Multiple toll-like receptors (TLRs) display differential bacterial and ligand specificity in the earthworm, Eisenia andrei.

Toll-like receptors (TLRs), an ancient and well-conserved group of pattern recognition receptors (PRRs), recognize conserved pathogen-associated molecular patterns. TLRs consist of three domains: the extracellular N-terminal domain, containing one or more leucine-rich repeats (LRRs), responsible for the recognizing and binding of antigens; the type-I transmembrane domain; and the intracellular domain known as the Toll/Interleukin-1 receptor (TIR) domain required for the downstream signaling pathway. We identified six new full-length complementary DNA (cDNA) sequences, Ean-TLR1/2/3/4/5/6. The deduced amino acid sequences indicate that Ean-TLRs consist of one signal peptide, one LRR N-terminal domain (Ean-TLR4/5), varying numbers of LRRs, one (Ean-TLR1/2/3/4/5) or two (Ean-TLR6) LRR C-terminal domains, one type-I transmembrane domain, and a TIR domain. In addition, a TIR domain alignment revealed that three conserved motifs, designated as Box 1, Box 2, and Box 3, contain essential amino acid residues for downstream signaling activity. Phylogenetic analysis of earthworm TLRs generated two separate evolutionary branches representing single (sccTLR) and multiple (mccTLR) cysteine cluster TLRs. Ean-TLR1/2/3/4 (sccTLR type) and Ean-TLR6 (mccTLR type) were clustered with corresponding types of previously reported earthworm TLRs as well as TLRs from Clitellata and Polychaete. As PRRs, earthworm TLRs should be capable of sensing a diverse range of pathogens. Except for Ean-TLR3, which was not responsive to any bacteria, earthworm TLR expression was significantly induced by Gram-positive but not Gram-negative bacteria. Moreover, it is likely that earthworms can differentiate between different species of Gram-positive bacteria via their TLR responses. The ligand specificity of earthworm TLRs suggests that their pathogenic ligand recognition is likely to be as specific and diverse as the mammalian TLR pathogen-sensing system.

Slit-Robo expression in the leech nervous system: insights into eyespot evolution.

BACKGROUND: Slit and Robo are evolutionarily conserved ligand and receptor proteins, respectively, but the number of slit and robo gene paralogs varies across recent bilaterian genomes. Previous studies indicate that this ligand-receptor complex is involved in axon guidance. Given the lack of data regarding Slit/Robo in the Lophotrochozoa compared to Ecdysozoa and Deuterostomia, the present study aims to identify and characterize the expression of Slit/Robo orthologs in leech development. RESULTS: We identified one slit (Hau-slit), and two robo genes (Hau-robo1 and Hau-robo2), and characterized their expression spatiotemporally during the development of the glossiphoniid leech Helobdella austinensis. Throughout segmentation and organogenesis, Hau-slit and Hau-robo1 are broadly expressed in complex and roughly complementary patterns in the ventral and dorsal midline, nerve ganglia, foregut, visceral mesoderm and/or endoderm of the crop, rectum and reproductive organs. Before yolk exhaustion, Hau-robo1 is also expressed where the pigmented eye spots will later develop, and Hau-slit is expressed in the area between these future eye spots. In contrast, Hau-robo2 expression is extremely limited, appearing first in the developing pigmented eye spots, and later in the three additional pairs of cryptic eye spots in head region that never develop pigment. Comparing the expression of robo orthologs between H. austinensis and another glossiphoniid leech, Alboglossiphonia lata allows to that robo1 and robo2 operate combinatorially to differentially specify pigmented and cryptic eyespots within the glossiphoniid leeches. CONCLUSIONS: Our results support a conserved role in neurogenesis, midline formation and eye spot development for Slit/Robo in the Lophotrochozoa, and provide relevant data for evo-devo studies related to nervous system evolution.

Interaction-and-Response Network for Distantly Supervised Relation Extraction.

Distantly supervised relation extraction (DSRE) aims to identify semantic relations from massive plain texts. A broad range of the prior research has leveraged a series of selective attention mechanisms over sentences in a bag to extract relation features without considering dependencies among the relation features. As a result, potential discriminative information existed in the dependencies is ignored, causing a decline in the performance of extracting entity relations. In this article, we focus on going beyond the selective attention mechanisms and propose a new framework termed interaction-and-response network (IR-Net) that adaptively recalibrates the features of sentence, bag, and group levels by explicitly modeling interdependencies among the features on each level. The IR-Net consists of a series of interactive and responsive modules throughout feature hierarchy, seeking to strengthen its power of learning salient discriminative features for distinguishing entity relations. We conduct extensive experiments on three benchmark DSRE datasets, including NYT-10, NYT-16, and Wiki-20m. The experimental results demonstrate that the IR-Net brings obvious improvements in performance when comparing ten state-of-the-art DSRE methods for entity relation extraction.

