Transcriptional upregulation of multiple earthworm chitinase genes following bacterial challenge suggests their implications in innate immunity.

BACKGROUND: Chitinase is a multi-functional enzyme that catalyzes the hydrolysis of ß-1,4-linkages between N-acetylglucosamines (GlcNAc) in chitin. Recent studies imply that earthworm chitinase is implicated in self-defense immunity against chitin-containing pathogens. However, a direct relationship of earthworm chitinase with innate immunity has not yet been established. OBJECTIVE: In this study, earthworm (Eisenia andrei) chitinase expression was examined following bacterial challenge by Bacillus subtilis. METHODS: RNA sequencing (RNA-seq) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to quantitatively evaluate mRNA expression changes in response to bacterial stimulation. RESULTS: Multiple chitinase-related mRNAs were found to be upregulated, among which EaChi3, EaChi4, and EaChi2 were upregulated by approximately eightfold, eightfold, and 2.5-fold, respectively. This strongly suggested that earthworm chitinases may act as inducible humoral effectors in earthworm innate immunity. The primary structures of all three chitinases contained an N-terminal glycol_18 domain with two chitin-binding and chitin-catalyzing domains, and a C-terminal proline, glycine, serine, threonine (PGST)-rich domain. In addition, EaChi2 had a chitin-binding peritrophin-A domain at the end of the C-terminus with 5 cysteine residues possibly contributing two intradomain disulfide bonds. Multiple sequence alignment of the catalytic domain centers of glycol_18 domain displayed highly conserved chitin-binding and chitin-catalyzing domains in which three essential amino acid residues (D, D, E) for catalyzing activity are well conserved except EaChi4. The critical glutamic acid (E) residue was substituted for glutamine (Q) in EaChi4 indicating that it is devoid of catalytic activity. CONCLUSIONS: To our knowledge, this is the first report providing direct evidence that multiple earthworm chitinases are bacteria-responsive, strongly suggesting that earthworm chitinases are inducible humoral effectors in earthworm innate immunity. In addition, our results possibly suggest that earthworm EaChi4 may function as a pattern recognition molecule modulating the downstream immune pathway.

Arginase Activity in Eisenia andrei Coelomocytes: Function in the Earthworm Innate Response.

Arginase is the manganese metalloenzyme catalyzing the conversion of l-arginine to l-ornithine and urea. In vertebrates, arginase is involved in the immune response, tissue regeneration, and wound healing and is an important marker of alternative anti-inflammatory polarization of macrophages. In invertebrates, data concerning the role of arginase in these processes are very limited. Therefore, in the present study, we focused on the changes in arginase activity in the coelomocytes of Eisenia andrei. We studied the effects of lipopolysaccharide (LPS), hydrogen peroxide (H2O2), heavy metals ions (e.g., Mn2+), parasite infection, wound healing, and short-term fasting (5 days) on arginase activity. For the first time in earthworms, we described arginase activity in the coelomocytes and found that it can be up-regulated upon in vitro stimulation with LPS and H2O2 and in the presence of Mn2+ ions. Moreover, arginase activity was also up-regulated in animals in vivo infected with nematodes or experiencing segment amputation, but not in fasting earthworms. Furthermore, we confirmed that the activity of coelomocyte arginase can be suppressed by l-norvaline. Our studies strongly suggest that similarly to the vertebrates, also in the earthworms, coelomocyte arginase is an important element of the immune response and wound healing processes.

Identification and expression of adenosine deaminases acting on tRNA (ADAT) during early tail regeneration of the earthworm.

