Coelomic Cytolytic Factor Research Articles

Pseudomonas putida HE alleviates glyphosate-induced toxicity in earthworm: Insights from the neurological, reproductive and immunological status

The proceeding study aimed to isolate glyphosate-degrading bacteria from soil and determine optimal degradation conditions through single-factor experiments and response surface methodology. The detoxifying efficacy of the isolate on glyphosate was assessed using earthworm model. The results indicate that Pseudomonas putida HE exhibited the highest glyphosate degradation rate. Optimal conditions for glyphosate degradation were observed at an inoculation percentage of approximately 5%, a pH of 7, and a temperature of 30 °C. Glyphosate induced notable neurotoxicity and reproductive toxicity in earthworms, evidenced by reduced activity of the neurotoxicity-associated enzyme AChE. Additionally, an increase in the activities of catalase, superoxide dismutase, and lactate dehydrogenase was observed. H&E staining revealed structural disruptions in the earthworm clitellum, with notable atrophy in the structure of spermathecae. Furthermore, glyphosate activation of earthworm immune systems led to increased expression of immune-related genes, specifically coelomic cytolytic factor and lysozyme. Notably, the introduction of strain HE mitigated the glyphosate toxicity to the earthworms mentioned above. P. putida HE was able to increase soil enzyme activities that were reduced due to glyphosate. The isolate P. putida HE, emerged as an effective and cost-efficient remedy for glyphosate degradation and toxicity reduction in natural settings, showcasing potential applications in real ecological settings.

THERAPEUTICS APPROACH IN SILICO USING A COELOMIC FLUID OF LUMBRICUS RUBELLUS AS COMBINED MAPLC3-TARGETED CHEMOTHERAPY IN ACUTE MYELOID LEUKEMIA

Leukemia ranked 11th in the incidence rate of all cancers worldwide, and acute myeloid leukemia (AML) has the highest leukemia mortality rate at 62%. Protein inhibition in the autophagy mechanism combined with cytarabine as the main chemotherapy treatment for AML can improve the effectiveness of drugs that have already developed resistance. Proteins in coelomic fluid (CFL) of Lumbricus rubellus, such as lumbrokinase, lysenin, and coelomic cytolytic factor-1 (CCF-1) are known to have potential in the treatment of cancer. The purpose of this study is to determine the potential of these proteins as candidates for co-chemotherapy agents through inhibiting microtubule-associated protein light chain 3 (MAPLC3), a protein target in the autophagy mechanism of AML, using a bioinformatics approach. To the best of our knowledge, this study was the first to explore these potential therapeutic proteins as co-chemotherapy agents for AML as well as their proposed molecular mechanisms. Their effectiveness can be predicted through in silico studies, specifically with a molecular docking method between the proteins and MAPLC3 as the target. The results show that hydrogen, hydrophobic, and electrostatics bonds are formed in lumbrokinase, lysenin, and CCF-1, and the combination of them with MAPLC3. There are several active sites on MAPLC3 that bind to lumbrokinase, lysenin, CCF-1 and the combination of them. The combination of cytarabine and CFL of Lumbricus rubellus inhibited the survival of HL-60 cells. This study revealed that lumbrokinase, lysenin, CCF-1 and the combination of them have the potential to partially inhibit MAPLC3, which then may inhibit the autophagy process.

Biochemical, transcriptomic, gut microbiome responses and defense mechanisms of the earthworm Eisenia fetida to salt stress

The accumulation of sodium chloride (NaCl) in soil is a worldwide problem with detrimental effects on the survival of soil animals. The effects of NaCl on earthworms remain unclear. Here, we show that the growth rate, cocoon production rate, annetocin precursor (ANN) mRNA level, and superoxide dismutase and catalase activities in earthworms were reduced under NaCl stress, whereas the mortality rate, reactive oxygen species (ROS) and malondialdehyde activity level increased. Histological damage to the earthworm body wall and intestine were observed under NaCl stress. NaCl stress increased DNA damage in the seminal vesicle and coelomocytes. The mRNA level of lumbrokinase, 1,3-beta-glucanse, coelomic cytolytic factor (CCF1), and alpha-amylase was significantly down-regulated, whereas that of earthworm excitatory peptides2 (EEP2) was up-regulated. From 16 S rRNA sequencing, the earthworm gut microbiota diversity decreased under NaCl stress. However, Verminephrobacter, Kluyvera, Lactobacillus, and Ochrobactrum increased under NaCl stress. These findings contribute to the risk assessment of the salt stress on soil organisms.