Peptidoglycan recognition proteins from the earthworm, Eisenia andrei: Differential inducibility and tissue-specific expression.

Several pattern recognition receptors (PRRs) involved in innate immunity have been identified and characterized in earthworms. Peptidoglycan recognition proteins (PGRPs) are highly conserved PRRs that activate effector pathways such as prophenoloxidase cascade and Toll-like receptor pathway. In addition, PGRPs function as an enzyme, N-acetylmuramoyl-l-alanine amidase (NAMLAA), to directly hydrolyze peptidoglycan. We identified four full-length complementary DNA (cDNA) sequences, Ean-PGRP1/2/3/4, in Eisenia andrei, an earthworm. Sequence and phylogenetic analyses indicate that earthworm PGRP orthologs resemble short PGRP member proteins. The subcellular localizations of four Ean-PGRPs lacking the transmembrane domain are predicted to be extracellular or cytoplasmic. All Ean-PGRPs contain a highly conserved PGRP domain with a conserved Zn2+ binding site including a tyrosine residue essential for active amidase activity. Three highly conserved amino-acid residues (His, Trp, and Thr) necessary for amidase activity are also present, indicating that the Ean-PGRPs can be predicted to have amidase activity. Furthermore, we demonstrate that the Ean-PGRP genes are differentially induced by certain bacterial species, suggesting that the innate immune system of earthworms is likely to be somewhat specific rather than entirely non-specific. Tissue expression patterns indicate that Ean-PGRP mRNAs are primarily expressed in the immune-competent tissues and that their expression is tissue-specific according to Ean-PGRP types, particularly for Ean-PGRP1.

Head transcriptome profiling of glossiphoniid leech (Helobdella austinensis) reveals clues about proboscis development.

Cephalization refers to the evolutionary trend towards the concentration of neural tissues, sensory organs, mouth and associated structures at the front end of bilaterian animals. Comprehensive studies on gene expression related to the anterior formation in invertebrate models are currently lacking. In this study, we performed de novo transcriptional profiling on a proboscis-bearing leech (Helobdella austinensis) to identify differentially expressed genes (DEGs) in the anterior versus other parts of the body, in particular to find clues as to the development of the proboscis. Between the head and the body, 132 head-specific DEGs were identified, of which we chose 11 to investigate their developmental function during embryogenesis. Analysis of the spatial expression of these genes using in situ hybridization showed that they were characteristically expressed in the anterior region of the developing embryo, including the proboscis. Our results provide information on the genes related to head formation and insights into the function of proboscis-related genes during organogenesis with the potential roles of genes not yet characterized.

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.

Behavioral variation according to feeding organ diversification in glossiphoniid leeches (Phylum: Annelida).

Adaptive radiation is a phenomenon in which various organs are diversified morphologically or functionally as animals adapt to environmental inputs. Leeches exhibit a variety of ingestion behaviors and morphologically diverse ingestion organs. In this study, we investigated the correlation between behavioral pattern and feeding organ structure of leech species. Among them, we found that Alboglossiphonia sp. swallows prey whole using its proboscis, whereas other leeches exhibit typical fluid-sucking behavior. To address whether the different feeding behaviors are intrinsic, we investigated the behavioral patterns and muscle arrangements in the earlier developmental stage of glossiphoniid leeches. Juvenile Glossiphoniidae including the Alboglossiphonia sp. exhibit the fluid ingestion behavior and have the proboscis with the compartmentalized muscle layers. This study provides the characteristics of leeches with specific ingestion behaviors, and a comparison of structural differences that serves as the first evidence of the proboscis diversification.

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

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

Identification and Spatiotemporal Expression of Adenosine Deaminases Acting on RNA (ADAR) during Earthworm Regeneration: Its Possible Implication in Muscle Redifferentiation.

