Transcriptomic profiling and the first spatial expression analysis of candidate genes in the salivary gland of the East Asian medicinal leech, Hirudo nipponia.

Hirudo nipponia, a blood-sucking leech native to East Asia, possesses a rich repertoire of active ingredients in its saliva, showcasing significant medical potential due to its anticoagulant, anti-inflammatory, and antibacterial effects against human diseases. Despite previous studies on the transcriptomic and proteomic characteristics of leech saliva, which have identified medicinal compounds, our knowledge of tissue-specific transcriptomes and their spatial expression patterns remains incomplete. In this study, we conducted an extensive transcriptomic profiling of the salivary gland tissue in H. nipponia based on de novo assemblies of tissue-specific transcriptomes from the salivary gland, teeth, and general head region. Through gene ontology (GO) analysis and hierarchical clustering, we discovered a novel set of anti-coagulant factors-i.e., Hni-Antistasin, Hni-Ghilanten, Hni-Bdellin, Hni-Hirudin-as well as a previously unrecognized immune-related gene, Hni-GLIPR1 and uncharacterized salivary gland specific transcripts. By employing in situ hybridization, we provided the first visualization of gene expression sites within the salivary gland of H. nipponia. Our findings expand on our understanding of transcripts specifically expressed in the salivary gland of blood-sucking leeches, offering valuable resources for the exploration of previously unidentified substances with medicinal applications.

Make it double: identification and characterization of a Tandem-Hirudin from the Asian medicinal leech Hirudinaria manillensis.

Haematophagous leeches express a broad variety of secretory proteins in their salivary glands, among them are hirudins and hirudin-like factors. Here, we describe the identification, molecular and initial functional characterization of Tandem-Hirudin (TH), a novel salivary gland derived factor identified in the Asian medicinal leech, Hirudinaria manillensis. In contrast to the typical structure of hirudins, TH comprises two globular domains arranged in a tandem-like orientation and lacks the elongated C-terminal tail. Similar structures of thrombin inhibitors have so far been identified only in kissing bugs and ticks. Expression of TH was performed in both cell-based and cell-free bacterial systems. A subsequent functional characterization revealed no evidence for a thrombin-inhibitory potency of TH.

Therapeutic effects of Hirudo medicinalis extract antigens on modulation of CD4+CD25+Foxp3 T cell activity in murine eimeriosis.

Eimeriosis is a common parasitic disease in the chicken industry. The aim of this study was to assess the protective role of Hirudo extract antigens (HEA) against murine eimeriosis induced by Eimeria papillate. The oocyst output, developmental stages, goblet cells and oxidative stress, were investigated. Immunohistochemistry was used to detect anti-apoptotic Bcl2 marker and the number of both CD4+ and CD25+ cells in jejunal tissue, while ELISA was used to quantify TGF-ß, IL-10 and IL-22 in jejunal tissue homogenate. Real-time PCR was also used to detect mRNA expression of mucin 2 (MUC2), inducible nitric oxide synthase (iNOS), IL-1ß, IFN-γ, TNF-α, IL-6, and FoxP3. The most effective dose (5 µg/mice) reduced the oocyst output by 82.95 ± 1.02% (P ˂ 0.001). Similarly, the same dose reduced the jejunal developmental stages by 66.67 ± 0.49% (P ˂ 0.001). Furthermore, HEA therapy increased the number of jejunal goblet cells by 12.8 ± 1 (P ˂ 0.001) and the expression of MUC2 by 0.83 ± 0.06 (P ˂ 0.001). In contrast, TNF-α, IFN-γ, IL-6, iNOS, and IL-1ß expression as well as apoptosis were reduced. The number of CD4+ and CD25+ in the jejunal tissue was increased (14.6 ± 1.2 (P ˂ 0.001), 6.84 ± 1 (P ˂ 0.01), respectively) after HEA therapy. The molecular analysis showed an increased expression of intestinal Foxp3 (3.2 ± 0.13 (P ˂ 0.001), while IL-22 was reduced (124 ± 10 (P ˂ 0.001)) versus an increase in TGF-ß (250 ± 17 (P ˂ 0.01)) and IL-10 (236 ± 16 (P ˂ 0.001)) after HEA treatment in comparison to the non-treated infected group. With respect to the infected group, HEA reduced lipid peroxidation (LPO) (15.7 ± 1.12 (P ˂ 0.001)) and nitric oxide (NO) (13 ± 1.3 (P ˂ 0.001)) but increased reduced glutathione (GSH) (3.7 ± 0.26 (P ˂ 0.001)). In conclusion, HEA therapy protected against intestinal tissue damage by activation of CD4+CD25+Foxp3 cells which showed anti-inflammatory action. Hence, HEA can be recommended as a therapeutic treatment for eimeriosis.

