Study on the virulence of Metarhizium anisopliae against Spodoptera frugiperda (J. E. Smith, 1797).

This study examined the impact of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) conidia on the eggs, larvae, pupae, and adults of Spodoptera frugiperda. The results showed that eggs, larvae, pupae, and adults exhibited mortality rates that were dependent on the dose. An increased amount of conidia (1.5 × 109 conidia/mL) was found to be toxic to larvae, pupae, and adults after 9 days of treatment, resulting in a 100% mortality rate in eggs, 98% in larvae, 76% in pupae, and 85% in adults. A study using earthworms as bioindicators found that after 3 days of exposure, M. anisopliae conidia did not cause any harmful effects on the earthworms. In contrast, the chemical treatment (positive control) resulted in 100% mortality at a concentration of 40 ppm. Histopathological studies showed that earthworm gut tissues treated with fungal conidia did not show significant differences compared with those of the negative control. The gut tissues of earthworms treated with monocrotophos exhibited significant damage, and notable differences were observed in the chemical treatment. The treatments with 70 and 100 µg/mL solutions of Eudrilus eugeniae epidermal mucus showed no fungal growth. An analysis of the enzymes at a biochemical level revealed a decrease in the levels of acetylcholinesterase, α-carboxylesterase, and ß-carboxylesterase in S. frugiperda larvae after exposure to fungal conidia. This study found that M. anisopliae is effective against S. frugiperda, highlighting the potential of this entomopathogenic fungus in controlling this agricultural insect pest.

Effects of cadmium and pyrene on earthworm-associated bacterial communities: Unveiling new perspectives for soil pollution management.

Earthworms are considered to be excellent bioindicators of soil pollution. In recent years, there has been increasing interest in examining the effects of soil pollution on earthworm-associated microbiomes, with a particular focus on the gut microbiomes. However, relatively little effort has been invested in comprehensively investigating other microbiomes associated with earthworms and their responses to soil pollution. To fill this gap, we systematically studied the effects of Cd, pyrene, and combined pollution on the bacterial community in different vermicompartments, i.e., burrow wall, gut, and cast, in both epigeic Eisenia fetida and anecic Metaphire guillelmi, using a 2D-terraria incubator and high-throughput sequencing techniques. The results showed that bacterial alpha diversity followed the order of burrow wall > cast > gut, and this did not vary with soil pollution or earthworm ecotypes. Moreover, the dominant phyla in the vermicompartments were similar across different pollution treatments. Principal coordinate analysis (PCoA) revealed that the bacterial communities in different vermicompartments and ecotypes of earthworm were separated from each other, whereas they were grouped together in polluted treatments and unpolluted conditions. These results imply that even in polluted soil, vermicompartment and earthworm ecotypes remain the most significant factors affecting earthworm-associated microbiomes. However, the impacts of soil pollution on the bacterial composition in each vermicompartment were still evident. A comprehensive analysis revealed that the gut bacterial communities are more sensitive to soil contamination than casts and burrow wall in different ecotypes. Additionally, linear discriminant analysis of effect size (LefSe) identified several bacteria in Gemmatimonadota, the Firmicutes phylum in the burrow walls, and Patescibacteria (phyla) in the gut as potential biomarkers for pyrene contamination in soil. This research provides a comprehensive understanding of the effects of soil pollution on earthworm-associated microbiomes, thereby enhancing our understanding of earthworm ecotoxicology and soil pollution management.

Dynamics of bacterial community in the foregut and hindgut of earthworms with the nutrition supplied by kitchen waste during vermicomposting.

Earthworm gut microbiota is vital in degrading bio-waste during vermicomposting. However, microbial dynamics in earthworm gut during this process are unclear. Thus, the aim is to firstly report the bacterial dynamics in both foregut and hindgut of earthworms over a 28 days' timeframe of vermicomposting by Eisenia foetida with the nutrition supplied by kitchen waste. Results showed that except the changing of the bacterial diversity, composition and structure, dynamics of the foregut and hindgut bacteria also differed during vermicomposting which related to the changes of nutrient provision. Day 3 was a turning point. The abundant bacteria of the top 20 % genera nearly did not overlap between the foregut and hindgut. In the end of vermicomposting, a remarkable stable bacterial structure appeared in the hindgut compared to somewhat muddled one in the foregut. Understanding the dynamics of earthworm gut microbiota enables the improvements to regulate the efficiency of organic waste vermicomposting.

