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

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

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

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

Injury-Induced Innate Immune Response During Segment Regeneration of the Earthworm, Eisenia andrei.

Regeneration of body parts and their interaction with the immune response is a poorly understood aspect of earthworm biology. Consequently, we aimed to study the mechanisms of innate immunity during regeneration in Eisenia andrei earthworms. In the course of anterior and posterior regeneration, we documented the kinetical aspects of segment restoration by histochemistry. Cell proliferation peaked at two weeks and remitted by four weeks in regenerating earthworms. Apoptotic cells were present throughout the cell renewal period. Distinct immune cell (e.g., coelomocyte) subsets were accumulated in the newly-formed blastema in the close proximity of the apoptotic area. Regenerating earthworms have decreased pattern recognition receptors (PRRs) (e.g., TLR, except for scavenger receptor) and antimicrobial peptides (AMPs) (e.g., lysenin) mRNA patterns compared to intact earthworms. In contrast, at the protein level, mirroring regulation of lysenins became evident. Experimental coelomocyte depletion caused significantly impaired cell divisions and blastema formation during anterior and posterior regeneration. These obtained novel data allow us to gain insight into the intricate interactions of regeneration and invertebrate innate immunity.

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

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

Cadmium-Related Effects on Cellular Immunity Comprises Altered Metabolism in Earthworm Coelomocytes.

The heavy metal cadmium (Cd) is known to modulate the immune system, challenging soil-dwelling organisms where environmental Cd pollution is high. Since earthworms lack adaptive immunity, we determined Cd-related effects on coelomocytes, the cellular part of innate immunity, which is also the site of detoxification processes. A proteomics approach revealed a set of immunity-related proteins as well as gene products involved in energy metabolism changing in earthworms in response to Cd exposure. Based on these results, we conducted extracellular flux measurements of oxygen and acidification to reveal the effect of Cd on coelomocyte metabolism. We observed a significantly changing oxygen consumption rate, extracellular acidification, as well as metabolic potential, which can be defined as the response to an induced energy demand. Acute changes in intracellular calcium levels were also observed, indicating impaired coelomocyte activation. Lysosomes, the cell protein recycling center, and mitochondrial parameters did not change. Taken together, we were able to characterize coelomocyte metabolism to reveal a potential link to an impaired immune system upon Cd exposure.

Transcriptomic analysis of oligochaete immune responses to myxosporeans infection: Branchiura sowerbyi infected with Myxobolus cultus.

The Myxozoa are endoparasites characterized by a two-host life cycle that typically involves invertebrates and vertebrates as definitive and intermediate hosts, respectively. However, little is known about invertebrate-myxosporean interactions, particularly about patterns of host immune defense. We used RNA-sequencing to identify genes that are possibly involved in the immune responses of the oligochaete Branchiura sowerbyi naturally infected with Myxobolus cultus. De novo assembly of the B. sowerbyi transcriptome yielded 119,031 unigenes, with an average length of 896 bp and an N50 length of 1754 bp. Comparative transcriptome analysis revealed 4059 differentially expressed genes (DEGs) between M. cultus-infected and uninfected B. sowerbyi groups, including 3802 upregulated genes and 257 downregulated genes. Among the B. sowerbyi immune factors implicated in the responses to M. cultus infection, DEGs related to lectins, ubiquitin-mediated proteolysis, phagocytosis, oxidative-antioxidative responses, proteases, and protease inhibitors were upregulated. The expression of some immune-related molecules such as calmodulin, heat shock proteins, antimicrobial peptides, lysenin, and serum amyoid A protein were also significantly upregulated. The expression patterns of 14 immune-related DEGs identified by RNA-seq were validated by quantitative real-time polymerase chain reaction. This study is the first attempt to characterize the B. sowerbyi transcriptome and identify immune-related molecules possibly associated with M. cultus infection. It is also the first report of invertebrate host-myxosporean interactions at the transcriptomic level. Our results will facilitate the elucidation of adaptive evolution mechanisms of myxosporean parasites in the definitive host and the genetic basis for differences in resistance of invertebrate hosts of different genotypes to a myxosporean species.

