Rhizosphere microbiomes derived from vermicompost alter gene expression and regulatory pathways in tomato (Solanum lycopersicum, L.).

The gut microbiome of worms from composting facilities potentially harbors organisms that are beneficial to plant growth and development. In this experiment, we sought to examine the potential impacts of rhizosphere microbiomes derived from Eisenia fetida worm castings (i.e. vermicompost) on tomato (Solanum lycopersicum, L.) plant growth and physiology. Our experiment consisted of a greenhouse trial lasting 17 weeks total in which tomato plants were grown with one of three inoculant treatments: a microbial inoculant created from vermicompost (V), a microbial inoculant created from sterilized vermicompost (SV), and a no-compost control inoculant (C). We hypothesized that living microbiomes from the vermicompost inoculant treatment would enhance host plant growth and gene expression profiles compared to plants grown in sterile and control treatments. Our data showed that bacterial community composition was significantly altered in tomato rhizospheres, but fungal community composition was highly variable in each treatment. Plant phenotypes that were significantly enhanced in the vermicompost and sterile vermicompost treatments, compared to the control, included aboveground biomass and foliar δ15N nitrogen. RNA sequencing revealed distinct gene expression changes in the vermicompost treatment, including upregulation of nutrient transporter genes such as Solyc06g074995 (high affinity nitrate transporter), which exhibited a 250.2-fold increase in expression in the vermicompost treatment compared to both the sterile vermicompost and control treatments. The plant transcriptome data suggest that rhizosphere microbiomes derived from vermicompost can influence tomato gene expression and growth-related regulatory pathways, which highlights the value of RNA sequencing in uncovering molecular responses in plant microbiome studies.

Unveiling potential roles of earthworms in mitigating the presence of virulence factor genes in terrestrial ecosystems.

Earthworms can redistribute soil microbiota, and thus might affect the profile of virulence factor genes (VFGs) which are carried by pathogens in soils. Nevertheless, the knowledge of VFG profile in the earthworm guts and its interaction with earthworm gut microbiome is still lacking. Herein, we characterized earthworm gut and soil microbiome and VFG profiles in natural and agricultural ecosystems at a national scale using metagenomics. VFG profiles in the earthworm guts significantly differed from those in the surrounding soils, which was mainly driven by variations of bacterial communities. Furthermore, the total abundance of different types of VFGs in the earthworm guts was about 20-fold lower than that in the soils due to the dramatic decline (also by approximately 20-fold) of VFG-carrying bacterial pathogens in the earthworm guts. Additionally, five VFGs related to nutritional/metabolic factors and stress survival were identified as keystones merely in the microbe-VFG network in the earthworm guts, implying their pivotal roles in facilitating pathogen colonization in earthworm gut microhabitats. These findings suggest the potential roles of earthworms in reducing risks related to the presence of VFGs in soils, providing novel insights into earthworm-based bioremediation of VFG contamination in terrestrial ecosystems.

Differential inducibility of transmembrane peptidoglycan recognition proteins (PGRPs) by bacterial challenges in the earthworm, Eisenia andrei.

Peptidoglycan recognition proteins (PGRPs) and Toll-like receptors (TLRs) are highly conserved pattern recognition receptors (PRRs). Earthworms possess genes encoding TLRs that specifically respond to Gram-positive bacteria. In addition, several PGRPs have been recently identified, which are predicted to exhibit amidase activity but lack receptor function. In lophotrochozoans, a membrane-bound PRR responsible for detecting Gram-negative bacteria remains unidentified. This study reveals several novel transmembrane peptidoglycan recognition proteins (Ean-PGRPLs) in earthworms, whose mRNA expression increases in response to Gram-negative but not Gram-positive bacteria. This indicates that Ean-PGRPLs may serve as a PRR associated with intracellular signaling for Gram-negative bacteria.

Unveiling resilience: coelomic fluid bacteria's impact on plant metabolism and abiotic stress tolerance.

Earthworms' coelomic fluid (CF) has been discovered to possess properties that promote plant development. In particular, the earthworm's coelomic fluid-associated bacteria (CFB) are the primary factor influencing the plants' response. To investigate this, we used bacteria isolated from the CF and selected based on different plant growth-promoting traits, in a mesocosm ecosystem that includes plants. This experiment aimed to assess their impact on the metabolism of plants growing under abiotic stress environments (alkaline soil and nitrogen (N), phosphate (P), and potassium (K) deficit) and compare the lipid profiles of plants under the various treatments. We used seven different bacterial species isolated from the CF of Aporrectodea molleri and as a plant model Zea mays L. For the metabolomic analysis method, we used gas chromatography-mass spectrometry lipidomic. After observing the metabolomic profiles, we found that a few molecular pathways are involved in how plants react to bacterial biostimulants. The bacterial isolates belonging to Pantoea vagans, Pseudomonas aeruginosa, Bacillus paramycoides, and Bacillus thuringiensis have led to a significant increase in synthesizing several metabolites belonging to various chemical categories. Contrary to predictions, abiotic stress did not cause a drop in the composition and concentration of lipids in plants treated with the CFB, demonstrating the rigidity of the protective mechanisms. The statistical analysis based on the Pearson method revealed a positive significant correlation between plant growth parameters (length of the aerial part, surface of the leaves, and biomass) and some metabolites belonging to fatty acids, carboxylic acids, benzene derivatives, and alkanes. Moreover, the standard metabolic components of all treatments in much higher concentrations during bacterial treatments than the control treatment suggests that the bacteria have stimulated the overexpression of these metabolic components. According to these results, we could assume that plants treated with CFB exhibit an adaptability of abiotic stress defense mechanisms, which may be attributed to the upregulation of genes involved in lipid biosynthesis pathways.

Effect of microplastic pollution on the gut microbiome of anecic and endogeic earthworms.