Adenosine deaminases acting on RNA (ADAR) catalyze the hydrolytic deamination of adenosine (A) to produce inosine (I) in double-stranded RNA substrates. A-to-I RNA editing has increasingly broad physiological significance in development, carcinogenesis, and environmental adaptation. Perionyx excavatus is an earthworm with potent regenerative potential; it can regenerate the head and tail and is an advantageous model system to investigate the molecular mechanisms of regeneration. During RNA sequencing analysis of P. excavatus regenerates, we identified an ADAR homolog (Pex-ADAR), which led us to examine its spatial and temporal expression to comprehend how Pex-ADAR is linked to regeneration. At first, in domain analysis, we discovered that Pex-ADAR only has one double-stranded RNA-binding domain (dsRBD) and a deaminase domain without a Z-DNA-binding domain (ZBD). In addition, a comparison of the core deaminase domains of Pex-ADAR with those of other ADAR family members indicated that Pex-ADAR comprises the conserved three active-site motifs and a glutamate residue for catalytic activity. Pex-ADAR also shares 11 conserved residues, a characteristic of ADAR1, supporting that Pex-ADAR is a member of ADAR1 class. Its temporal expression was remarkably low in the early stages of regeneration before suddenly increasing at 10 days post amputation (dpa) when diverse cell types and tissues were being regenerated. In situ hybridization of Pex-ADAR messenger RNA (mRNA) indicated that the main expression was observed in regenerating muscle layers and related connective tissues. Taken together, the present results demonstrate that an RNA-editing enzyme, Pex-ADAR, is implicated in muscle redifferentiation during earthworm regeneration.

Characterization of Perionyx excavatus Development and Its Head Regeneration.

Regeneration is a biological process restoring lost or amputated body parts. The capability of regeneration varies among organisms and the regeneration of the central nervous system (CNS) is limited to specific animals, including the earthworm Perionyx excavatus. Thus, it is crucial to establish P. excavatus as a model system to investigate mechanisms of CNS regeneration. Here, we set up a culture system to sustain the life cycle of P. excavatus and characterize the development of P. excavatus, from embryo to juvenile, based on its morphology, myogenesis and neurogenesis. During development, embryos have EdU-positive proliferating cells throughout the whole body, whereas juveniles maintain proliferating cells exclusively in the head and tail regions, not in the trunk region. Interestingly, juveniles amputated at the trunk, which lacks proliferating cells, are able to regenerate the entire head. In this process, a group of cells, which are fully differentiated, reactivates cell proliferation. Our data suggest that P. excavatus is a model system to study CNS regeneration, which is dependent on the dedifferentiation of cells.

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

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

Spatiotemporal Expression of Anticoagulation Factor Antistasin in Freshwater Leeches.

Antistasin, which was originally discovered in the salivary glands of the Mexican leech Haementeria officinalis, was newly isolated from Helobdella austinensis. To confirm the temporal expression of antistasin during embryogenesis, we carried out semi-quantitative RT-PCR. Hau-antistasin1 was uniquely expressed at stage 4 of the cleavage and was strongly expressed in the late stages of organogenesis, as were other antistasin members. In order to confirm the spatial expression of antistasin, we performed fluorescence in situ hybridization in the late stages of organogenesis. The expression of each antistasin in the proboscis showed a similar pattern and varied in expression in the body. In addition, the spatial expression of antistasin orthologs in different leeches showed the possibility of different function across leech species. Hau-antistasin1 was expressed in the same region as hedgehog, which is a known mediator of signal transduction pathway. Hau-antistasin1 is probably a downstream target of Hedgehog signaling, involved in segment polarity signal pathway.

Identification and expression pattern of a new digestive invertebrate-type lysozyme from the earthworm.

BACKGROUND: The invertebrate type (i-type) lysozyme not showing a clear homology with the known types of lysozyme was first demonstrated from a marine bivalve, conch and earthworm by N-terminal sequence. An i-type lysozyme isolated from the earthworm found to be up-regulated upon bacterial challenge, suggesting this lysozyme to function as an inducible immune factor. However, information on the i-type lysozyme related with digestive function is very limited in the earthworm. OBJECTIVE: The objective of this study is to investigate the molecular characteristics and function of the new i-type lysozyme from the earthworm. METHODS: To identify a new i-type lysozyme, multiple amino acid sequence alignment and phylogenetic analyses were employed. Its mRNA expression pattern was observed by fluorescent in situ hybridization (FISH). RESULTS: A new i-type lysozyme (Ea-iLys) from an earthworm, Eisenia andrei with the open reading frame of 678 bp (226 amino acid residues) appeared to comprise conserved 14 cysteine residues for disulfide bridges and amino acid residues for the enzyme activities of lysozyme and isopeptidase, of which mRNA expression is mainly localized in the lining of midgut epithelium. No significant expression signal was detected in immune competent sites such as chloragogue tissue, typhlosole region, body coelom and muscle layers. CONCLUSION: Our results suggest that this enzyme primarily acts as a digestive enzyme rather than an innate immune factor.

Temporal and spatial expression of the Fox gene family in the Leech Helobdella austinensis.