Toxicity sharing model of earthworm intestinal microbiome reveals shared functional genes are more powerful than species in resisting pesticide stress.

Earthworm intestinal bacteria and indigenous soil bacteria work closely during various biochemical processes and play a crucial role in maintaining the internal stability of the soil environment. However, the response mechanism of these bacterial communities to external pesticide disturbance is unknown. In this study, soil and earthworm gut contents were metagenomically sequenced after exposure to various concentrations of nitrochlorobenzene (0-1026.7 mg kg-1). A high degree of similarity was found between the microbial community composition and abundance in the worm gut and soil, both of which decreased significantly (P < 0.05) under elevated pesticide stress. The toxicity sharing model (TSM) showed that the toxicity sharing capacity was 97.4-125.7 % and 100.4-130.2 % for Egenes (genes in the worm gut) and Emet(degradation genes in the worm gut) in the earthworm intestinal microbiome, respectively. This indicated that the earthworm intestinal microbiome assisted in relieving the pesticide toxicity of the indigenous soil microbiome. This study showed that the TSM could quantitatively describe the toxic effect of pesticides on the earthworm intestinal microbiome. It provides a new analytical model for investigating the ecological alliance between earthworm intestinal microbiome and indigenous soil microbiome under pesticide stress while contributing a more profound understanding of the potential to use earthworms to mitigate pesticide pollution in soils and develop earthworm-based soil remediation techniques.

Metagenomic Insights into the Gut Microbiota of Eudrilus eugeniae (Kinberg) and Its Potential Roles in Agroecosystem.

Gut microbiomes, a consortium of microorganisms that live in the animal gut, are highly engineered microbial communities. It makes a major contribution to digestive health, metabolism management, and the development of a strong immune system in the host. The present study was taken up to answer the long-running question about the existence of truly indigenous microflora of the epigeic earthworm gut. This is due to the general difficulties of culturing many of the microorganisms found in soil or earthworms' gut. Keeping this fact in a view, the metagenomics approach using 16S rRNA marker gene incorporated with amplicon-based sequencing was used to explore microbiota of commercially overriding, diversely fed epigeic earthworm Eudrilus eugeniae (Kinberg) in three varied habitats viz., artificial soil (AS), organic agricultural farm soil (OAFS) and conventional agriculture farm soil (CAFS). There are predominant bacteria that belong to different phyla such as Proteobacteria (29.72-76.81%), Actinobacteria (11.06-34.42%), Firmicutes (6.02-19.81%), and Bacteroidetes (2.40-9.22%) present in the gut of E. eugeniae. The alpha diversity (Observed species, Chao1, ACE, Shannon, Simpson, and Fisher alpha) indices showed that OAFS had significantly higher alpha diversity than AS and CAFS groups. The core microbiota analysis showed that OAFS and AS groups had a relatively similar bacterial panel in comparison to the CAFS group. Various statistical tools i.e. MetagenomeSeq, LEfSe, and Random Forest analysis were performed and the findings demonstrated prevalence of the most significant bacterial genera; Aeromonas, Gaiella, and Burkholderia in CAFS group. Nonetheless, in AS and OAFS groups, the common existence of Anaerosporobacter and Aquihabitans were found respectively. Metagenomic functional prediction revealed that earthworms' gut microbial communities were actively involved in multiple organic and xenobiotics compound degradation-related pathways. This is the first research to use high-throughput 16S rRNA gene amplicon sequencing to show the gut microbiota of E. eugeniae in diverse agricultural systems. The findings suggest the configuration of the gut microbiota of earthworms and its potential role in the soil ecosystem depends on the microbial communities of the soil.

Host taxonomy determines the composition, structure, and diversity of the earthworm cast microbiome under homogenous feeding conditions.