Spectral responses across a dorsal-ventral array of dermal sensilla in the medicinal leech.

How do animals use visual systems to extract specific features of a visual scene and respond appropriately? The medicinal leech, Hirudo verbana, is a predatory, quasi-amphibious annelid with a rich sensorium that is an excellent system in which to study how sensory cues are encoded, and how key features of visual images are mapped into the CNS. The leech visual system is broadly distributed over its entire body, consisting of five pairs of cephalic eyecups and seven segmentally iterated pairs of dermal sensilla in each mid-body segment. Leeches have been shown to respond behaviorally to both green and near ultraviolet light (UV, 365-375 nm). Here, we used electrophysiological techniques to show that spectral responses by dermal sensilla are mapped across the dorsal-ventral axis, such that the ventral sensilla respond strongly to UV light, while dorsal sensilla respond strongly to visible light, broadly tuned around green. These results establish how key features of visual information are initially encoded by spatial mapping of photo-response profiles of primary photoreceptors and provide insight into how these streams of information are presented to the CNS to inform behavioral responses.

3D Reconstruction of HvRNASET2 Molecule to Understand Its Antibacterial Role.

Recent studies performed on the invertebrate model Hirudo verbana (medicinal leech) suggest that the T2 ribonucleic enzyme HvRNASET2 modulates the leech's innate immune response, promoting microbial agglutination and supporting phagocytic cells recruitment in challenged tissues. Indeed, following injection of both lipoteichoic acid (LTA) and Staphylococcus aureus in the leech body wall, HvRNASET2 is expressed by leech type I granulocytes and induces bacterial aggregation to aid macrophage phagocytosis. Here, we investigate the HvRNASET2 antimicrobial role, in particular assessing the effects on the Gram-negative bacteria Escherichia coli. For this purpose, starting from the three-dimensional molecule reconstruction and in silico analyses, the antibacterial activity was evaluated both in vitro and in vivo. The changes induced in treated bacteria, such as agglutination and alteration in wall integrity, were observed by means of light, transmission and scanning electron microscopy. Moreover, immunogold, AMPs (antimicrobial peptides) and lipopolysaccharide (LPS) binding assays were carried out to evaluate HvRNASET2 interaction with the microbial envelopes and the ensuing ability to affect microbial viability. Finally, in vivo experiments confirmed that HvRNASET2 promotes a more rapid phagocytosis of bacterial aggregates by macrophages, representing a novel molecule for counteracting pathogen infections and developing alternative solutions to improve human health.

The Hirudo Medicinalis Microbiome Is a Source of New Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are considered a promising new class of anti-infectious agents. This study reports new antimicrobial peptides derived from the Hirudo medicinalis microbiome identified by a computational analysis method applied to the H. medicinalis metagenome. The identified AMPs possess a strong antimicrobial activity against Gram-positive and Gram-negative bacteria (MIC range: 5.3 to 22.4 µM), including Staphylococcus haemolyticus, an opportunistic coagulase-negative pathogen. The secondary structure analysis of peptides via CD spectroscopy showed that all the AMPs except pept_352 have mostly disordered structures that do not change under different conditions. For peptide pept_352, the α-helical content increases in the membrane environment. The examination of the mechanism of action of peptides suggests that peptide pept_352 exhibits a direct membranolytic activity. Furthermore, the cytotoxicity assay demonstrated that the nontoxic peptide pept_1545 is a promising candidate for drug development. Overall, the analysis method implemented in the study may serve as an effective tool for the identification of new AMPs.

Draft genome sequences of Hirudo medicinalis and salivary transcriptome of three closely related medicinal leeches.

BACKGROUND: Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today. RESULTS: We annotated the Hirudo medicinalis genome and performed RNA-seq on salivary cells isolated from three closely related leech species, H. medicinalis, Hirudo orientalis, and Hirudo verbana. Differential expression analysis verified by proteomics identified salivary cell-specific gene expression, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants have been found to be expressed not only in salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis. CONCLUSIONS: Here we report a genome draft of Hirudo medicinalis and describe identification of novel salivary proteins and new homologs of genes encoding known anticoagulants in transcriptomes of three medicinal leech species. Our data provide new insights in genetics of blood-feeding lifestyle in leeches.