BACKGROUND: RNA editing is a widespread phenomenon in all metazoans. One of the common RNA editing event is the chemical conversion of adenosine to inosine (A-to-I) catalyzed by adenosine deaminases acting on tRNA (ADAT). During D. melanogaster development, the ADAT1 transcript was found to localize mainly to the central nervous system including brain and ventral nerve cord during brain development. Although an earthworm adenosine deaminases acting on mRNA (ADAR) has been identified and its possible implication in earthworm regeneration has been investigated, there is little accumulated information on ADAT and tRNA editing in the annelid including terrestrial earthworms. OBJECTIVE: This study aimed to investigate the molecular characteristics and the expression pattern of earthworm ADAT during tail regeneration to understand its physiological significance. METHODS: Nucleotide sequence of Ean-ADAT was retrieved from the genome assembly of Eisenia andrei via Basic Local Alignment Search Tool (BLAST). The genome assembly of Eisenia andrei was downloaded from National Genomics Data Center ( http://bigd.big.ac.cn/gwh/ ). The alignment and phylogenetic relationship of the core deaminase domains of ADATs and ADARs were analyzed. Its temporal expression during early tail regeneration was measured using real-time PCR. RESULTS: The open reading frame of Ean-ADAT consists of 1719 nucleotides encoding 573 amino acids. Domain analysis indicates that Ean-ADAT has a deaminase domain composed of 498 amino acids and a predicted nuclear localization signal at the N-terminal. Its subcellular localization was predicted to be nuclear. The core deaminase region of Ean-ADAT encompasses the three active-site motifs, including zinc-chelating residues and a glutamate residue for catalytic activity. In addition, Ean-ADAT shares highly conserved RNA recognition region flanking the third cysteine of the deaminase motif with other ADAT1s even from the yeast. Multiple sequence alignment and phylogenetic analysis indicate that Ean-ADAT shows greater similarity to vertebrate ADARs than to yeast Tad1p. Ean-ADAT mRNA expression began to remarkably decrease before 12 h post-amputation, showing a tendency to gradual decrease until 7 dpa and then it slightly rebounded at 10 dpa. CONCLUSIONS: Our results demonstrate that Ean-ADAT belongs to a class of ADAT1s and support the hypothesis of a common evolutionary origin for ADARs and ADATs. The temporal expression of Ean-ADAT could suggest that its activity is unrelated to the molecular mechanisms of dedifferentiation.

Acute toxicity and sublethal effects of sodium laureth sulfate on oxidative stress enzymes in benthic oligochaete worm, Tubifex tubifex.

The present study was performed to determine the acute toxicity of sodium laureth sulfate (SLES) and its sublethal effects on oxidative stress enzymes in benthic oligochaete worm Tubifex tubifex. The results showed that 96 h median lethal concentration (LC50) value of SLES for Tubifex tubifex is 21.68 mg/l. Moreover exposed worms showed abnormal behaviours including incremented erratic movement, mucus secretion, and decreased clumping tendency at acute level. Percentage of autotomy additionally increased significantly (P < 0.05) with the increasing dose of toxicant at 96 h exposure. Sublethal concentrations of SLES (10% and 30% of 96 h LC50 value) caused paramount alterations in the oxidative stress enzymes. Superoxide dismutase (SOD), reduced glutathione (GSH), glutathione S-transferase (GST), and glutathione peroxidase (GPx) exhibited a striking initiatory increment followed by a resulting descending pattern. Moreover, during exposure times, catalase (CAT) activity and malondialdehyde (MDA) level increased markedly with incrementing concentrations of SLES. However, the effects of sodium laureth sulfate on Tubifex tubifex were characterized and portrayed by the development of a correlation matrix and an integrated biomarker response (IBR) assessment. These results indicate that exposure to this anionic surfactant alters the survivability and behavioral response at acute level and modifies changes in oxidative stress enzymes at sublethal level in Tubifex tubifex.

Toxic mechanism of pyrene to catalase and protective effects of vitamin C: Studies at the molecular and cell levels.

Polycyclic aromatic hydrocarbons, distributing extensively in the soil, would potentially threaten the soil organisms (Eisenia fetida) by triggering oxidative stress. As a ubiquitous antioxidant enzyme, catalase can protect organisms from oxidative damage. To reveal the potential impact of polycyclic aromatic hydrocarbon pyrene (Pyr) on catalase (CAT) and the possible protective effect of Ascorbic acid (vitamin C), multi-spectral and molecular docking techniques were used to investigate the influence of structure and function of catalase by pyrene. Fluorescence and circular dichroism analysis showed that pyrene would induce the microenvironmental changes of CAT amino acid residues and increase the α-helix in the secondary structure. Molecular simulation results indicated that the main binding force of pyrene around the active center of CAT is hydrogen bonding force. Furthermore, pyrene inhibited catalase activity to 69.9% compared with the blank group, but the degree of inhibition was significantly weakened after vitamin C added into the research group. Cell level experiments showed that pyrene can increase the level of ROS in the body cavity cell of earthworms, and put the cells under the threat of potential oxidative damage. Antioxidants-vitamin C has a protective effect on catalase and maintains the stability of intracellular ROS levels to a certain extent.