Host evolutionary history is a key factor shaping the earthworm cast microbiome, although its effect can be shadowed by the earthworm's diet. To untangle dietary from taxon effects, we raised nine earthworm species on a uniform diet of cow manure and compared cast microbiome across species while controlling for diet. Our results showed that, under controlled laboratory conditions, earthworm microbiomes are species-specific, more diverse than that of the controlled diet, and mainly comprised of native bacteria (i.e. not acquired from the diet). Furthermore, diet has a medium to large convergence effect on microbiome composition since earthworms shared 16%-74% of their bacterial amplicon sequence variants (ASV). The interspecies core microbiome included 10 ASVs, while their intraspecies core microbiomes were larger and varied in ASV richness (24%-48%) and sequence abundance across earthworm species. This specificity in core microbiomes and variable degree of similarity in bacterial composition suggest that phylosymbiosis could determine earthworm microbiome assembly. However, lack of congruence between the earthworm phylogeny and the microbiome dendrogram suggests that a consistent diet fed over several generations may have weakened potential phylosymbiotic effects. Thus, cast microbiome assembly in earthworms seem to be the result of an interplay among host phylogeny and diet.

Bacterial Keystone Taxa Regulate Carbon Metabolism in the Earthworm Gut.

As important ecosystem engineers in soils, earthworms strongly influence carbon cycling through their burrowing and feeding activities. Earthworms do not perform these roles in isolation, because their intestines create a special habitat favorable for complex bacterial communities. However, how the ecological functioning of these earthworm-microbe interactions regulates carbon cycling remains largely unknown. To fill this knowledge gap, we investigated the bacterial community structure and carbon metabolic activities in the intestinal contents of earthworms and compared them to those of the adjacent soils in a long-term fertilization experiment. We discovered that earthworms harbored distinct bacterial communities compared to the surrounding soil under different fertilization conditions. The bacterial diversity was significantly larger in the adjacent soils than that in the earthworm gut. Three statistically identified keystone taxa in the bacterial networks, namely, Solirubrobacterales, Ktedonobacteraceae, and Jatrophihabitans, were shared across the earthworm gut and adjacent soil. Environmental factors (pH and organic matter) and keystone taxa were important determinants of the bacterial community composition in the earthworm gut. Both PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) and FAPROTAX (Functional Annotation of Prokaryotic Taxa) predicted that carbon metabolism was significantly higher in adjacent soil than in the earthworm gut, which was consistent with the average well color development obtained by the Biolog assay. Structural equation modeling combined with correlation analysis suggested that pH, organic matter, and potential keystone taxa exhibited significant relationships with carbon metabolism. This study deepens our understanding of the mechanisms underlying keystone taxa regulating carbon cycling in the earthworm gut. IMPORTANCE The intestinal microbiome of earthworms is a crucial component of the soil microbial community and nutrient cycling processes. If we could elucidate the role of this microbiome in regulating soil carbon metabolism, we would make a crucial contribution to understanding the ecological role of these gut bacterial taxa and to promoting sustainable agricultural development. However, the ecological functioning of these earthworm-microbe interactions in regulating carbon cycling has so far not been fully investigated. In this study, we revealed, first, that the bacterial groups of Solirubrobacterales, Ktedonobacteraceae, and Jatrophihabitans were core keystone taxa across the earthworm gut and adjacent soil and, second, that the environmental factors (pH and organic carbon) and keystone taxa strongly affected the bacterial community composition and exhibited close correlations with microbial carbon metabolism. Our results provide new insights into the community assembly of the earthworm gut microbiome and the ecological importance of potential keystone taxa in regulating carbon cycling dynamics.

Comparative Study on Bacterial Population Dynamics of Foregut, Midgut, and Hindgut Content of Perionyx excavatus (Perrier) Isolated from Eco-friendly, Non-hazardous Vermicompost.