Fluoroquinolone antibiotics disturb the defense system, gut microbiome, and antibiotic resistance genes of Enchytraeus crypticus

Antibiotic residues from animal manure cause soil pollution and can pose a threat to soil animals. In this study, the toxicological effects of fluoroquinolone antibiotics on Enchytraeus crypticus, including defence response, gut microbiome, and antibiotic resistance genes (ARGs), were studied. The cytochrome P450 enzyme activity and reactive oxygen species levels increased, activating the defense response. The superoxide dismutase and glutathione S-transferase activity, and the expression of immune defense molecules such as coelomic cytolytic factor, lysozyme, bactericidal protein fetidins and lysenin changed. Furthermore, the diversity of the gut microbiome decreased, and the relative abundance of Bacteroidetes decreased significantly at the phylum level but increased in pathogenic and antibiotic-secreting bacteria (Rhodococcus and Streptomyces) at the genus level. However, the soil microbiome was not significantly different from that of the control group. The relative abundance of ARGs in the gut and soil microbiome significantly increased with enrofloxacin concentration, and the fluoroquinolone ARGs were significantly increased in both the soil (20.85-fold, p < 0.001) and gut (11.72-fold, p < 0.001) microbiomes. Subtypes of ARGs showed a positive correlation with Rhodococcus, which might increase the risk of disease transmission and the probability of drug-resistant pathogens. Furthermore, mobile genetic elements significantly promote the spread of ARGs.

Insilico Study of Earthworm CCF1 Peptides in Earthworm

The earthworms are known to possess a robust immune system that can combat pollutants and pathogens to which they are constantly exposed in their environment (Ghosh in Environ Sci Pollut Res 25:6196, 2018). Amongst the different components of its immune system, the coelomic cytolytic factor (CCF-1), is an important molecule that plays active role in immune system in earthworm and is of pharmaceutical importance, however its role in immune defence is not known. Reports suggest that CCF-1 has lytic role like human TNF-α, with role in hemolysis, bacterial and tumor cell lysis (Bilej et al. in Immunol Lett 45(1–2):123–128, 1995; Hua et al. in Pharm Biol 49:269–275, 2011) and lysis of Trypanosoma cruzi (T cruzi) thereby activating the prophenoloxidase pathway (Olivares Fontt et al. in Dev Comp Immunol 26(1):27–34, 2002). They have been reported to reveal convergent evolution with human TNF-α (Beschin et al. in Nature 400(6745):627–628, 1999). Due to its such important functions, even though it obtained from primitive organism like earthworm, CCF-1 finds importance both as an innate immune molecule conferring earthworm immunity and is of probable therapeutic importance and forms the object of the current study. In this study insilco approaches and molecular modelling was adopted to explore properties, structure and predicted cellular signaling network that would be affected based on the predicted functions of CCF1 being a vital innate immune molecule of earthworm.

Mutual interactions of E. andrei earthworm and pathogens during the process of vermicomposting.

Vermicomposting is a process by which earthworms together with microorganisms degrade organic wastes into a humus-like material called vermicompost. This process does not include a thermophilic stage, and therefore, the possible presence of pathogens represents a potential health hazard. To elucidate the effect of earthworms in the selective reduction of pathogens, grape marc substrate was artificially inoculated with Escherichia coli, Enterococcus spp., thermotolerant coliform bacteria (TCB), and Salmonella spp., and their reduction during vermicomposting was monitored. Various defense mechanisms eliminating microorganisms in the earthworm gut were assumed to be involved in the process of pathogen reduction. Therefore, we followed the expression of three pattern recognition receptors (coelomic cytolytic factor (CCF), lipopolysaccharide-binding protein (LBP), and Toll-like receptor (v-TLR)), two antimicrobial molecules (fetidin/lysenins and lysozyme), and heat shock protein HSP70. We detected the significant decrease of some defense molecules (fetidin/lysenins and LBP) in all pathogen-inoculated substrates, and the increase of CCF and LBP in the Salmonella spp.-inoculated substrate. At the same time, the reduction of pathogens during vermicomposting was assessed. We observed the accelerated reduction of E. coli, Enterococcus spp., and TCB in pathogen-inoculated substrates with earthworms compared to that without earthworms. Moreover, the differences between the microbiome of grape marc substrate and earthworm intestines were determined by high throughput sequencing. This analysis revealed that the bacterial composition of grape marc substrate differed from the composition of the content of earthworm intestines, suggesting the elimination of specific bacterial species during food passage through the gut.