Acute toxicity of organophosphate pesticide profenofos, pyrethroid pesticide λ cyhalothrin and biopesticide azadirachtin and their sublethal effects on growth and oxidative stress enzymes in benthic oligochaete worm, Tubifex tubifex.

The present study was aimed to assess the acute toxicity of organophosphate pesticide, profenofos; synthetic pyrethroid pesticide, λ cyhalothrin and biopesticide, azadirachtin and their sublethal effects on growth rate and oxidative stress biomarkers in Tubifex tubifex in vivo. The results showed that 96 h LC50 value of profenofos, λ cyhalothrin and azadirachtin to Tubifex tubifex are 0.59, 0.13 and 82.15 mg L-1 respectively. Pesticide treated worms showed several behavioral abnormalities including increased mucus secretion, erratic movements, wrinkling activity and decreased clumping tendency during acute exposure. The percentage of autotomy increased significantly (p < 0.05) with the increasing concentration of the pesticides at 96 h of exposure. Sublethal concentrations of profenofos (0.059 and 0.118 mg L-1), λ cyhalothrin (0.013 and 0.026 mg L-1) and azadirachtin (8.2 and 16.4 mg L-1) caused significant alterations in growth rate and oxidative stress enzymes in T. tubifex during 14 days exposure period. The growth rate of the pesticide exposed worms decreased significantly (P < 0.05) in a concentration and duration-dependent manner. Superoxide dismutase (SOD), reduced glutathione (GSH), glutathione-s-transferase (GST) and glutathione peroxidase (GPx) demonstrated a noteworthy (p < 0.05) initial induction followed by a subsequent reduction, while catalase (CAT) and malondialdehyde (MDA) exhibited noteworthy induction (p < 0.05) all through the exposure time. Through principal component analysis, correlation matrix, and integrated biomarker response, the effects of profenofos, λ cyhalothrin and azadirachtin on T. tubifex were distinguished. These results indicate that exposure to profenofos, λ cyhalothrin and azadirachtin affect survivability, change the behavioral responses, reduce the growth rate and induce oxidative stress enzymes in T. tubifex.

Elucidating pesticide sensitivity of two endogeic earthworm species through the interplay between esterases and glutathione S-transferases.

Earthworms are common organisms in soil toxicity-testing framework, and endogeic species are currently recommended due to their ecological role in agroecosystem. However, little is known on their pesticide metabolic capacities. We firstly compared the baseline activity of B-esterases and glutathione-S-transferase in Allolobophora chlorotica and Aporrectodea caliginosa. Secondly, vulnerability of these species to pesticide exposure was assessed by in vitro trials using the organophosphate (OP) chlorpyrifos-ethyl-oxon (CPOx) and ethyl-paraoxon (POx), and by short-term (7 days) in vivo metabolic responses in soil contaminated with pesticides. Among B-esterases, acetylcholinesterase (AChE) activity was abundant in the microsomal fraction (80% and 70% of total activity for A. caliginosa and A. chlorotica, respectively). Carboxylesterase (CbE) activities were measured using three substrates to examine species differences in isoenzyme and sensitivity to both in vitro and in vivo exposure. CbEs were mainly found in the cytosolic fraction (80% and 60% for A. caliginosa and A. chlorotica respectively). GST was exclusively found in the soluble fraction for both species. Both OPs inhibited B-esterases in a concentration-dependent manner. In vitro trials revealed a pesticide-specific response, being A. chlorotica AChE more sensitive to CPOx compared to POx. CbE activity was inhibited at the same extent in both species. The 7-d exposure showed A. chlorotica less sensitive to both OPs, which contrasted with outcomes from in vitro experiments. This non-related functional between both approaches for assessing pesticide toxicity suggests that other mechanisms linked with in vivo OP bioactivation and excretion could have a significant role in the OP toxicity in endogeic earthworms.