The ideal condition of earthworm gut promotes growth and multiplication of beneficial soil microorganisms eliminating pathogens and converts organic wastes into nutrients rich compost. The present study has been carried out to determine the population dynamics of earthworm gut bacteria and to find out relative abundance of different functional bacterial groups in the foregut, midgut, and hindgut of earthworm Perionyx excavatus. To assess bacterial diversity, a viable plate count method was adopted. In the different gut region of earthworm, aerobic heterotrophic, amylolytic, Bacillus, Gram-negative, proteolytic, fat hydrolyzing, nitrate-reducing, nitrifying, asymbiotic nitrogen-fixing, Azotobacter, and phosphate solubilizing bacterial populations ranged from 22.2 to 241.6 × 106, 8.0 to 171.60 × 106, 1.83 to 2.79 × 106, 10.68 to 23.04 × 104, 3.70 to 5.52 × 104, 59.60 to 208.40 × 104, 1.86 to 7.34 × 104, 10.94 to 19.78 × 104, 0.80 to 3.42 × 104, 7.83 to 13.70 × 104, 1.31 to 2.67 × 104 cfu/ml gut suspension, respectively. The results of the one-way ANOVA revealed that the bacterial load of most of the bacterial groups was significantly higher (p < 0.05) in the hindgut region, followed by midgut and foregut. Only the density of the proteolytic group was significantly higher (p < 0.05) in the midgut region followed by foregut and hindgut. Starch hydrolyzing bacteria constitute the largest group of bacteria in the gut content. From principal component analysis, two components were extracted with the eigenvalues of 8.485 and 1.132. Agglomerative hierarchical cluster analysis revealed that the bacterial populations were clustered into four different groups. Quantitative variation among bacterial groups in earthworm's gut seems to determine the soil health and composting efficiency; from this point of view, the present study will provide a better understanding about different functional bacterial groups of earthworm's guts and might be helpful in sustainable agriculture and waste management.

Medicinal Perspective of Antibacterial Bioactive Agents in Earthworms (Clitellata, Annelida): A Comprehensive Review.

Humoral practice is a fundamental natural biological phenomenon in earthworm defensive system which protects them from infectious bacteria and irritating agents by different mechanisms. The defensive system of earthworms is highly complicated because they lack antibodies in their blood circulatory system but their body extracts and coelomic fluid comprise of different bioactive agents (i.e. peptides and proteins) that defend these worms. There are various groups of bioactive agents such as proteases (name depends on proteins/peptide function or formal earthworm species name), metabolites (total 59 metabolites found in Eisenia fetida), metal binding proteins (2 proteins such as Ca2+ binding calmodulin and metallothionein), active proteins (include lysozyme, lysenin and eiseniapore etc.), antimicrobial peptides (antibacterial vermi-peptides family (AVPF), antimicrobial peptide I (PP-I), coelomic cytolytic factor (CCF, CCF-I and CCF like protein), fetidin, lysenin, lumbricin (lumbricin I, lumbricin PG, and lumbricusin), organic acids (fatty acids, succinic acids, and lauric acid) and other organic compounds (such as purine and vitamin D). The presence of above mentioned molecules confer therapeutic potential that affect energy intake and involve in decreasing oxidative stress, metabolic disturbances and pro-inflammatory conditions. The future perspectives of earthworm bioactive compounds are concerned with the development of provisional standards, purification and classification for utilizations in pharma industry.

Metagenomic Sequencing Reveals that the Assembly of Functional Genes and Taxa Varied Highly and Lacked Redundancy in the Earthworm Gut Compared with Soil under Vanadium Stress.

Exploring the ecological mechanism of microbial community assembly in soil and the earthworm gut in a vanadium polluted environment could help us understand the effects of vanadium stress on microbial diversity maintenance and function, as well as the mechanism of microbial mitigation of vanadium stress. Combining metagenomic sequencing and abundance distribution models, we explored the assembly of earthworm intestinal bacteria and native soil bacteria after 21 days of earthworm exposure to a gradient level of vanadate (0 to 300 mg kg-1) in soil. Stochastic processes dominated the assembly of both genes and taxa in earthworm gut and soil. Both the composition of taxa and functional genes in earthworm gut varied highly with the vanadium concentration, while in soil, only the taxa changed significantly, whereas the functional genes were relatively stable. The functional redundancy in soil, but not in the earthworm gut, was confirmed by a Mantel test and analysis of similarities (ANOSIM) test. In addition, vanadium detoxifying gene (VDG)-carrying taxa were more diverse but less abundant in soil than in the worm gut; and VDGs were more abundant in soil than in the worm gut. Their wider niche breadth indicated that VDG-carrying taxa were generalists in soil, in contrast to their role in the worm gut. These results suggested that earthworm intestinal and soil microbes adopted different strategies to counteract vanadium stress. The results provide new insights into the effects of soil vanadium stress on the assembly of earthworm gut and soil microbiota from both bacterial taxa and genetic function perspectives. IMPORTANCE Metagenomic sequencing revealed the variation of functional genes in the microbial community in soil and earthworm gut with increasing vanadium concentrations, which provided a new insight to explore the effect of vanadium stress on microbial community assembly from the perspective of functional genes. Our results reinforced the view that functional genes and taxa do not appear to have a simple corresponding relationship. Taxa are more sensitive compared with functional genes, suggesting the existence of bacterial functional redundancy in soil, but not in the earthworm gut. These observations indicate different assembly patterns of earthworm intestinal and soil bacteria under vanadium stress. Thus, it is important and necessary to include genetic functions to comprehensively understand microbial community assembly.