ITRAQ-based quantitative proteomic analysis of the earthworm Eisenia fetida response to Escherichia coli O157:H7

Soil environment contaminated by Escherichia coli O157:H7 which come from the waste of infected animals. Earthworms can live in the pathogens-polluted soil by their innate immunity. How the proteins of earthworms E. fetida will response to E. coli O157:H7-contaminated-soil still unclear? To identify the defense proteins under E. coli O157:H7 stress, we performed a proteomic analysis of earthworm under E. coli O157:H7 exposure through an iTRAQ technology. In total, we found 283 non-redundant proteins, including fibrinolytic protease 1, lombricine kinase, lysozyme, gelsolin, coelomic cytolytic factor-1, antimicrobial peptide lumbricin-l, lysenin, and et al. The proteins participate in metabolic processes, transcription, defense response to bacterium, translation, response to stress, and transport. The study will contribute to understand why earthworm can live in the pathogens-polluted environment.

LBP/BPI homologue in Eisenia andrei earthworms

LBP/BPIs are pattern recognition receptors that are often present in vertebrates and in invertebrates, and they play a defense role against pathogens. We have identified 1698 bp cDNA sequence from the Eisenia andrei earthworm with predicted amino acid sequence that shares homology with the LBP/BPI family (EaLBP/BPI). Sequence analysis of EaLBP/BPI proved the existence of two conserved domains with the potential ability to bind LPS. The predicted molecular mass of the EaLBP/BPI protein is 53.5 kDa, and its high basicity (pI 9.8) is caused by its high arginine content. Constitutive transcription of the Ealbp/bpi gene was shown in all tested tissues, with the highest level in coelomocytes and seminal vesicles; the lowest level was detected in the intestine. On the contrary, another earthworm LPS-binding molecule CCF (coelomic cytolytic factor) was expressed only in the intestine and coelomocytes. In E. andrei coelomocytes, the transcription of Ealbp/bpi gene was up-regulated in response to bacterial stimulation, reaching a maximum at 8 and 16 h post stimulation with Bacillus subtilis and Escherichia coli, respectively.

The effect of dibenzo-p-dioxin- and dibenzofuran-contaminated soil on the earthworm Eisenia andrei

Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) belong to the group of persistent organic pollutants, highly toxic environmental pollutants that include hydrophobic compounds with the tendency to bioaccumulate. Earthworms (Eisenia andrei) were exposed to PCDD/Fs-contaminated soil, and changes in their lipophilic structures and the gene expression of their defense molecules were followed. Damage to the intestinal wall and adjacent chloragogenous tissue was observed. Further, the up-regulation of the expression of several genes was detected. On the basis of these results, the mechanism of the impact of PCDD/Fs on earthworms has been proposed. Dioxins that accumulate in the lipophilic structures cause an increase in reactive oxidative species that triggers oxidative stress followed by the gene expression of two molecules that play a role in protection against oxidant toxicity, calreticulin (CRT) and Hsp70. Moreover, the effect of microbial biomass on the expression of coelomic cytolytic factor (CCF), a pattern recognition receptor, was also observed.

Cellular and molecular responses of E. fetida cœlomocytes exposed to TiO2 nanoparticles

An in vitro approach using cœlomocytes of Eisenia fetida was investigated to evaluate toxicity of TiO2 nanoparticles. Cœlomocytes were exposed to well-dispersed suspension of small aggregates (130 nm) of TiO2 nanoparticles (1–25 μg/ml) during 4, 12 and 24 h. Intracellular localisation suggested that the main route of uptake was endocytosis. Cellular responses showed that TiO2 nanoparticles were not cytotoxic and had no effect on phagocytosis at any of the four concentrations for each time tested. Concerning molecular responses, an increase of fetidin and metallothionein mRNA expression was observed starting from 4 h of exposure. In contrast, expression of coelomic cytolytic factor mRNA decreased for 10 and 25 μg/ml after 4 h. Superoxide dismutase, catalase and glutathione-S-transferase expression were not modified suggesting that oxidative stress was not induced by TiO2 in our experimental conditions. This in vitro approach showed that TiO2 nanoparticles were taken up by cœlomocytes and they could modify the molecular response of immune and detoxification system.