Developmental and Immune Role of a Novel Multiple Cysteine Cluster TLR From Eisenia andrei Earthworms.

Earthworms are not endowed with adaptive immunity and they are rely on the tools of innate immunity. Cells of the innate immune system utilize pattern recognition receptors, such as Toll-like receptors, to detect the pathogen-associated molecular patterns (PAMPs). The first earthworm TLR was isolated from Eisenia andrei earthworms (EaTLR), which belongs to the single cysteine cluster TLR (sccTLR). Here, we identified a new multiple cysteine cluster TLR (mccTLR) in E. andrei earthworms. Phylogenetic DNA analysis revealed that it has no variability within one earthworm as well as in the population. By screening of the tissue expression profile, the TLR was expressed primarily in earthworm seminal vesicles and receptacles suggesting a connection to sperm cells. Seminal vesicles are often heavily infected by gregarine parasites. As a sign of immune response, a strong melanization reaction is visible around parasites. Stimulation experiments with profilin from related parasite Toxoplasma gondii, led to the upregulation of mccEaTLR in the earthworm seminal vesicles. Also, profilin activated prophenoloxidase cascade, the efficient mechanism of innate immunity. However, its involvement in the NF-κB signaling was not proven. Further, we provide evidence that the antibiotics metronidazole and griseofulvin destroyed the developing spermatocytes. The observed decrease in the mccEaTLR mRNA levels after the antibiotic treatment of parasites is caused by the decline of sperm cells numbers rather than by diminution of the parasites. Since earthworms with extensively reduced parasite load had a similar amount of mccEaTLR mRNA, presumably, earthworm sperm cells have a certain level of mccEaTLR expressed as a standard, which can be augmented by particular antigenic stimulation. Also, mccEaTLR was expressed mainly in the early stages of earthworm development and presumably is primarily involved in early embryonic development. Expression of mccEaTLR in seminal vesicles correlates with the expression of endothelial monocyte-activation polypeptide II. High-throughput sequencing of gregarine DNA from seminal vesicles of individual earthworms resulted in great diversity of the observed genotypes. Phylogenetically, all observed OTUs belong to the clade of earthworm gregarines suggesting host specificity. Overall, mccEaTLR is supposed to play a function role in early embryonic development and potentially it participates in immune response against parasites.

Identification of novel lumbricin homologues in Eisenia andrei earthworms.

Lumbricin and its orthologue antimicrobial peptides were typically isolated from annelids. In this report, mRNA for lumbricin and -serendipitously- a novel lumbricin-related mRNA sequence were identified in Eisenia andrei earthworms. The determined mRNA sequences of E. andrei lumbricin and lumbricin-related peptide consist of 477 and 575 nucleotides. The precursors of proline-rich E. andrei lumbricin and the related peptide contain 63 and 59 amino acids, respectively. Phylogenetic analysis indicated close relationship with other annelid lumbricins. Highest expression of both mRNAs appeared in the proximal part of the intestine (pharynx, gizzard), while other tested organs had moderate (body wall, midgut, ovary, metanephridium, seminal vesicles, ventral nerve cord) or low (coelomocytes) levels. During ontogenesis their expression revealed continuous increase in embryos. Following 48 h of in vivo Gram-positive bacteria challenge both mRNAs were significantly elevated in coelomocytes, while Gram-negative bacteria or zymosan stimulation had no detectable effects.

Earthworm coelomocyte extracellular traps: structural and functional similarities with neutrophil NETs.

Invertebrate immunity is associated with natural mechanisms that include cellular and humoral elements, similar to those that play a role in vertebrate innate immune responses. Formation of extracellular traps (ETs) is a newly discovered mechanism to combat pathogens, operating not only in vertebrate leucocytes but also in invertebrate immune cells. The ET components include extracellular DNA (exDNA), antimicrobial proteins and histones. Formation of mammalian ETs depends on enzymes such as neutrophil elastase, myeloperoxidase, the citrullination of histones and protease activity. It was confirmed that coelomocytes-immunocompetent cells of the earthworm Eisenia andrei-are also able to release ETs in a protease-dependent manner, dependent or independent of the formation of reactive oxygen species and rearrangement of the cell cytoskeleton. Similar to vertebrate leukocytes (e.g., neutrophil), coelomocytes are responsible for many immune functions like phagocytosis, cytotoxicity and secretion of humoral factors. ETs formed by coelomocyte analogues to neutrophil ETs consist of exDNA, histone H3 and attached to these structures proteins, e.g., heat shock proteins HSP27. The latter fact confirms that mechanisms of ET release are conserved in evolution. The study on Annelida adds this animal group to the list of invertebrates capable of ET release, but most importantly provides insides into innate mechanisms of ET formation in lower animal taxa.