Exploration of microbial communities in the guts and casts of Eudrilus eugeniae, Perionyx excavatus, and Eisenia fetida.

Earthworms and their casts have been widely used for organic waste degradation and plant growth promotion. The microbial communities that reside in the guts and casts of earthworms markedly influence both applications. In the present study, next-generation sequencing was applied to identify the microbial communities in the guts and casts of three epigeic earthworm species, Eudrilus eugeniae, Perionyx excavatus, and Eisenia fetida, reared under two different feeding conditions. A total of 580 genera belonging to 43 phyla were identified. By comparing bacterial diversity among samples divided into groups based on the earthworm species, sample types, and conditions, the beta diversity analysis supported the impact of the sample type and suggested that there was significant dissimilarity of the bacterial diversity between the gut and cast. Besides, bacterial Phylum compositions within the group were compared. The result showed that the top three high relative frequency phyla found in the casts were the same regardless of earthworm species, while those found in the gut depended on both the condition and earthworm species. Focusing on the cellulolytic and plant growth-promoting bacteria, certain cellulolytic bacteria, Paenibacillus, Comamonas, and Cytophaga, were found only in the cast. Citrobacter and Streptomyces aculeolatus were detected only in the guts of earthworms reared in the bedding containing vegetables and bedding alone, respectively. Besides, Actinomadura and Burkholderia were detected only in the gut of E. eugeniae and E. fetida, respectively. The results proved that the microbial composition was affected by sample type, condition, and earthworm species. In addition, the proportion of these beneficial bacteria was also influenced by these factors. Hence, the information from this study can be used as a guide for selecting earthworm species or their casts for more efficient organic waste decomposition and plant growth promotion.

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.

Earthworms drastically change fungal and bacterial communities during vermicomposting of sewage sludge.

Wastewater treatment plants produce hundreds of million tons of sewage sludge every year all over the world. Vermicomposting is well established worldwide and has been successful at processing sewage sludge, which can contribute to alleviate the severe environmental problems caused by its disposal. Here, we utilized 16S and ITS rRNA high-throughput sequencing to characterize bacterial and fungal community composition and structure during the gut- and cast-associated processes (GAP and CAP, respectively) of vermicomposting of sewage sludge. Bacterial and fungal communities of earthworm casts were mainly composed of microbial taxa not found in the sewage sludge; thus most of the bacterial (96%) and fungal (91%) taxa in the sewage sludge were eliminated during vermicomposting, mainly through the GAP. Upon completion of GAP and during CAP, modified microbial communities undergo a succession process leading to more diverse microbiotas than those found in sewage sludge. Consequently, bacterial and fungal community composition changed significantly during vermicomposting. Vermicomposting of sewage resulted in a stable and rich microbial community with potential biostimulant properties that may aid plant growth. Our results support the use of vermicompost derived from sewage sludge for sustainable agricultural practices, if heavy metals or other pollutants are under legislation limits or adequately treated.

Impact of Altitudinal Variation on the Phytochemical Profile, Anthelmintic and Antimicrobial Activity of Two Pinus Species.

Active components from natural sources are the current focus in most pharmacological research to provide new therapeutic agents for clinical use. Essential oils from the Pinus species have been traditionally used in medicine. This study aimed to investigate the chemical profile of two Pinus species, Pinus halepensis L. and Pinus pinea Mill, from different altitudes in Libya and study the effect of environmental conditions on the biological activities of essential oils. A clevenger apparatus was used to prepare the essential oils by hydrodistillation. Analyses were done using GC/MS. Anthelmintic and antimicrobial activities were tested against the earthworm Allolobophora caliginosa, gram-positive bacteria, gram-negative bacteria, and fungi. Different chemical profiles were observed among all tested essential oils, and terpenes were the most dominant class. All studied essential oils from the Pinus species exhibited a remarkable anthelmintic activity compared to the standard piperazine citrate drug. Pinus halepensis from both altitudes showed broad-spectrum antimicrobial activity against all tested microorganisms, while Pinus pinea was effective against only Escherichia coli. From these findings, one can conclude that there are variations between studied species. The essential oil compositions are affected by environmental factors, which consequently affect the anthelmintic and antimicrobial activity.