C60 exposure induced tissue damage and gene expression alterations in the earthworm Lumbricus rubellus

Effects of C60 exposure (0, 15 or 154 mg/kg soil) on the earthworm Lumbricus rubellus were assessed at the tissue and molecular level, in two experiments. In the first experiment, earthworms were exposed for four weeks, and in the second lifelong. In both experiments, gene expression of heat shock protein 70 (HSP70) decreased. For catalase and glutathione-S-transferase (GST), no significant trends in gene expression or enzyme activity were observed. Gene expression of coelomic cytolytic factor-1 (CCF-1) did not alter in earthworms exposed for four weeks, but was significantly down-regulated in the lifelong exposure. Histology of earthworms exposed to C60 in both experiments showed a damaged cuticle, with underlying pathologies of epidermis and muscles, as well as effects on the gut barrier. However, tissue repair was also observed in these earthworms. Overall, these data show that sub-lethal C60 exposure to earthworms via the soil affects gene expression and causes tissue pathologies.

A two-dimensional electrophoresis reference map of the earthworm &amp;lt;i&amp;gt;Eisenia fetida&amp;lt;/i&amp;gt;

The molecular mechanisms underlying innate immunity in the earthworm E. fetida remain unclear. For the recognition of innate immunity in the earthworm E. fetida, a detailed knowledge of this proteome is a prerequisite. The absence of a high-resolution E. fetida proteome map prompted us to determine E. fetida protein spots that can be visualised on 2-D protein gels. In this study, we present a preliminary description of the whole earthworm E. fetida proteome. A highly detailed two-dimensional gel electrophoresis (2-DE) map of the E. fetida proteome was established and approximately 1500 protein spots were detected from the earthworm sample when applying a 500 μg protein 2-DE in the pH range 3.0 - 10.0. We present a 2-DE proteome map of E. fetida, identifying 76 different proteins by matrix-assisted laser desorption/ionization-tandem time of flight mass spectrometry (MALDI-TOF/ TOF-MS) analysis. These identified proteins, including heat shock protein 90 (Hsp90), chaperonine protein HSP60, caspase-8, fibrinolytic protease 0, gelsolin-like protein, lombricine kinase, coelomic cytolytic factor1 (CCF 1), manganous superoxide dismutase (MnSOD), triosephosphate isomerase, extracellular globin-4, lysenin, and intermediate filament protein, glyceraldehyde-3- phosphate dehydrogenase, et al., are involved in several processes, including transcripttion, translation, the tricarboxylic acid cycle, the cellular amino acid metabolic process, protein amino acid phosphorylation, glycolysis, and the glucose metabolic process. These 2-DE data will enhance future comparisons of immunity, toxicology, biotic processes and other challenges, thereby allowing for further study of the molecular mechanisms in response to environmental stressors, and it will be useful to investigate environmental proteomics in invertebrate earthworms.

Analysis of earthworm Eisenia fetida proteomes during cadmium exposure: An ecotoxicoproteomics approach

Identification of differential proteomic responses to cadmium (Cd) would provide a means for better understanding the survival mechanisms of the earthworm, Eisenia fetida, living in a Cd-polluted environment, and the stress responses can be assessed by ecotoxiptoromics approaches. Extracts of whole earthworm collected at days 7, 14, 21, and 28 after Cd exposure were analyzed by 2-DE and quantitative image analysis. In total, 143 proteins demonstrated significant regulation in at least at one time point, and 56 proteins were identified by MALDI-TOF/TOF-MS and database searching. Compared with control samples, 28 protein spots were up-regulated and 28 were down-regulated during at least one time point. The identified proteins, including chaperonine protein HSP60, caspase-8, calcium ion-binding protein, zinc ion-binding protein, actin-binding protein, proteolysis, fibrinolytic protease, glutamate dehydrogenase, gelsolin-like protein, lombricine kinase, coelomic cytolytic factor 1, manganous superoxide dismutase, extracellular globin-4, lysenin, intermediate filament protein, and tubulin, are involved in several processes, including transcription, translation, the tricarboxylic acid cycle, the cellular amino acid metabolic process, protein amino acid phosphorylation, glycolysis, and the glucose metabolic process. Thus, our study provides a functional profile of the Cd-responsive proteins in earthworms.