X-ray crystallographic structural studies of α-amylase I from Eisenia fetida.

The earthworm Eisenia fetida possesses several cold-active enzymes, including α-amylase, ß-glucanase and ß-mannanase. E. fetida possesses two isoforms of α-amylase (Ef-Amy I and II) to digest raw starch. Ef-Amy I retains its catalytic activity at temperatures below 10°C. To identify the molecular properties of Ef-Amy I, X-ray crystal structures were determined of the wild type and of the inactive E249Q mutant. Ef-Amy I has structural similarities to mammalian α-amylases, including the porcine pancreatic and human pancreatic α-amylases. Structural comparisons of the overall structures as well as of the Ca2+-binding sites of Ef-Amy I and the mammalian α-amylases indicate that Ef-Amy I has increased structural flexibility and more solvent-exposed acidic residues. These structural features of Ef-Amy I may contribute to its observed catalytic activity at low temperatures, as many cold-adapted enzymes have similar structural properties. The structure of the substrate complex of the inactive mutant of Ef-Amy I shows that a maltohexaose molecule is bound in the active site and a maltotetraose molecule is bound in the cleft between the N- and C-terminal domains. The recognition of substrate molecules by Ef-Amy I exhibits some differences from that observed in structures of human pancreatic α-amylase. This result provides insights into the structural modulation of the recognition of substrates and inhibitors.

Shift in phagocytosis, lysosomal stability, lysozyme activity, apoptosis and cell cycle profile in the coelomocytes of earthworm of polluted soil near a tannery field of India.

Calcutta Leather Complex of the state of West Bengal, India has been designated as an industrially active zone with around 400 active tannery units. This area spanning 4.5 km2 is surrounded by human habitation. The soil of this region is contaminated with metal pollutants and exhibited an alteration in selected physicochemical parameters, namely cation exchange capacity, moisture content, pH, total nitrogen, total organic carbon and water holding capacity. Metaphire posthuma, a common variety of endogeic earthworm inhabiting this region is thus continuously exposed to these toxic metals. Coelomocytes, the chief immune effector cells of earthworm presented a shift in phagocytosis, lysosomal membrane stability, lysozyme and phosphatase activity, physiological apoptosis and cell cycle profile of M. posthuma sampled from the soil of tannery industry. Presence of high concentration of toxic metals and change in the physicochemical characteristics of soil led to a state of cellular stress and immunocompromisation in M. posthuma, a common inhabitant of soil of this region. Experimental endpoints bear ecotoxicological significance as biomarkers of physiological stress in earthworm for monitoring the health of soil around this tannery industrial zone.

Benzotriazole alleviates copper mediated lysosomal membrane damage and antioxidant defense system responses in earthworms (Eisenia fetida).

Benzotriazole (BTR) is a common corrosion inhibitor used to protect copper (Cu) and Cu alloys. To reveal the combined subacute toxicity of BTR and Cu at environmental levels on terrestrial animals, the activity of antioxidative enzymes and the glutathione levels in earthworms (Eisenia fetida) of the single or co-exposure treatments were determined. The activity of both antioxidant enzymes and non-enzymatic antioxidants was affected by BTR in earthworms. Moreover, the analyses of lysosomal neutral red retention time and total antioxidant capacity indicated a detoxification effect of BTR on Cu-induced impairments of the antioxidant defense capacity in earthworms. The apoptotic rate of coelomocytes in earthworms of the co-exposure treatment was lower than that in earthworms treated with Cu only, indicating that BTR alleviates Cu mediated lysosomal membrane damage and antioxidant defense system responses in earthworms.

Antioxidant Enzyme Activity and Lipid Peroxidation in Aporrectodea caliginosa Earthworms Exposed to Silver Nanoparticles and Silver Nitrate in Spiked Soil.