Sphingobacterium lumbrici sp. nov., a novel bacterium isolated from wormcast of Eisenia foetida.

A novel Gram-stain-negative, rod-shaped, non-motile, yellowish bacterium, designated strain 1.3611T, was isolated from the wormcast of Eisenia foetida. The strain grew optimally at 30-37 ℃, at pH 7.0 and with 0-1.0 % (w/v) NaCl. Based on the results of 16S rRNA gene sequence and phylogenetic analyses, strain 1.3611T showed the highest degree of 16S rRNA gene sequence similarity to Sphingobacterium olei HAL-9T (97.0 %), followed by Sphingobacterium alkalisoli Y3L14T (95.8 %). The respiratory quinone of strain 1.3611T was menaquinone-7 (MK-7) and its major cellular fatty acids were iso-C15 : 0 (41.3 %), summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c, 22.1 %) and iso-C17 : 0 3-OH (16.2 %). The major polar lipids were sphingophospholipid, phosphatidylethanolamine, four unidentified glycolipids, two unidentified phospholipids and five unidentified polar lipids. The genomic DNA G+C content was 39.0 mol%. The digital DNA-DNA hybridization and average nucleotide identity values between the genomes of strain 1.3611T and S. olei HAL-9T were 37.9 and 88.9 %, respectively. According to the phenotypic and chemotaxonomic phylogenetic results, strain 1.3611T should represent a novel species of the genus Sphingobacterium, for which the name Sphingobacterium lumbrici sp. nov. is proposed, with strain 1.3611T (=KCTC 62980T=CCTCC AB 2018349T) as the type strain.

Amynthas corticis genome reveals molecular mechanisms behind global distribution.

Earthworms (Annelida: Crassiclitellata) are widely distributed around the world due to their ancient origination as well as adaptation and invasion after introduction into new habitats over the past few centuries. Herein, we report a 1.2 Gb complete genome assembly of the earthworm Amynthas corticis based on a strategy combining third-generation long-read sequencing and Hi-C mapping. A total of 29,256 protein-coding genes are annotated in this genome. Analysis of resequencing data indicates that this earthworm is a triploid species. Furthermore, gene family evolution analysis shows that comprehensive expansion of gene families in the Amynthas corticis genome has produced more defensive functions compared with other species in Annelida. Quantitative proteomic iTRAQ analysis shows that expression of 147 proteins changed in the body of Amynthas corticis and 16 S rDNA sequencing shows that abundance of 28 microorganisms changed in the gut of Amynthas corticis when the earthworm was incubated with pathogenic Escherichia coli O157:H7. Our genome assembly provides abundant and valuable resources for the earthworm research community, serving as a first step toward uncovering the mysteries of this species, and may provide molecular level indicators of its powerful defensive functions, adaptation to complex environments and invasion ability.

Multiple heavy metal tolerance and removal by an earthworm gut fungus Trichoderma brevicompactum QYCD-6.

Fungal bioremediation is a promising approach to remove heavy-metal from contaminated water. Present study examined the ability of an earthworm gut fungus Trichoderma brevicompactum QYCD-6 to tolerate and remove both individual and multi-metals. The minimum inhibitory concentration (MIC) of heavy metals [Cu(II), Cr(VI), Cd(II) and Zn(II)] against the fungus was ranged 150-200 mg L-1 on composite medium, and MIC of Pb(II) was the highest with 1600 mg L-1 on potato dextrose (PD) medium. The Pb(II) presented the highest metal removal rate (97.5%) which mostly dependent on bioaccumulation with 80.0%, and synchronized with max biomass (6.13 g L-1) in PD medium. However, on the composite medium, the highest removal rate was observed for Cu(II) (64.5%). Cellular changes in fungus were reflected by TEM analysis. FTIR and solid-state NMR analyses indicated the involvement of different functional groups (amino, carbonyl, hydroxyl, et al.) in metallic biosorption. These results established that the earthworm-associated T. brevicompactum QYCD-6 was a promising fungus for the remediation of heavy-metal wastewater.