Comparative Proteomic Analysis of Differentially Expressed Proteins in the Earthworm Eisenia fetida during Escherichia coli O157:H7 Stress

Escherichia coli O157:H7 is an intestine-inhabiting bacterium associated with many severe disease outbreaks worldwide. It may enter the soil environment with the excreta of infected animals (e.g., horses, cattle, chickens) and humans. Earthworms can protect themselves against invading pathogens because of their efficient innate defense system. Identification of differential proteomic responses to E. coli O157:H7 may provide a better understanding of the survival mechanisms of the earthworm Eisenia fetida that lives in E. coli O157:H7-polluted environments. Whole earthworm extracts, collected at days 7, 14, 21, and 28 after E. coli O157:H7 stress, were analyzed by two-dimensional gel electrophoresis and quantitative image analysis. In total, 124 proteins demonstrated significant regulation at least at one time point, and 52 proteins were identified by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry and database searching. Compared with control samples, 11 protein spots were up-regulated and 41 were down-regulated for at least one time point. The identified proteins, including heat shock protein 90, fibrinolytic protease 0, gelsolin-like protein, lombricine kinase, coelomic cytolytic factor-1, manganous superoxide dismutase, catalase, triosephosphate isomerase, extracellular globin-4, lysenin, intermediate filament protein, and glyceraldehyde-3-phosphate dehydrogenase, are involved in several processes, including transcription, translation, the tricarboxylic acid cycle, and the glucose metabolic process. Thus, our study provides a functional profile of the E. coli O157:H7-responsive proteins in earthworms. We suggest that the variable levels and trends in these spots on the gel may be useful as biomarker profiles to investigate E. coli O157:H7 contamination levels in soils.

Growth and stress responses of the earthworm Eisenia fetida to Escherichia coli O157:H7 in an artificial soil

Escherichia coli O157:H7 is an intestine-inhabiting bacterium associated with many severe disease outbreaks worldwide. It may enter the soil environment with the excreta of infected animals (including horses, cattle and chickens) and human. Earthworms are able to protect themselves against invading pathogens due to their efficient innate defense system. To better understand the mechanisms earthworms ( Eisenia fetida) utilize to defend and eliminate Escherichia coli O157:H7, we examined the changes in the growth rate, as well as antioxidant and antimicrobial functions of earthworms in response to various concentrations of Escherichia coli O157:H7 in an artificial soil. Our results show that earthworms can inhibit the growth of Escherichia coli O157:H7. Upon exposure to Escherichia coli O157:H7 from 0 d to 24 d, coelomic cytolytic factor (CCF) in earthworms is induced, the reactive oxygen species (ROSs) and superoxide dismutase (SOD) are also elevated. Under lower bacterial concentrations (10 5–10 6 CFU g −1), these ROSs can be rapidly scavenged by superoxide dismutase (SOD) to avoid peroxidation damage, but when the bacterial concentrations are high (10 7–10 8 CFU g −1), excess amount of ROSs then cause accumulation of lipid peroxidation molecular malondialdehyde (MDA). In addition, exposure to Escherichia coli O157:H7 can induce the gene expression of antimicrobial peptide lumbricin I in the tissues of the earthworms. In conclusion, antioxidant systems and antimicrobial immune function may play important roles in the defense of the earthworms Eisenia fetida against Escherichia coli O157:H7.

An invertebrate TNF functional analogue activates macrophages via lectin-saccharide interaction with ion channels

The invertebrate pattern-recognition protein named coelomic cytolytic factor (CCF) and the mammalian cytokine tumor necrosis factor (TNF) share functional analogies that are based on a similar saccharide recognition specificity. In particular, CCF and TNF have been shown to interact with ion channels on the surface of vertebrate cells via N,N'-diacetylchitobiose lectin-like activity. In the present study, we show that CCF-induced membrane depolarization results in the release of TNF, IL-6 and nitric oxide (NO) by macrophages via nuclear factor-kappaB signaling. Interestingly, our data suggest that TNF contributes, through lectin-saccharide interaction, to the secretion of IL-6 and NO induced by CCF. This experimental non-physiological setting based on the interaction of an invertebrate defense lectin with vertebrate cells involved in the innate immune response may have highlighted an evolutionarily ancient mechanism of macrophage activation in vertebrates.