Silver nanoparticles (AgNPs) from industrial use, discharged via the land application of sewage sludge, are interacting with soil biota, including earthworms. In affected organisms, excessive production of reactive oxygen species can result in lipid peroxidation, shifting the balance between oxidants and antioxidants to cause oxidative stress. We determined selected lower-tier biomarkers such as antioxidant responses and lipid peroxidation in Aporrectodea caliginosa earthworms exposed to soils spiked with AgNPs or silver nitrate (AgNO3 ). Aporrectodea caliginosa were exposed to AgNPs at 0 (control), 0.3, 3, 30, and 300 mg/kg or Ag+ (as AgNO3 ) at 0, 0.03, 0.3, 3, and 10 mg/kg in soil for 4 wk. At 1, 2, 3, and 4 wk, the activity of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, as well as lipid peroxidation (malondialdehyde content), increased as a function of concentration, with a much larger response for Ag+ than AgNPs. Given the likelihood of ever-increasing AgNP concentrations in soil, where AgNPs can transform to ionic Ag (Ag+ ), our findings of antioxidant response to oxidative stress in a common indicator organism even at an environmentally realistic exposure concentration of 0.03 mg/kg demonstrate that AgNPs may affect soil fertility and, thus, agricultural production. Evaluating selected lower-tier biomarkers offers a meaningful assessment of AgNPs and Ag+ effects on terrestrial earthworms. Environ Toxicol Chem 2020;39:1257-1266. © 2020 SETAC.

Structural Evolution of the Glacier Ice Worm Fo ATP Synthase Complex.

The segmented annelid worm, Mesenchytraeus solifugus, is a permanent resident of temperate, maritime glaciers in the Pacific northwestern region of North America, displaying atypically high intracellular ATP levels which have been linked to its unusual ability to thrive in hydrated glacier ice. We have shown previously that ice worms contain a highly basic, carboxy terminal extension on their ATP6 regulatory subunit, likely acquired by horizontal gene transfer from a microbial dietary source. Here we examine the full complement of F1F0 ATP synthase structural subunits with attention to non-conservative, ice worm-specific structural modifications. Our genomics analyses and molecular models identify putative proton shuttling domains on either side of the F0 hemichannel, which predictably function to enhance proton flow across the mitochondrial membrane. Other components of the ice worm ATP synthase complex have remained largely unchanged in the context of Metazoan evolution.

Effect of phenanthrene on the biological characteristics of earthworm casts and their relationships with digestive and anti-oxidative systems.

Earthworms as ecosystem engineers partially improve soil properties by egesting casts. Our previous study confirmed that soil pollution affects the physico-chemical properties of earthworm casts. It is still unclear whether the biological properties (e.g. cellulase, urease, and acid and alkaline phosphatase activities, as well as microbial biomass carbon) of casts are affected by foreign substances in soil. The present study aimed to investigate the effect of phenanthrene (PHE) on the biological characteristics of earthworm (Eisenia fetida) casts. Furthermore, correlations between cast properties and the digestive and antioxidant systems were explored by the determination of digestive enzyme (urease, protease, acid and alkaline phosphatase) activities, antioxidant indexes [superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD)], as well as malondialdehyde (MDA) content. Exposure at a range of PHE doses (0, 2, 5, 10, and 20 mg kg-1) for 15 d resulted in the following observations: (1) Compared with urease, as well as acid and alkaline phosphatase activities, cellulase activities in both soil and casts were sensitive to PHE, and could potentially act as biomarkers to provide early-warning signals for soil pollution. (2) Microbial biomass carbon in casts was modified, but with no clear pattern. (3) Cellulase and POD activities, as well as MDA content in earthworms, increased with elevated exposure to PHE in soil. Protease, SOD, and CAT activities exhibited a biphasic dose response to PHE, while acid and alkaline phosphatase activities were inhibited under treatment conditions. (4) Correlation analysis suggested that microbial biomass carbon in casts significantly and positively correlated with cellulase and acid phosphatase activities of earthworms, but negatively correlated with protease activities. A significant but weak negative correlation between alkaline phosphatase activities in casts and POD activities was also observed. Based on these results, we concluded that PHE content in soil modified some biological properties of casts, by partially affecting the earthworm's digestive and antioxidant systems. This study advances our knowledge of earthworm ecology in polluted soil by providing a better understanding of their ecological functions.

Upregulation of cellulase activity and mRNA levels by bacterial challenge in the earthworm Eisenia andrei, supporting the involvement of cellulases in innate immunity.