Microplastic (MP) pollution constitutes an emerging type of pollution threatening both aquatic and terrestrial ecosystems. The impact on aquatic ecosystems has been extensively studied, but the effect on terrestrial ecosystems and their inhabitants is mostly underexplored. In this study, we explored the effect of MP pollution on gut bacterial microbiome of endogeic (Aporrectodea caliginosa) and anecic (Lumbricus terrestris) earthworms. The experiments were performed in sandy soil with 0.2% of low-density polyethylene MPs (LDPE MPs). We observed that the endogeic earthworms had 100% survival, while anecic earthworms survived 25 days in the control (i.e. in absence of MPs) and 21 days in the treatment with LDPE MPs. The main driver of shifts in the diversity and composition of the bacterial communities in the gut of tested earthworms was the lifestyle of the worms, followed by the presence of MPs. The bacterial microbiome diversity was significantly different among the two types of earthworms, and the highest bacterial diversity was found in the gut of the endogeic earthworms. The effect of MPs on gut bacterial microbiome was clearly observed in the changes in the relative abundance of several phyla and families of the bacterial communities in both types of earthworms, although it was most evident in the anecic earthworms. The Actinobacteriota, Proteobacteria, and Firmicutes were the main groups enhanced in the MP treatments, suggesting enrichment of the bacterial communities with potential plastic degraders.

Novel report on soil infection with Metarhizium rileyi against soil-dwelling life stages of insect pests.

Entomopathogenic fungi are the most effective control remedy against a wide range of medical and agricultural important pests. The present study aimed to isolate, identify, and assess the virulence of Metarhizium rileyi against Spodoptera litura and Spodoptera frugiperda pupae under soil conditions. The biotechnological methods were used to identify the isolate as M. rileyi. The fungal conidial pathogenicity (2.0 × 107, 2.0 × 108, 2.0 × 109, 2.0 × 1010, and 2.0 × 1011 conidia/mL-1) was tested against prepupae of S. litura and S. frugiperda at 3, 6, 9, and 12 days after treatments. Additionally, the artificial soil-conidial assay was performed on a nontarget species earthworm Eudrilus eugeniae, using M. rileyi conidia. The present results showed that the M. rileyi caused significant mortality rates in S. litura pupae (61-90%), and S. litura pupae were more susceptible than S. frugiperda pupae (46%-73%) at 12 day posttreatment. The LC50 and LC90 of M. rileyi against S. litura, were 3.4 × 1014-9.9 × 1017 conidia/mL-1 and 6.6 × 105-4.6 × 1014 conidia/mL-1 in S. frugiperda, respectively. The conidia of M. rileyi did not exhibit any sublethal effect on the adult stage of E. eugeniae, and Artemia salina following a 12-day treatment period. Moreover, in the histopathological evaluation no discernible harm was observed in the gut tissues of E. eugeniae, including the lumen and epithelial cells, as well as the muscles, setae, nucleus, mitochondria, and coelom. The present findings provide clear evidence that M. rileyi fungal conidia can be used as the foundation for the development of effective bio-insecticides to combat the pupae of S. litura and S. frugiperda agricultural pests.

Assessment of microbial flora and pesticidal effect of vermicast generated from Azadirachta indica (neem) for developing a biofertilizer-cum-pesticide as a single package.

The soil comprising organic matter, nutrients, serve as substrate for plant growth and various organisms. In areas where there are large plantations, there is a huge leaf litter fall. The leaf litter upon decomposition releases nutrients and helps in nutrient recycling, for which the soil engineers such as earthworms, ants and termites are important key players. In this context, the present study was conducted to assess the characteristics of the vermicast obtained by vermicomposting neem leaf litter in terms of microbial flora, plant growth promoting properties and antagonistic activities of the vermicast against phytopathogens. Vermicomposting of neem leaf litter was done using two epigeic earthworm species Eisenia fetida and Eudrilus eugeniae. The vermicast exhibited antagonistic potential against plant pathogens. Out of the four vermiwash infusions studied, the 75 % formulation reduced the disease incidence against mealybug by 82 % in the tree Neolamarkia cadamba. The result of the study suggests that vermicast made from neem leaf litter may be a potent combination of a biofertilizer and a pesticide.

Earthworm gut bacteria facilitate cadmium immobilization through the formation of CdS nanoparticles.

Gut bacteria of earthworm Amynthas hupeiensis exhibit significant potential for the in-situ remediation of cadmium (Cd)-contaminated soil. However, the mechanisms by which these gut bacteria immobilize and tolerate Cd remain elusive. The composition of the gut bacterial community was characterized by high-throughput sequencing. Cd-tolerant bacteria were isolated from the gut, and their roles in Cd immobilization, as well as their tolerance mechanisms, were explored through chemical characterization and transcriptome analysis. The predominant taxa in the gut bacterial community included unclassified Enterobacteriaceae, Citrobacter, and Bacillus, which were distinctly different from those in the surrounding soil. Notably, the most Cd-tolerant gut bacterium, Citrobacter freundii DS strain, immobilized 63.61% of Cd2+ within 96 h through extracellular biosorption and intracellular bioaccumulation of biosynthetic CdS nanoparticles, and modulation of solution pH and NH4+ concentration. Moreover, the characteristic signals of CdS were also observed in the gut content of A. hupeiensis when the sterilized Cd-contaminated soil was inoculated with C. freundii. The primary pathways involved in the response of C. freundii to Cd stress included the regulation of ABC transporters, bacterial chemotaxis, cell motility, oxidative phosphorylation, and two-component system. In conclusion, C. freundii facilitates Cd immobilization both in vitro and in vivo, thereby enhancing the host earthworm's adaptation to Cd-contaminated soil.

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.

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.

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.

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.