Comparative study of the CCF-like pattern recognition protein in different Lumbricid species

Coelomic fluid of the Lumbricid Eisenia fetida contains a 42-kDa pattern recognition protein named coelomic cytolytic factor (CCF) that binds microbial cell wall components and triggers the activation of the prophenoloxidase cascade, an important invertebrate defense pathway. Here we report on the sequence characterization of CCF-like molecules of other Lumbricids: Aporrectodea caliginosa, Aporrectodea icterica, Aporrectodea longa, Aporrectodea rosea, Dendrobaena veneta, Lumbricus rubellus and Lumbricus terrestris, and show that CCF from E. fetida has a broader saccharide-binding specificity, being the only one recognizing N, N′-diacetylchitobiose. We suggest that the broad recognition repertoire of E. fetida CCF reflects a particular microbial environment this species lives in.

Anticipating innate immunity without a Toll

Earthworm innate immunity depends upon small and large leukocytes (coelomocytes) that synthesize and secrete humoral antimicrobial molecules (e.g. lysenin, fetidin, eiseniapore, coelomic cytolytic factor [CCF]; Lumbricin I). Small coelomocytes (cytotoxic) are positive (CD11a, CD45RA, CD45RO, CDw49b, CD54, β 2-m and Thy-1 [CD90]; CD24; TNF-α) but negative using other mammalian markers. Large coelomocytes (phagocytic) are uniformly negative. Specific earthworm anti-EFCC 1, 2, 3, 4 mAbs are negative for Drosophila melanogaster hemocytes and mammalian cells but positive those of earthworms. Coelomocytes contain several lysosomal enzymes involved in phagocytosis and a pattern recognition molecule (CCF) that may trigger the prophenoloxidase cascade a crucial innate immune response. Earthworms and other invertebrates possess natural, non- specific, non- clonal, and non- anticipatory immune response governed by germ line genes. Toll and Toll-like receptor signaling is essential for phagocytosis and antimicrobial peptide synthesis and secretion in insects and vertebrates but has not yet been shown to be essential in earthworm innate responses.

The Invertebrate Defence Molecule Coelomic Cytolytic Factor, a Functional Analog of the Cytokine Tumour Necrosis Factor‐α, Interacts with Mammalian Cells through its Lectin‐Like Domain

Coelomic cytolytic factor (CCF) is a 42 kDa invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm Eisenia foetida (Oligochaeta, Annelida). CCF displays a number of similarities with the mammalian cytokine tumour necrosis factor‐α (TNF‐α) as a result of a shared N,N′‐diacetylchitobiose lectin‐like domain. However, these similarities are solely functional and are not based on any (DNA or amino acid) sequence homology, thus suggesting a form of convergent evolution. In particular, the lectin‐like domain of TNF‐α has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride‐sensitive ion channels. This nonreceptor‐mediated activity of TNF‐α has been reported to be involved in the resorption of oedema. Likewise, the lectin‐like domain of CCF also induces membrane depolarization in mammalian cells. Here, we show that CCF appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin‐like domain. This lectin‐like domain of CCF interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. Additionally, we suggest that the JNK/SAPK and Erk1/2 pathways are involved in CCF‐induced macrophage activation. These results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing CCF in the treatment of oedema.

Effect of experimental microbial challenge on the expression of defense molecules in Eisenia foetida earthworm

Earthworms are able to protect themselves against invading pathogens due to efficient innate defense mechanisms. Currently, two types of antimicrobial factors including lysozyme-like molecule and factors with hemolytic activity, as well as a pattern recognition protein named coelomic cytolytic factor (CCF) have been identified in Eisenia foetida earthworms. However, the modulations of these defense molecules during in vivo immune response have not been addressed. In this study, we investigated the effect of experimental challenge with live Gram-negative and Gram-positive bacteria and with β-1,3-glucan on the expression of CCF and the hemolytic factor fetidin. In parallel, we followed levels of hemolytic activity and lysozyme-like activity in the coelomic fluid of challenged earthworms. We show that the biosynthesis of CCF, but not fetidin, is up-regulated upon microbial stimulation. Parenteral administration of bacteria or microbial polysaccharides in earthworms results, in the coelomic fluid, in augmented level of CCF, increased lysozyme-like activity and decreased hemolytic activity. The decreased hemolytic activity of the coelomic fluid reflects the increase of the whole protein content in the absence of synthesis of hemolytic proteins.