Environmental pollutants, pathogens and immune system in earthworms.

Earthworms also known as farmer's friends are natural tillers of soil. They belong to Phylum Annelida and class Oligochaeta. Acid soils with organic matter and surface humus maintain the largest fauna of worms and earthworms. Due to their habitat in soil, they are constantly exposed to microbes and pollution generated by anthropogenic sources. Studies have revealed that damage of the immune system of earthworms can lead to alterations of both morphological and cellular characteristics of worms, activation of signalling pathways and can strongly influence their survival. Therefore, the understanding of the robust immune system in earthworms has become very important from the point of view of understanding its role in combating pathogens and pollutants and its role in indicating the soil pollution. In this article, we have outlined the (i) components of the immune system and (ii) their function of immunological responses on exposure to pollutants and pathogens. This study finds importance from the point of view of ecotoxicology and monitoring of earthworm health and exploring the scope of earthworm immune system components as biomarkers of pollutants and environmental toxicity. The future scope of this review remains in understanding the earthworm immunobiology and indicating strong biomarkers for pollution.

Bisphenol A in artificial soil: Effects on growth, reproduction and immunity in earthworms.

The application of biosolids in agricultural fields is increasing annually. They contain not only nutrients but also xenobiotics, such as Bisphenol A (BPA). These compounds are not regulated in the use of biosolids in agriculture, which highlights the need to assess their effects on soil life, of which earthworms are most abundant of the animal representatives. In this study the effect of BPA on life-history parameters, such as mortality, growth and reproduction, and on immunity, is evaluated for Dendrobaena veneta and Eisenia fetida. Sublethal concentrations were evaluated by a modified OECD artificial soil test. Decline in growth with increasing concentration of BPA was detected during the first two weeks and the opposite effect for the next two, although these differences were only significant at the highest concentration. Reproduction traits were only significantly different for E. fetida, for which the number of juveniles decreased at higher concentrations, thus showing different sensitivity in both species. By using a contact test, the potentially harmful effect of direct contact with BPA was shown to be much higher than in soil (resembling natural) conditions. Finally, results indicate that BPA may not affect the immune system of these animals, at least in terms of coelomocyte viability.

Lethal and sub-lethal evaluation of Indigo Carmine dye and byproducts after TiO2 photocatalysis in the immune system of Eisenia andrei earthworms.

The Indigo carmine (IC) dye has been widely used in textile industries, even though it has been considered toxic for rats, pigs and humans. Owing to its toxicity, wastes containing this compound should be treated to minimize or eliminate their toxic effects on the biota. As an alternative to wastewater treatment, advanced oxidative processes (AOPs) have been highlighted due to their high capacity to destruct organic molecules. In this context, this study aimed to evaluate Indigo Carmine toxicity to soil organisms using the earthworm Eisenia andrei as a model-organism and also verify the efficiency of AOP in reducing its toxicity to these organisms. To this end, lethal (mortality) and sub-lethal (loss or gain of biomass, reproduction, behavior, morphological changes and immune system cells) effects caused by this substance and its degradation products in these annelids were evaluated. Morphological changes were observed even in organisms exposed to low concentrations, while mortality was the major effect observed in individuals exposed to high levels of indigo carmine dye. The organisms exposed to the IC during the contact test showed mortality after 72h of exposure (LC50 = 75.79mgcm-2), while those exposed to photoproducts showed mortality after 48h (LC50 = 243min). In the chronic study, the organisms displayed a mortality rate of 14%, while those exposed to the photoproduct reached up to 32.7%. A negative influence of the dye on the reproduction rate was observed, while by-products affected juvenile survival. A loss of viability and alterations in the cellular proportion was verified during the chronic test. However, the compounds did not alter the behavior of the annelids in the leak test (RL ranged from 20% to 30%). Although photocatalysis has been presented as an alternative technology for the treatment of waste containing the indigo carmine dye, this process produced byproducts even more toxic than the original compounds to E. andrei.