To investigate whether earthworm cellulases contribute to the innate immune system, the responsiveness of cellulase activity and mRNA expression to bacterial challenge was examined by zymography and RNA sequencing. A zymographic analysis revealed that the activity levels of earthworm cellulases were upregulated in response to either a bacterial (Bacillus subtilis or Escherichia coli) or LPS challenge. After the challenge, significant increases in cellulase 1 and cellulase 2 activity levels were observed within 8-16 and 16-24 h, respectively. In the coelomic fluid, both activities were significantly upregulated at 8 h post-injection with B. subtilis. Based on RNA sequencing, cellulase-related mRNAs encoding beta-1,4-endoglucanases were upregulated by 3-fold within 6 h after B. subtilis injection. Our results clearly demonstrated that earthworm cellulases are upregulated by bacterial challenge at the mRNA and protein levels. These results support the view that earthworm cellulases act as inducible humoral effectors of innate immunity against bacterial infection.

[Effects of enrofloxacin and Cu combined pollution on the activities of digestive enzymes of earthworm in soil.] / æ©è¯ºæ²™æ˜Ÿå’Œé“œå¤åˆæ±¡æŸ“å¯¹èš¯èš“æ¶ˆåŒ–é ¶æ´»æ€§çš„å½±å“.

The effects of single and combined pollution of enrofloxacin and Cu on the digestive enzymes of earthworms were studied, based on the actual pollution of caused by the application of livestock feces in farmland soil. Results showed that single enrofloxacin (0.1-4 mg·kg-1, 28 d) did not affect protease, but inhibited cellulase and alkaline phosphatase, with an induced effect on acid phosphatase. Single Cu pollution (20-200 mg·kg-1, 28 d) had inhibitory effects on the four digestive enzymes in earthworms. The effects of combined exposures on the digestive enzymes were mainly negative, showing a synergistic increasing character of toxicity in cellulase and acid phosphatase activities. The response dynamics of digestive enzymes to exposure duration was regulatory response (3 d)-intense response (7 d)-reaction recovery (14 d)-chronic exposure (28 d). Chronic exposure results showed that the combined treatments containing high-dose pollutant (200 mg·kg-1 Cu or 4 mg·kg-1 ENR) had more ecological risk.

Comparison of Earthworm Bioluminescent Systems.

The results of a comparative study of the luciferin-luciferase systems of seven species of bioluminescing oligochaetes-Henlea petushkovi, Henlea rodionovae, Fridericia heliota (Enchytraeidae), Microscolex phosphoreus (Acanthodrilidae), Pontodrilus litoralis (Megascolecidae), Eisenia lucens, and Avelona ligra (Lumbricidae)-are presented.

Phthalate induced oxidative stress and DNA damage in earthworms (Eisenia fetida).

Phthalates (phthalic acid esters) have been widely applied as plasticizers. They are ubiquitous contaminants in soils, thereby posing a threat to human health. In this study, ecotoxicological effects of three typical PAEs (dimethyl phthalate-DMP, di-n-octyl phthalate-DOP and butyl benzyl phthalate-BBP) were investigated. As a biological indicator, earthworms (Eisenia fetida) were exposed to phthalates at various doses (0, 0.1, 1, 10 and 50 mg/kg) for different times (7, 14, 21, and 28 d). We evaluated the effects of phthalates on reactive oxygen species (ROS) generation, antioxidant enzymes (superoxide dismutase-SOD, peroxidase-POD and catalase-CAT) activities, glutathione S-transferase enzyme (GST) activity, malondialdehyde (MDA) content and DNA damage. Results showed that ROS content increased with increasing phthalates, whereas ROS content generally increased and then decreased with exposure time. However, antioxidant enzymes activities in earthworms displayed different trends. The GST activity in high-dose treatment group was significantly activated. For DMP and DOP, lipid peroxidation mainly occurred between 14 and 28 d, while for BBP, it primarily existed after 7 d and then disappeared after 28 d. Besides, comet assay indicated that there was a dose-response relationship between the DNA damage and phthalate dose, following DMP > DOP > BBP. Given their toxicity, it is important to understand the mechanisms associated with their eco-toxicity and to reduce their adverse impacts on the environment.

Toxic effects of new ethyl-carbamates on the morphology, mortality and acetylcholinesterase activity of Eisenia foetida.