Hirudins of the Asian medicinal leech, Hirudinaria manillensis: same same, but different.

Blood coagulation in vertebrates is a complex mechanism that involves the precisely coordinated and regulated action of a cascade of factors in order to prevent excessive blood loss upon wounding. Any blood sucking ectoparasite, however, has to circumvent this mechanism to ensure the uptake of an adequate blood meal. Inhibitors of blood coagulation in the saliva are hence widespread among these animals. Thrombin as a key factor of blood coagulation is a prominent target of such inhibitors, and hirudin is probably the best known among the thrombin inhibitors. Hirudin was originally described in the genus Hirudo, but occurs in other leech genera like Hirudinaria and Macrobdella as well. Besides several isoforms of hirudin, a new class of putative leech saliva components, the hirudin-like factors (HLFs), was identified in both genera Hirudo and Hirudinaria. Here, we describe the expression, purification, and functional characterization of three HLFs (HLF5, 6, and 8, respectively) and two additional hirudins (HM3 and HM4) of Hirudinaria manillensis. While HLF6 lacked any inhibitory activity on thrombin, HLF5 as well as HLF8 clearly exhibited anticoagulatory properties. The inhibitory activity of HLF5 and HLF8, however, was much lower compared with both HM3 and HM4 of Hirudinaria manillensis as well as the hirudin variants 1 (HV1) and 2 (HV2) of Hirudo medicinalis. Neither an inhibition of trypsin nor a platelet aggregation was caused by HLF8. Our data indicates the presence of two classes (rather than isoforms) of hirudins in Hirudinaria manillensis with markedly different inhibitory activity on human thrombin.

Transcriptomic analysis of the salivary gland of medicinal leech Hirudo nipponia.

Hirudo nipponia (known as Shui Zhi in Chinese) is a well-known Chinese medicine with numerous active ingredients in its body, especially in its saliva. This native Chinese blood-sucking leech has been used for therapeutic purposes since before 100 AD. Modern Chinese physicians use it for a wide range of diseases. Genomic data and molecular information about the pharmacologically active substances produced by this medicinal leech are presently unavailable despite this organism's medicinal importance. In this study, we performed transcriptome profiling of the salivary glands of medicinal leech H. nipponia using the Illumina platform. In total, 84,657,362 clean reads were assembled into 50,535 unigenes. The obtained unigenes were compared to public databases. Furthermore, a unigene sequence similarity search and comparisons with the whole transcriptome of medical leech were performed to identify potential proteins. Finally, more than 21 genes were predicted to be involved in anticoagulatory, antithrombotic, antibacterial, anti-inflammatory and antitumor processes, which might play important roles in the treatment of various diseases. This study is the first analysis of a sialotranscriptome in H. nipponia. The transcriptome profile will shed light on its genetic background and provide a useful tool to deepen our understanding of the medical value of H. nipponia.

An annotated CNS transcriptome of the medicinal leech, Hirudo verbana: De novo sequencing to characterize genes associated with nervous system activity.

The medicinal leech is one of the most venerated model systems for the study of fundamental nervous system principles, ranging from single-cell excitability to complex sensorimotor integration. Yet, molecular analyses have yet to be extensively applied to complement the rich history of electrophysiological study that this animal has received. Here, we generated the first de novo transcriptome assembly from the entire central nervous system of Hirudo verbana, with the goal of providing a molecular resource, as well as to lay the foundation for a comprehensive discovery of genes fundamentally important for neural function. Our assembly generated 107,704 contigs from over 900 million raw reads. Of these 107,704 contigs, 39,047 (36%) were annotated using NCBI's validated RefSeq sequence database. From this annotated central nervous system transcriptome, we began the process of curating genes related to nervous system function by identifying and characterizing 126 unique ion channel, receptor, transporter, and enzyme contigs. Additionally, we generated sequence counts to estimate the relative abundance of each identified ion channel and receptor contig in the transcriptome through Kallisto mapping. This transcriptome will serve as a valuable community resource for studies investigating the molecular underpinnings of neural function in leech and provide a reference for comparative analyses.

