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.

Structural insights into thrombolytic activity of destabilase from medicinal leech.

Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes. It has two different enzymatic activities: microbial cell walls destruction (muramidase activity), and dissolution of the stabilized fibrin (isopeptidase activity). Both activities are known to be inhibited by sodium chloride at near physiological concentrations, but the structural basis remains unknown. Here we present two crystal structures of destabilase, including a 1.1 Å-resolution structure in complex with sodium ion. Our structures reveal the location of sodium ion between Glu34/Asp46 residues, which were previously recognized as a glycosidase active site. While sodium coordination with these amino acids may explain inhibition of the muramidase activity, its influence on previously suggested Ser49/Lys58 isopeptidase activity dyad is unclear. We revise the Ser49/Lys58 hypothesis and compare sequences of i-type lysozymes with confirmed destabilase activity. We suggest that the general base for the isopeptidase activity is His112 rather than Lys58. pKa calculations of these amino acids, assessed through the 1 µs molecular dynamics simulation, confirm the hypothesis. Our findings highlight the ambiguity of destabilase catalytic residues identification and build foundations for further research of structure-activity relationship of isopeptidase activity as well as structure-based protein design for potential anticoagulant drug development.

Life without blood: Molecular and functional analysis of hirudins and hirudin-like factors of the Asian non-hematophagous leech Whitmania pigra.

BACKGROUND: Several leech species of the genera Hirudo, Hirudinaria, and Whitmania are widely used in traditional Chinese medicine (TCM) for the oral treatment of disorders associated with blood stasis. Among them, the non-hematophagous leech Whitmania pigra expresses a variety of components that have the potential to act on the vertebrate blood coagulation system. OBJECTIVE: Whether the thrombin inhibitor hirudin, probably the most prominent leech-derived anticoagulant, is actually present in Whitmania pigra, is still a matter of debate. To answer that open question was the aim of the study. METHODS: We identified several putative hirudin-encoding sequences in transcriptome data of Whitmania pigra. Upon gene synthesis and molecular cloning the respective recombinant proteins were expressed in Escherichia coli, purified, processed, and eventually functionally characterized for thrombin-inhibitory potencies in coagulation assays. RESULTS: We were successful in the identification and functional characterization of several putative hirudins in Whitmania pigra. Some, but not all, of these factors are indeed thrombin inhibitors. Whitmania pigra hence expresses both hirudins (factors that inhibit thrombin) and hirudin-like factors (that do not or only very weakly inhibit thrombin). Furthermore, we revealed the exon/intron structures of the corresponding genes. Coding sequences of some putative hirudins of Whitmania pigra were present also in transcriptome datasets of Hirudo nipponia, a hematophagous leech that is likewise used in TCM. CONCLUSIONS: Based on both structural and functional data we provide very strong evidence for the expression of hirudins in Whitmania pigra. This is the first description of hirudins in a non-hematophagous leech.

A study of the influence of lead pollution on the anticoagulant activity of Whitmania pigra based on pharmacodynamics and metabolomics research.

Whitmania pigra Whitman (leech, also called Shuizhi in China, abbreviated as SZ), which has been used as a traditional Chinese medicine in the treatment of blood stasis syndrome (BSS) for a long time, is vulnerable to lead pollution in aquaculture environments. SZ has good anticoagulant activity. However, there are few studies on the influence of lead pollution on it. Therefore, we carried out the following researches to explore the influence of lead pollution on the anticoagulant activity of SZ and its mechanism. Firstly, the acute blood stasis model of rats was established by subcutaneous injection of adrenaline hydrochloride and ice water bath. Then unpolluted SZ (UPS) and lead-polluted SZ (LPS) were extracted. Next, the blood stasis model rats were administrated by gavage and the rats in normal control (NC) group and blood stasis model (BM) group were given the same amount of normal saline. Finally, the blood of the rats was collected to detect the coagulation function and hemorheology indexes. The metabolomics of rat plasma was studied by ultra-high-performance liquid chromatography coupled with orbitrap mass spectrometry (UPLC-Orbitrap-MS) technology. Principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and Hierarchical clustering analysis (HCA) were used to perform metabolomics analysis. MetPA analysis was used to search for related metabolic pathways. The results of coagulation function and hemorheology showed that lead pollution could decrease the anticoagulant activity of SZ. The OPLS-DA score plots indicated that the plasma metabolites of rats in LPS group were close to BM group, while UPS group tended to be close to NC group both in the positive and negative ion mode. Hierarchical cluster analysis (HCA) suggested that UPS group and NC group were clustered into a branch, while LPS group and BM group were clustered into a branch. To sum up, lead pollution will reduce the anticoagulant activity of SZ. And lead pollution reduces the anticoagulant activity of SZ probably by influencing the metabolic pathways such as sphingolipid metabolism, amino acid metabolism and energy metabolism in rats.

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.