The toxicity of the ixodicidal carbamates ethyl-4-bromophenyl carbamate (LQM 919), ethyl-4-chlorophenyl carbamate (LQM 996) and propoxur on Eisenia foetida adults was evaluated to estimate their ecotoxic potential. The earthworm mortality and weight loss produced by the three evaluated carbamates showed a concentration-dependent effect (p < 0.0001) in the contact filter paper test (CFPT). In the artificial soil test (AST), mortality increased in relation to the exposure time (p < 0.0001) and the concentration (p < 0.01) of the carbamates. Only the earthworms exposed in the CFPT showed morphological alterations. According to the LC50 obtained in the CFPT, the three carbamates were classified as very toxic and, according to the LC50 obtained in the AST, the three carbamates were classified as highly toxic for E. foetida. The values of ki and kd indicated that LQM 919 and LQM 996 are weak inhibitors with lower affinity for the acetylcholinesterase of E. foetida than that of propoxur. The concentrations in the CFPT and AST at which 100% mortality was observed in E. foetida were 64- and 4-fold higher, respectively, than the egg hatching inhibitory concentration 99% reported for ticks.

Biochar increases pesticide-detoxifying carboxylesterases along earthworm burrows.

Herein, we examined whether synergistic effects of earthworms (Lumbricus terrestris) and pine needle-derived biochar result in biochar-coated burrows with enhanced carboxylesterase (CE) activity (a pesticide-detoxifying enzyme). Biochar was placed at the top of soil columns at two doses (2.5 and 5% w/w dry mass), with an additional biochar-free treatment as control. Carboxylesterase and dehydrogenase activities were measured in the burrow walls sampled at three depths (0-4, 8-12, and 18-22 cm). Biochar was recovered from these samples to confirm its vertical transport and enzymatic activation. We tested whether biochar protected CE activity against desiccation stress of burrow wall samples. Likewise, the role of earthworm mucus in stabilizing CE onto biochar surface was also investigated by measuring the enzyme activity in fresh biochar particles previously incubated in the presence of earthworm mucus and purified esterase. Finally, we checked for the sensitivity of biochar-bound CE activity against selected organophosphorus pesticides. The main results were: i) co-application of earthworms and biochar caused a significant increase of CE activity in the first 12 cm of the soil column, ii) recovered biochar particles displayed CE activity which was significantly higher in the particles collected from the bottom of columns, iii) soil desiccation decreased the enzymatic activity, although such effect depended on biochar treatment and column depth (22-77% inhibition). Nevertheless, CE activity still was higher in the samples from the 5%-biochar treatment than activity in control and 2.5%-biochar treatments, iv) earthworm mucus favored the retention of CE onto the biochar surface, and v) the activity of biochar-bound CE was sensitive to inhibition by chlorpyrifos-oxon. These results suggest that the joint application of L. terrestris and biochar may be a suitable vermiremediation strategy to inactivate OP pesticides.

Effects of biochar on the earthworm (Eisenia foetida) in soil contaminated with and/or without pesticide mesotrione.

The plant-derived biochars act as soil conditioners, and thus may influence biological interactions in the soil environment. However, their unintended negative and positive effects on soil organisms remain largely understudied. Therefore, we investigated the effect of 0, 1, 3, and 10% of wheat straw-derived biochar amendments on earthworm (Eisenia foetida) activity in the soil contaminated with and/or without pesticide mesotrione (10 mg/kg dry soil) after 28 days of incubation. The pesticide mesotrione did not affect earthworm growth or reproduction; however, it induced oxidative stress and DNA damage. Although biochar application significantly decreased the concentration of mesotrione in earthworms, it delayed the degradation of pesticide in the soil environment. Compared to zero amendment, the amendment of 1 and 3% of biochar significantly increased (P < 0.05) the earthworm weight and reduced the toxicity effects of mesotrione on earthworms. However, the application of 10% biochar significantly decreased (P < 0.05) earthworm growth and caused DNA damage even in the absence of mesotrione. This study suggests that it is necessary to investigate the effects of different levels of biochar amendments on earthworms and other soil organisms in agricultural fields to develop a broader understanding about the use of biochar and its consequences on soil health.