The Forkhead box (Fox) gene family is an evolutionarily ancient gene family named after the Drosophila melanogaster forkhead gene (fkh). Fox genes are highly conserved transcription factors critical for embryogenesis and carcinogenesis. In the current study, we report a whole-genome survey of Fox genes and their expression patterns in the leech Helobdella austienesis. Phylogenetic analysis suggests that some Fox genes of leeches are correlated with other Lophotrochozoa and vertebrate Fox genes. Here we have performed semiquantitative reverse transcription polymerase chain reaction and whole-mount in situ hybridization of Fox genes throughout the embryonic development of H. austinensis. We found that each one of the leech Fox genes (FoxA1, FoxA3, FoxC, FoxL2, FoxO1, and FoxO2) is expressed in a specific set of cells or tissue type. From Stages 9-11, Hau-FoxA1 was expressed in the foregut of the anterior region, and Hau-FoxL2 was expressed in mesodermal muscle fiber. Hau-FoxA3 was temporally expressed in the ventral neuroectoderm. At Stage 11, Hau-FoxC was expressed in the foregut. Hau-FoxO genes have a ubiquitous expression. Our results provide more insight on the evolutionary linkage and role of the Fox gene function in Bilateria.

Expression patterns of duplicated snail genes in the leech Helobdella.

snail gene family members are zinc-finger transcription factors with key roles in morphogenesis. Involvement of snail family genes in mesoderm formation has been observed in insects and mammals. The snail genes are also involved in cell motility, neural differentiation, cell fate, survival decision, and left-right identity. The functions of snail genes have been studied primarily among ecdysozoans and deuterostomes, with relatively little work carried out in lophotrochozoans. In this study, we isolated two snail homologs (Hau-snail1 and Hau-snail2) from the leech Helobdella austinensis. We characterized the temporal and spatial expression patterns of these two genes by semi-quantitative RT-PCR and in situ hybridization. The expression of Hau-snail1 and Hau-snail2 correlates with ventral nerve cord (VNC) development, segmental mesoderm, and with a ring of cells that comes to lie at the base of the leech proboscis, respectively, showing similarity to the divergent expression of duplicated snail genes in polychaetes. Our results do not support the function of lophotrochozoan snail genes in mesoderm specification.

Spatiotemporal expression of a twist homolog in the leech Helobdella austinensis.

Genes of the twist family encode bHLH transcription factors known to be involved in the regulation and differentiation of early mesoderm. Here, we report our characterization of Hau-twist, a twist homolog from the leech Helobdella austinensis, a tractable lophotrochozoan representative. Hau-twist was expressed in segmental founder cells of the mesodermal lineage, in subsets of cells within the mesodermal lineage of the germinal plate, in circumferential muscle fibers of a provisional integument during segmentation and organogenesis stages and on the ventral side of the developing proboscis. Thus, consistent with other systems, our results suggest that twist gene of the leech Helobdella might function in mesoderm differentiation.

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.

Long noncoding RNA linc00598 regulates CCND2 transcription and modulates the G1 checkpoint.

Data derived from genomic and transcriptomic analyses have revealed that long noncoding RNAs (lncRNAs) have important roles in the transcriptional regulation of various genes. Recent studies have identified the mechanism underlying this function. To date, a variety of noncoding transcripts have been reported to function in conjunction with epigenetic regulator proteins. In this study, we investigated the function of linc00598, which is transcribed by a genomic sequence on chromosome 13, downstream of FoxO1 and upstream of COG6. Microarray analysis showed that linc00598 regulates the transcription of specific target genes, including those for cell cycle regulators. We discovered that linc00598 regulates CCND2 transcription through modulation of the transcriptional regulatory effect of FoxO1 on the CCND2 promoter. Moreover, we observed that knockdown of linc00598 induced G0/G1 cell cycle arrest and inhibited proliferation. These data indicate that linc00598 plays an important role in cell cycle regulation and proliferation through its ability to regulate the transcription of CCND2.

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.

Additional duplicated Hox genes in the earthworm: Perionyx excavatus Hox genes consist of eleven paralog groups.

Annelida is a lophotrochozoan phylum whose members have a high degree of diversity in body plan morphology, reproductive strategies and ecological niches among others. Of the two traditional classes pertaining to the phylum Annelida (Polychaete and Clitellata), the structure and function of the Hox genes has not been clearly defined within the Oligochaeta class. Using a PCR-based survey, we were able to identify five new Hox genes from the earthworm Perionyx excavatus: a Hox3 gene (Pex-Hox3b), two Dfd genes (Pex-Lox6 and Pex-Lox18), and two posterior genes (Pex-post1 and -post2a). Our result suggests that the eleven earthworm Hox genes contain at least four paralog groups (PG) that have duplicated. We found the clitellates-diagnostic signature residues and annelid signature motif. Also, we show by semi-quantitative RT-PCR that duplicated Hox gene orthologs are differentially expressed in six different anterior-posterior body regions. These results provide essential data for comparative evolution of the Hox cluster within the Annelida.