Lysenin family proteins in earthworm coelomocytes - Comparative approach.

Sphingomyelin-binding proteins of the lysenin family were originally identified in earthworms belonging to the genus Eisenia comprised of at least two distinct species, E. andrei and E. fetida, until recently considered subspecies or morphotypes of E. foetida (sic). In the present study the presence of lysenin and lysenin-related protein 2 (LRP-2, known also as fetidin) was detected in coelomocytes retrieved from all investigated adult specimens of E. andrei, and E. fetida. They were accompanied by LRP-3 and LRP-1 in some specimens of E. andrei and E. fetida, respectively. Lysenins were not observed in a third composting lumbricid species, Dendrobaena veneta, which served as a convenient negative reference for techniques and procedures used in the study. The pore-forming potential of soluble and cellular fractions of coelomic fluid was studied towards sheep red blood cells and sphingomyelin-rich liposomes. After experimental depletion the potential was restored in parallel with restoration of chloragocyte-derived eleocytes in both E. andrei and E. fetida.

A new earthworm cellulase and its possible role in the innate immunity.

A new endogenous cellulase (Ean-EG) from the earthworm, Eisenia andrei and its expression pattern are demonstrated. Based on a deduced amino acid sequence, the open reading frame (ORF) of Ean-EG consisted of 1368 bps corresponding to a polypeptide of 456 amino acid residues in which is contained the conserved region specific to GHF9 that has the essential amino acid residues for enzyme activity. In multiple alignments and phylogenetic analysis, the deduced amino acid sequence of Ean- EG showed the highest sequence similarity (about 79%) to that of an annelid (Pheretima hilgendorfi) and could be clustered together with other GHF9 cellulases, indicating that Ean-EG could be categorized as a member of the GHF9 to which most animal cellulases belong. The histological expression pattern of Ean-EG mRNA using in situ hybridization revealed that the most distinct expression was observed in epithelial cells with positive hybridization signal in epidermis, chloragogen tissue cells, coelomic cell-aggregate, and even blood vessel, which could strongly support the fact that at least in the earthworm, Eisenia andrei, cellulase function must not be limited to digestive process but be possibly extended to the innate immunity.

Transcriptomic and proteomic insights into innate immunity and adaptations to a symbiotic lifestyle in the gutless marine worm Olavius algarvensis.

BACKGROUND: The gutless marine worm Olavius algarvensis has a completely reduced digestive and excretory system, and lives in an obligate nutritional symbiosis with bacterial symbionts. While considerable knowledge has been gained of the symbionts, the host has remained largely unstudied. Here, we generated transcriptomes and proteomes of O. algarvensis to better understand how this annelid worm gains nutrition from its symbionts, how it adapted physiologically to a symbiotic lifestyle, and how its innate immune system recognizes and responds to its symbiotic microbiota. RESULTS: Key adaptations to the symbiosis include (i) the expression of gut-specific digestive enzymes despite the absence of a gut, most likely for the digestion of symbionts in the host's epidermal cells; (ii) a modified hemoglobin that may bind hydrogen sulfide produced by two of the worm's symbionts; and (iii) the expression of a very abundant protein for oxygen storage, hemerythrin, that could provide oxygen to the symbionts and the host under anoxic conditions. Additionally, we identified a large repertoire of proteins involved in interactions between the worm's innate immune system and its symbiotic microbiota, such as peptidoglycan recognition proteins, lectins, fibrinogen-related proteins, Toll and scavenger receptors, and antimicrobial proteins. CONCLUSIONS: We show how this worm, over the course of evolutionary time, has modified widely-used proteins and changed their expression patterns in adaptation to its symbiotic lifestyle and describe expressed components of the innate immune system in a marine oligochaete. Our results provide further support for the recent realization that animals have evolved within the context of their associations with microbes and that their adaptive responses to symbiotic microbiota have led to biological innovations.