Role of the tissue free amino acids in adaptation of medicinal leeches Hirudo medicinalis L., 1758 to extreme climatic conditions.

The first comparison of the spectra of free amino acids in tissues of the medicinal leeches H. medicinalis from different climatic and geographical Eurasian areas has been performed. Adaptation of H. medicinalis to extreme climatic conditions occurs via intensification of the amino acid metabolism resulting from a significant increase in the content of essential amino acids. Accumulation of arginine, histidine, and lysine (3.6-, 3.9-, and 2.0-fold increases, respectively) has proved to play a special protective role in adaptation of H. medicinalis to the low positive temperatures.

Hm-MyD88 and Hm-SARM: two key regulators of the neuroimmune system and neural repair in the medicinal leech.

Unlike mammals, the CNS of the medicinal leech can regenerate damaged neurites, thus restoring neural functions after lesion. We previously demonstrated that the injured leech nerve cord is able to mount an immune response promoting the regenerative processes. Indeed neurons and microglia express sensing receptors like Hm-TLR1, a leech TLR ortholog, associated with chemokine release in response to a septic challenge or lesion. To gain insights into the TLR signaling pathways involved during these neuroimmune responses, members of the MyD88 family were investigated. In the present study, we report the characterization of Hm-MyD88 and Hm-SARM. The expression of their encoding gene was strongly regulated in leech CNS not only upon immune challenge but also during CNS repair, suggesting their involvement in both processes. This work also showed for the first time that differentiated neurons of the CNS could respond to LPS through a MyD88-dependent signalling pathway, while in mammals, studies describing the direct effect of LPS on neurons and the outcomes of such treatment are scarce and controversial. In the present study, we established that this PAMP induced the relocalization of Hm-MyD88 in isolated neurons.

Microglia of medicinal leech (Hirudo medicinalis) express a specific activation marker homologous to vertebrate ionized calcium-binding adapter molecule 1 (Iba1/alias aif-1).

The Ionized calcium-Binding Adapter molecule 1 (Iba1), also known as Allograft Inflammatory Factor 1 (AIF-1), is a 17 kDa cytokine-inducible protein, produced by activated macrophages during chronic transplant rejection and inflammatory reactions in Vertebrates. In mammalian central nervous system (CNS), Iba1 is a sensitive marker associated with activated macrophages/microglia and is upregulated following neuronal death or brain lesions. The medicinal leech Hirudo medicinalis is able to regenerate its CNS after injury, leading to a complete functional repair. Similar to Vertebrates, leech neuroinflammatory processes are linked to microglia activation and recruitment at the lesion site. We identified a gene, named Hmiba1, coding a 17.8 kDa protein showing high similarity with Vertebrate AIF-1. The present work constitutes the first report on an Iba1 protein in the nervous system of an invertebrate. Immunochemistry and gene expression analyses showed that HmIba1, like its mammalian counterpart, is modulated in leech CNS by mechanical injury or chemical stimuli (ATP). We presently demonstrate that most of leech microglial cells migrating and accumulating at the lesion site specifically expressed the activation marker HmIba1. While the functional role of Iba1, whatever species, is still unclear in reactive microglia, this molecule appeared as a good selective marker of activated cells in leech and presents an interesting tool to investigate the functions of these cells during nerve repair events.

Calreticulin contributes to C1q-dependent recruitment of microglia in the leech Hirudo medicinalis following a CNS injury.

BACKGROUND: The medicinal leech is considered as a complementary and appropriate model to study immune functions in the central nervous system (CNS). In a context in which an injured leech's CNS can naturally restore normal synaptic connections, the accumulation of microglia (immune cells of the CNS that are exclusively resident in leeches) has been shown to be essential at the lesion to engage the axonal sprouting. HmC1q (Hm for Hirudo medicinalis) possesses chemotactic properties that are important in the microglial cell recruitment by recognizing at least a C1q binding protein (HmC1qBP alias gC1qR). MATERIAL AND METHODS: Recombinant forms of C1q were used in affinity purification and in vitro chemotaxis assays. Anti-calreticulin antibodies were used to neutralize C1q-mediated chemotaxis and locate the production of calreticulin in leech CNS. RESULTS: A newly characterized leech calreticulin (HmCalR) has been shown to interact with C1q and participate to the HmC1q-dependent microglia accumulation. HmCalR, which has been detected in only some microglial cells, is consequently a second binding protein for HmC1q, allowing the chemoattraction of resident microglia in the nerve repair process. CONCLUSIONS: These data give new insight into calreticulin/C1q interaction in an immune function of neuroprotection, suggesting another molecular target to use in investigation of microglia reactivity in a model of CNS injury.