Characterization of a monoacylglycerol lipase in the medicinal leech, Hirudo verbana.

Endocannabinoids are a class of lipid neuromodulators found throughout the animal kingdom. Among the endocannabinoids, 2-arachydonoyl glycerol (2-AG) is the most prevalent endocannabinoid and monoacylglycerol lipase (MAGL) is a serine hydrolase primarily responsible for metabolizing 2-AG in mammals. In the medicinal leech, Hirudo verbana, 2-AG has been found to be an important and multi-functional modulator of synaptic transmission and behavior. However, very little is known about the molecular components of its synthesis and degradation. In this study we have identified cDNA in Hirudo that encodes a putative MAGL (HirMAGL). The encoded protein exhibits considerable sequence and structural conservation with mammalian forms of MAGL, especially in the catalytic triad that mediates 2-AG metabolism. Additionally, HirMAGL transcripts are detected in the Hirudo central nervous system. When expressed in HEK 293 cells HirMAGL segregates to the plasma membrane as expected. It also exhibits serine hydrolase activity that is blocked when a critical active site residue is mutated. HirMAGL also demonstrates the capacity to metabolize 2-AG and this capacity is also prevented when the active site is mutated. Finally, HirMAGL activity is inhibited by JZL184 and MJN110, specific inhibitors of mammalian MAGL. To our knowledge these findings represent the first characterization of an invertebrate form of MAGL and show that HirMAGL exhibits many of the same properties as mammalian MAGL's that are responsible for 2-AG metabolism.

Metabolomics-based study reveals the effect of lead (Pb) in the culture environment on Whitmania pigra.

Whitmania pigra, called Mahuang (MH) in Chinese, has been used as a traditional Chinese medicine for many years and is susceptible to Pb exposure in aquaculture environments. To understand the impact of Pb in the culture environment on MHs, we carried out a 50-day culture of MHs in environments with different levels of Pb pollution. Then, tissue samples of MHs reared in the different Pb-polluted environments were collected and analysed by UPLC-Q/TOF-MS. The results showed that the Pb residue in MHs increased with increasing Pb in the culture environment. There was no significant difference in MH Pb content (P < 0.05) between the low-Pb residue group (PbL) and the blank control group (BC), and those of the middle-Pb residue group (PbM) and the high-Pb residue group (PbH) were significantly different from that of the BC group. Metabolomics results showed significant changes in 24 metabolites in the PbL, PbM and PbH groups, some of which were dose-dependent. These metabolites were mainly lipids, nucleotides, and dipeptides, which are involved in metabolic pathways such as glycerophospholipid metabolism, sphingolipid metabolism, and nucleotide metabolism. Overall, the results proved that metabolomics can be an effective tool to understand the effects of Pb on the metabolic responses of MHs.

A novel protease-activated receptor 1 inhibitor from the leech Whitmania pigra.

Whitmania pigra has been used as a traditional Chinese medicine (TCM) for promoting blood circulation, alleviating blood coagulation, activating meridians and relieving stasis for several hundred years. However, the therapeutic components of this species, especially proteins and peptides were poorly exploited. Until now only a few of them were obtained by using chromatographic isolation and purification. In recent decade, transcriptome techniques were rapidly developed, and have been used to fully reveal the functional components of many animal venoms. In the present study, the cDNA of the salivary gland of Whitmania pigra was sequenced by illumina and the transcriptome was assembled by using Trinity. The proteome were analysed by LC-MS/MS. Based on the data of the transcriptome and the proteome, a potential antiplatelet protein named pigrin was found. Pigrin was cloned and expressed using P. pastoris GS115. The antiplatelet andantithrombotic bioactivities of pigrin were tested by using aggregometer and the rat arterio-venous shunt thrombosis model, respectively. Thebleeding time of pigrin was measured by a mice tail cutting method. The docking of pigrin and protease-activated receptor 1 (PAR1) or collagen were conducted using the ZDOCK Server. Pigrin was able to selectively inhibit platelet aggregation stimulated by PAR1 agonist and collagen. Pigrin attenuated thrombotic formation in vivo in rat, while did not prolong bleeding time at its effective dosage. There are significant differences in the key residues participating in binding of Pigrin-Collagen complex from Pigrin-PAR1 complex. In conclusion,a novel PAR1 inhibitor pigrin was found from the leech Whitmania pigra. This study helped to elucidate the mechanism of the leech for the treatment of cardiovascular disorder.

[Effects of stocking density and exchanging water frequency on growth and inner quality of Whitmania pigra].

The effects of stocking density and exchanging water frequency on growth, digestive enzyme activity, anti-oxidative enzyme and inner quality of Whitmania pigra Whitman were evaluated with corresponding measures. The results showed that the eventual biomass, specific growth rate, gained weight rate, activities of amylase, lipase, protease, SOD, CAT, and ALP correlated positively with stocking density and negatively with exchanging water frequency (P<0.05). Exchanging water frequency had negative correlation with ammonia nitrogen, nitrite, and hydrogen sulfide while revealed positive correlation with dissolved oxygen in the water. Stocking density and exchanging water frequency showed no significant effects on the contents of moisture, total ash, and acid-insoluble ash. It suggested that the optimum stocking density was 7.5 million per hectare and the appropriate exchanging water interval was 72 h.