Phenotypic and functional characterization of earthworm coelomocyte subsets: Linking light scatter-based cell typing and imaging of the sorted populations.

Flow cytometry is a common approach to study invertebrate immune cells including earthworm coelomocytes. However, the link between light-scatter- and microscopy-based phenotyping remains obscured. Here we show, by means of light scatter-based cell sorting, both subpopulations (amoebocytes and eleocytes) can be physically isolated with good sort efficiency and purity confirmed by downstream morphological and cytochemical applications. Immunocytochemical analysis using anti-EFCC monoclonal antibodies combined with phalloidin staining has revealed antigenically distinct, sorted subsets. Screening of lectin binding capacity indicated wheat germ agglutinin (WGA) as the strongest reactor to amoebocytes. This is further evidenced by WGA inhibition assays that suggest high abundance of N-acetyl-d-glucosamine in amoebocytes. Post-sort phagocytosis assays confirmed the functional differences between amoebocytes and eleocytes, with the former being in favor of bacterial engulfment. This study has proved successful in linking flow cytometry and microscopy analysis and provides further experimental evidence of phenotypic and functional heterogeneity in earthworm coelomocyte subsets.

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.

Sensing microorganisms in the gut triggers the immune response in Eisenia andrei earthworms.

The tube-within-tube body plan of earthworms is appropriate for studying the interactions of microorganisms with the immune system of body cavities such as the digestive tract and coelom. This study aims to describe the immune response on the molecular and cellular level in the coelomic cavity and the gut of the earthworm Eisenia andrei after experimental microbial challenge by administering two bacterial strains (Escherichia coli and Bacillus subtilis) or yeast Saccharomyces cerevisiae to the environment. The changes in mRNA levels of defense molecules (pattern recognition receptor CCF, lysozyme, fetidin/lysenins) in the coelomocytes and gut tissue were determined by quantitative PCR. The immune response at a cellular level was captured in histological sections, and the expression of CCF was localized using in situ hybridization. Coelomocytes respond to the presence of bacteria in the coelomic cavity by increasing the mRNA levels of defense molecules, especially CCF. The immune response in gut tissue is less affected by microbial stimulation because the epithelial cells of gut exhibit basically strong mRNA synthesis of ccf as a defense against the continuous microbial load in the gut lumen. The cellular immune response is mediated by coelomocytes released from the mesenchymal lining of the coelomic cavity. These combined immune mechanisms are necessary for the survival of earthworms in the microbially rich environment of soil.

Gene expression of TLR homologues identified by genome-wide screening of the earthworm Dendrobaena veneta.

TLRs represent one of the most important components of innate immunity. Currently, these receptors have been extensively studied in vertebrates and insects, but our knowledge for annelids is very limited. Therefore, the aim of our study was to identify earthworm TLR homologs by genome-wide screening, and to determine the expression of candidate genes as a response to Gram-positive and Gram-negative bacteria. Using a combination of deep pyrosequencing and quantitative PCR we found six candidate genes, for which all were expressed in Dentrobaena veneta. Two of the candidates showed significant response to bacterial exposure. In conclusion, TLRs seem to have a role in earthworm immunology.

Gene expression profiling of coelomic cells and discovery of immune-related genes in the earthworm, Eisenia andrei, using expressed sequence tags.

The coelomic cells of the earthworm consist of leukocytes, chlorogocytes, and coelomocytes, which play an important role in innate immunity reactions. To gain insight into the expression profiles of coelomic cells of the earthworm, Eisenia andrei, we analyzed 1151 expressed sequence tags (ESTs) derived from the cDNA library of the coelomic cells. Among the 1151 ESTs analyzed, 493 ESTs (42.8%) showed a significant similarity to known genes and represented 164 unique genes, of which 93 ESTs were singletons and 71 ESTs manifested as two or more ESTs. From the 164 unique genes sequenced, we found 24 immune-related and cell defense genes. Furthermore, real-time PCR analysis showed that levels of lysenin-related proteins mRNA in coelomic cells of E. andrei were upregulated after the injection of Bacillus subtilis bacteria. This EST data-set would provide a valuable resource for future researches of earthworm immune system.