Gap junction-dependent homolog avoidance in the developing CNS.

Oppositely directed projections of some homologous neurons in the developing CNS of the medicinal leech (Hirudo verbana), such as the AP cells, undergo a form of contact-dependent homolog avoidance. Embryonic APs extend axons within the connective nerve toward adjacent ganglia, in which they meet and form gap junctions (GJs) with the oppositely directed axons of their segmental homologs, stop growing, and are later permanently retracted (Wolszon et al., 1994a,b). However, early deletion of an AP neuron leads to resumed growth and permanent maintenance of the projections of neighboring APs. Here we test the hypothesis that a GJ-based signaling mechanism is responsible for this instance of homolog avoidance. We demonstrate that selective knockdown of GJ gene Hve-inx1 expression in single embryonic APs, by expressing a short-hairpin interfering RNA, leads to continued growth of the projections of the cell toward, into, and beyond adjacent ganglia. Moreover, the projections of the APs in adjacent ganglia also resume growth, mimicking their responses to cell deletion. Continued growth was also observed when two different INX1 mutant transgenes that abolish dye coupling between APs were expressed. These include a mutant transgene that effectively downregulates all GJ plaques that include the INX1 protein and a closed channel INX1 mutant that retains the adhesive cellular binding characteristic of INX1 GJs but not the open channel pore function. Our results add GJ intercellular communication to the list of molecular signaling mechanisms that can act as mediators of growth-inhibiting cell-cell interactions that define the topography of neuronal arbors.

[Use of the most recent reagent (CuFL) for stimulation of NO synthesis by the medicinal leech salivary cell secretion in the cultures of human endothelium cells (HUVEC) and in rat cardiomiocytes].

The medicinal leech salivary cell secretion (SCS) may stimulate NO-production in cultures of human endothelium cells (HUVEC) and rat cardiomiocytes (RCM). This effect was detected using a NO specific reagent, - the complex Cu2+ with a fluorescein derivative (Cu-Fl). NO had also been detected in the cells by fluorescent electronic microscopy and determined quantitatively in the cells and in culture fluid by the fluorescence method. SCS stimulated NO synthesis in HUVEC cells (but not in RCM) is accompanied by NO release into intercellular space. Localization of NO synthesis centers is presented and it is shown that the increase in NO levels during the SCS action on HUVEC and RCM is associated with the increase in the activity of eNOS/nNOS, but not iNOS. In endothelial cells SCS activates nitrosylation processes, assessed by the increase of nitrite-ions in the culture medium. It is therefore important to use Cu-Fl, other than Griss-reagent, during the first hour of analysis of NO synthesis. The NO-depended mechanism of SCS action on endothelial cells might be a factor in providing of its positive action in hirudotheraphy.

Bacterial symbiont and salivary peptide evolution in the context of leech phylogeny.

The evolutionary history of leeches is employed as a general framework for understanding more than merely the systematics of this charismatic group of annelid worms, and serves as a basis for understanding blood-feeding related correlates ranging from the specifics of gut-associated bacterial symbionts to salivary anticoagulant peptides. A variety of medicinal leech families were examined for intraluminal crop bacterial symbionts. Species of Aeromonas and Bacteroidetes were characterized with DNA gyrase B and 16S rDNA. Bacteroidetes isolates were found to be much more phylogenetically diverse and suggested stronger evidence of phylogenetic correlation than the gammaproteobacteria. Patterns that look like co-speciation with limited taxon sampling do not in the full context of phylogeny. Bioactive compounds that are expressed as gene products, like those in leech salivary glands, have 'passed the test' of evolutionary selection. We produced and bioinformatically mined salivary gland EST libraries across medicinal leech lineages to experimentally and statistically evaluate whether evolutionary selection on peptides can identify structure-function activities of known therapeutically relevant bioactive compounds like antithrombin, hirudin and antistasin. The combined information content of a well corroborated leech phylogeny and broad taxonomic coverage of expressed proteins leads to a rich understanding of evolution and function in leech history.

Multiple changes in peptide and lipid expression associated with regeneration in the nervous system of the medicinal leech.