Effects of tetracycline on the relationship between the microbial community and oxidative stress in earthworms based on canonical correlation analysis.

In this study, Eisenia fetida was taken as the test organism and tetracycline was taken as the stress compound. The artificial soil test was conducted to study the utilization intensity of different carbon sources (the Biolog-microplate supplied) by microorganisms under different stress times and stress concentrations. The changes in the in vivo key enzymes activities of earthworms and oxidative stress indicators, such as malondialdehyde (MDA), were explored. The canonical correlation analysis method was the first used to establish a analysis-model to explore the relationship between the functional diversity of microbial community and the oxidative stress in earthworms in vivo under different stress times and concentrations. Research shows: 1) after tetracycline stress, in the earthworm, the CAT, POD, SOD, GPX were related to the microbes that use carbohydrate carbon sources; the GST and AChE were related to the microbes that use polymer carbon sources; the MDA was related to the microbes that use carbon sources: amino acid, carboxylic acid and phenolic acid. 2) Under low concentrations of tetracycline stress, there was no significant relationship between the functional diversity of the microbial communities and the effects of oxidative stress at this concentration. The high concentration of tetracycline can be utilized to screen probiotics that alleviate the effects of oxidative stress. 3) The utilization of carbon sources by microbial community in the earthworm after stress can be used as biomarker of ecotoxicology. It provides a basic theoretical for adding beneficial carbon sources to combat oxidative damage in vivo.

Do combined nanoscale polystyrene and tetracycline impact on the incidence of resistance genes and microbial community disturbance in Enchytraeus crypticus?

It has been proved that nanoplastics can effectively adsorb pollutants and thus influence their behavior and availability. The combined toxic effects of nanoplastic and its adsorbed pollutant on the soil fauna are still not well known. We used high-throughput quantitative PCR to explore the effects of oral nanoscale polystyrene and tetracycline exposure on antibiotic resistance genes in the soil invertebrate Enchytraeus crypticus, and used bacterial 16S rRNA gene amplification sequencing to examine the response of the microbiome of E. crypticus. After 14 days of tetracycline and nanoscale polystyrene exposure, we terminated exposure and monitored the restoration of ARGs and microbiome in the E. crypticus. Results showed that the number of ARGs, especially macrolide-lincosamide-streptogramin B (MLSB), tetracycline ARGs, as well as multidrug ARGs, increased with exposure to nanoscale polystyrene and tetracycline. The abundance of Aminoglycoside and Beta_Lactamase ARGs in E. crypticus also significantly increased. The exposure significantly perturbed the abundance of families Microbacteriaceae, Streptococcaceae, Enterobacteriaceae, Rhodocyclaceae and Sphinomonadaceae. After terminating exposure for 14 days, the diversity and abundance of ARGs were not completely restored, while the microbiome was not permanently changed but reversibly impacted.

The bacterial community structures in response to the gut passage of earthworm (Eisenia fetida) feeding on cow dung and domestic sludge: Illumina high-throughput sequencing-based data analysis.

Diets are shown to be capable of shaping the gut microbiota of earthworm, while the effects of distinct foods on bacterial communities of different digestive tracts of earthworm are unknown. For this purpose, cow dung (CD) and domestic sludge (DS) were chosen as diets for earthworms (Eisenia fetida), and different gut contents, namely gizzard + foregut area, hindgut, and mature vermi-compost were sampled for Illumina sequencing analysis. We found that there existed significant reductions in bacterial diversity and abundance in the gizzard + foregut area, where there were stable bacteria with the ability of biodegradation of xenobiotics, such as Amycolatopsis, Methylobacterium, Ralstonia, Ochrobactrum, and Sphingomonas. The decreases could be recovered in the hindgut and mature vermi-compost to different extents, suggesting that a bottleneck effect on the bacterial community occurred in the gizzard + foregut area. Beta-Proteobacteria was the most abundant subclass regardless of the different diets, and bacteria affiliated with gamma-, delta- and epsilon-subclasses were taken as food by the earthworms. Vermi-composts based on the various diets should be used differently according to different aims.