown ecosystem impacts. Forest soils in urban areas sequester trace elements and earthworms may be bioaccumulating them. This study examined Cd, Cr, Co, Cu, Mn, Ni, Pb, V, and Zn in soils and earthworm tissues at 28 urban forest sites in and surrounding Poughkeepsie, NY, USA. Megascolecidae were present at 22 sites with means of 12 to 27 individuals m-2 and 4 to 12 dry weight g m-2. Urban forest soils within commercial uses had Mn, Pb, and Zn concentrations higher than within residential and agricultural uses. Earthworm trace element concentrations were poorly predicted by their respective soil concentrations, except for Pb. Urban forests in commercial uses and land-preserves, earthworm Cd and Pb concentrations were at or above concentrations known to negatively impact small mammal and bird health ( > 10 mg kg-1) with Co and V approaching toxic concentrations.

[Study on proteins in Guangdilong by nano LC/orbitrap fusion lumos HR-MS].

In the present study,fresh Guangdilong( GD),originating from Pheretima aspergillum,was taken as the object. The total proteins from GD were firstly separated by SDS-PAGE according to their molecular weights and in-gel digestion was then performed.After that,the peptides were analyzed by nano LC/orbitrap fusion lumos high resolution mass spectrometry( nano LC/orbitrap fusion lumos HR-MS). Protein identification was implemented by comparison with Annelida. fasta database using Proteome Discoverer software.As a result,386 proteins were tentatively identified,including chain F,globin B chain,glyceraldehyde-3-phosphate dehydrogenase,fibrinolytic protein,and so on. Most of the proteins took part in cell structure and energy metabolism,and fibrinolytic protein and lombricine kinase might be related to fibrinolytic activity. Protein classification based on gene ontology was carried out using PANTHER and KEGG for metabolic pathway enrichment. The results indicated that these proteins were related to diverse signal transduction pathways,including metabolic pathways,central carbon metabolism,biosynthesis of amino acids,ribosome,glycolysis,citrate cycle( TCA cycle),and so on. This study would lay the foundation for the further research on the proteins in GD and also their functions.

Metabolic Profiling of Chloroacetanilide Herbicides in Earthworm Coelomic Fluid Using 1H NMR and GC-MS.

Earthworms ( Eisenia fetida) are vital members of the soil environment. Because of their sensitivity to many contaminants, monitoring earthworm metabolism may be a useful indicator of environmental stressors. Here, metabolic profiles of exposure to five chloroacetanilide herbicides and one enantiomer (acetochlor, alachlor, butachlor, racemic metolachlor, S-metolachlor, and propachlor) are observed in earthworm coelomic fluid using proton nuclear magnetic resonance spectroscopy (NMR) and gas chromatography-mass spectrometry (GC-MS). Multiblocked-orthogonal partial least-squares-discriminant analysis (MB-OPLS-DA) and univariate analysis were used to identify metabolic perturbations in carnitine biosynthesis, carbohydrate metabolism, lipid metabolism, nitrogen metabolism, and the tricarboxylic acid cycle. Intriguingly, stereospecific metabolic responses were observed between racemic metolachlor and S-metolachlor exposed worms. These findings support the utility of coelomic fluid in monitoring metabolic perturbations induced by chloroacetanilide herbicides in nontarget organisms and reveal specificity in the metabolic impacts of herbicide analogues in earthworms.

Development of a method for the simultaneous determination of multi-class pesticides in earthworms by liquid chromatography coupled to tandem electrospray mass spectrometry.

Agricultural intensification, and in particular the use of pesticides, leads over the years to a loss of biodiversity and a decline of ecosystem services in cultivated zones and agricultural landscapes. Among the animal communities involved in the functioning of agro-ecosystems, earthworms are ubiquitous and recognized as indicators of land uses and cultural practices. However, little data is available on the levels of pesticides in such organisms in natura, which would allow estimating their actual exposure and the potentially resulting impacts. Thus, the objective of this study was to develop a sensitive analytical methodology to detect and quantify 27 currently used pesticides in earthworms (Allolobophora chlorotica). A modified QuEChERS extraction was implemented on individual earthworms. This step was followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The whole analytical method was validated on spiked earthworm blank samples, with regard to linearity (from 1 to 100 method limit of quantification, r2 > 0.95), intra-day precision (relative standard deviation (RSD) < 15%), inter-day precision (RSD < 20%), recoveries (mainly in the range 70-110%), and limits of detection and of quantification (inferior to 5 ng/g for most of the pesticides). The developed method was successfully applied to determine the concentrations of pesticides in nine individuals collected in natura. Up to five of the selected pesticides have been detected in one individual. Graphical abstract.

High-throughput multipesticides residue analysis in earthworms by the improvement of purification method: Development and application of magnetic Fe3 O4 -SiO2 nanoparticles based dispersive solid-phase extraction.

As a key representative organism, earthworms can directly illustrate the influence of pesticides on environmental organisms in soil ecosystems. The present work aimed to develop a high-throughput multipesticides residue analytical method for earthworms using solid-liquid extraction with acetonitrile as the solvent and magnetic material-based dispersive solid-phase extraction for purification. Magnetic Fe3 O4 nanoparticles were modified with a thin silica layer to form Fe3 O4 -SiO2 nanoparticles, which were fully characterized by field-emission scanning electron microscopy, transmission electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffractometry, and vibrating sample magnetometry. The Fe3 O4 -SiO2 nanoparticles were used as the separation media in dispersive solid-phase extraction with primary secondary amine and ZrO2 as the cleanup adsorbents to eliminate matrix interferences. The amounts of nanoparticles and adsorbents were optimized for the simultaneous determination of 44 pesticides and six metabolites in earthworms by liquid chromatography with tandem mass spectrometry. The method performance was systematically validated with satisfactory results. The limits of quantification were 20 µg/kg for all analytes studied, while the recoveries of the target analytes ranged from 65.1 to 127% with relative standard deviation values lower than 15.0%. The developed method was subsequently utilized to explore the bioaccumulation of bitertanol in earthworms exposed to contaminated soil, verifying its feasibility for real sample analysis.