BACKGROUND: The adult medicinal leech central nervous system (CNS) is capable of regenerating specific synaptic circuitry after a mechanical lesion, displaying evidence of anatomical repair within a few days and functional recovery within a few weeks. In the present work, spatiotemporal changes in molecular distributions during this phenomenon are explored. Moreover, the hypothesis that neural regeneration involves some molecular factors initially employed during embryonic neural development is tested. RESULTS: Imaging mass spectrometry coupled to peptidomic and lipidomic methodologies allowed the selection of molecules whose spatiotemporal pattern of expression was of potential interest. The identification of peptides was aided by comparing MS/MS spectra obtained for the peptidome extracted from embryonic and adult tissues to leech transcriptome and genome databases. Through the parallel use of a classical lipidomic approach and secondary ion mass spectrometry, specific lipids, including cannabinoids, gangliosides and several other types, were detected in adult ganglia following mechanical damage to connected nerves. These observations motivated a search for possible effects of cannabinoids on neurite outgrowth. Exposing nervous tissues to Transient Receptor Potential Vanilloid (TRPV) receptor agonists resulted in enhanced neurite outgrowth from a cut nerve, while exposure to antagonists blocked such outgrowth. CONCLUSION: The experiments on the regenerating adult leech CNS reported here provide direct evidence of increased titers of proteins that are thought to play important roles in early stages of neural development. Our data further suggest that endocannabinoids also play key roles in CNS regeneration, mediated through the activation of leech TRPVs, as a thorough search of leech genome databases failed to reveal any leech orthologs of the mammalian cannabinoid receptors but revealed putative TRPVs. In sum, our observations identify a number of lipids and proteins that may contribute to different aspects of the complex phenomenon of leech nerve regeneration, establishing an important base for future functional assays.

Protein-lipid particles of medicinal leech salivary gland secretion; their size and morphology.

The relative location of proteins and lipids in particles of medicinal leech salivary gland secretion (SGS) is revealed for the first time. Their sizes and morphology are described. Using scanning electron microscopy and transmission electron microscopy, it was determined that SGS consists of particles of different sizes and form. This picture is supported by confocal laser scanning microscopy of SGS preparations treated with fluorescein isothiocyanate. After incubation with nonionic detergents (Brij 35 and Tween 20), transmission electron microscopy revealed the dissociation of fragments composing protein-lipid particles (PLP), and in this case an increase in free protein concentration determined by a modification of the Lowry method was observed. Perylene probing of lipids in SGS preparations showed that they are concentrated mainly inside PLP and are almost absent on the surface. Cholesterol was detected during SGS probing using the cholesteryl-Bodipy (hydrophobic fluorescent analog of cholesterol) on surface sections during confocal analysis of electron microphotographs of SGS. This analysis detected PLP structures in SGS resembling caveoles full of cholesterol. SGS, preliminary frozen at -70 degrees C, transformed into a multitude of similar small particles visualized by transmission electron microscopy, whose fixed distribution resembled water crystal structure.

Evidence for a novel chemotactic C1q domain-containing factor in the leech nerve cord.

In vertebrates, central nervous system (CNS) protection is dependent on many immune cells including microglial cells. Indeed, activated microglial cells are involved in neuroinflammation mechanisms by interacting with numerous immune factors. Unlike vertebrates, some lophotrochozoan invertebrates can fully repair their CNS following injury. In the medicinal leech Hirudo medicinalis, the recruitment of microglial cells at the lesion site is essential for sprouting of injured axons. Interestingly, a new molecule homologous to vertebrate C1q was characterized in leech, named HmC1q (for H. medicinalis) and detected in neurons and glial cells. In chemotaxis assays, leech microglial cells were demonstrated to respond to human C1q. The chemotactic activity was reduced when microglia was preincubated with signaling pathway inhibitors (Pertussis Toxin or wortmannin) or anti-human gC1qR antibody suggesting the involvement of gC1qR in C1q-mediated migration in leech. Assays using cells preincubated with NO chelator (cPTIO) showed that C1q-mediated migration was associated to NO production. Of interest, by using anti-HmC1q antibodies, HmC1q released in the culture medium was shown to exhibit a similar chemotactic effect on microglial cells as human C1q. In summary, we have identified, for the first time, a molecule homologous to mammalian C1q in leech CNS. Its chemoattractant activity on microglia highlights a new investigation field leading to better understand leech CNS repair mechanisms.