Effect of medicinal leeches' antigens on the proliferative response of human blood mononuclear cells and cytokine production in vitro.

UNLABELLED: The purpose of research is to study the influence of plant mitogens and antigens of the water-salt extract from the bodies of Hirudo verbana, H. medicinalis, H. orientalis on the reaction of lymphocyte blast-transformation and the synthesis of pro-inflammatory cytokines (IL-1ß, IL-8, TNF-α) in patients' cell culture supernatants before and after hirudotherapy. RESEARCH METHODS: the reaction of lymphocyte blast-transformation; the determination of proinflammatory cytokines by ELISA. After hirudotherapy increased values of the reaction of lymphocyte blasttransformation have got bigger in all stimulated types of cell culture, but reactivity on medicinal leeches antigens proceeded to unproductive immunogenesis (apoptosis and necrosis). In supernatants of mitogen- and antigen-stimulated lymphocyte cultures induced high levels of pro-inflammatory cytokines after hirudotherapy have decreased adequately to apoptotic induction of lymphocytes by medicinal leeches' antigens.

The medicinal leech genome encodes 21 innexin genes: different combinations are expressed by identified central neurons.

Gap junctional proteins are important components of signaling pathways required for the development and ongoing functions of all animal tissues, particularly the nervous system, where they function in the intracellular and extracellular exchange of small signaling factors and ions. In animals whose genomes have been sufficiently sequenced, large families of these proteins, connexins, pannexins, and innexins, have been found, with 25 innexins in the nematode Caenorhabditis elegans Starich et al. (Cell Commun Adhes 8: 311-314, 2001) and at least 37 connexins in the zebrafish Danio rerio Cruciani and Mikalsen (Biol Chem 388:253-264, 2009). Having recently sequenced the medicinal leech Hirudo verbana genome, we now report the presence of 21 innexin genes in this species, nine more than we had previously reported from the analysis of an EST-derived transcriptomic database Dykes and Macagno (Dev Genes Evol 216: 185-97, 2006); Macagno et al. (BMC Genomics 25:407, 2010). Gene structure analyses show that, depending on the leech innexin gene, they can contain from 0 to 6 introns, with closely related paralogs showing the same number of introns. Phylogenetic trees comparing Hirudo to another distantly related leech species, Helobdella robusta, shows a high degree of orthology, whereas comparison to other annelids shows a relatively low level. Comparisons with other Lophotrochozoans, Ecdyzozoans and with vertebrate pannexins suggest a low number (one to two) of ancestral innexin/pannexins at the protostome/deuterostome split. Whole-mount in situ hybridization for individual genes in early embryos shows that ∼50% of the expressed innexins are detectable in multiple tissues. Expression analyses using quantitative PCR show that ∼70% of the Hirudo innexins are expressed in the nervous system, with most of these detected in early development. Finally, quantitative PCR analysis of several identified adult neurons detects the presence of different combinations of innexin genes, a property that may underlie the participation of these neurons in different adult coupling circuits.

The receptor protein tyrosine phosphatase HmLAR1 is up-regulated in the CNS of the adult medicinal leech following injury and is required for neuronal sprouting and regeneration.

LAR-like receptor protein tyrosine phosphatases (RPTPs), which are abundantly expressed in the nervous systems of most if not all bilaterian animals thus far examined, have been implicated in regulating a variety of critical neuronal processes. These include neuronal pathfinding, adhesion and synaptogenesis during development and, in adult mammals, neuronal regeneration. Here we explored a possible role of a LAR-like RPTP (HmLAR1) in response to mechanical trauma in the adult nervous system of the medicinal leech. In situ hybridization and QPCR analyses of HmLAR1 expression in individual segmental ganglia revealed a significant up-regulation in receptor expression following CNS injury, both in situ and following a period in vitro. Furthermore, we observed up-regulation in the expression of the leech homologue of the Abelson tyrosine kinase, a putative signaling partner to LAR receptors, but not among other tyrosine kinases. The effects on neuronal regeneration were assayed by comparing growth across a nerve crush by projections of individual dorsal P neurons (P(D)) following single-cell injection of interfering RNAs against the receptor or control RNAs. Receptor RNAi led to a significant reduction in HmLAR1 expression by the injected cells and resulted in a significant decrease in sprouting and regenerative growth at the crush site relative to controls. These studies extend the role of the HmLARs from leech neuronal development to adult neuronal regeneration and provide a platform to investigate neuronal regeneration and gene regulation at the single cell level.

Localized RNAi and ectopic gene expression in the medicinal leech.

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls. The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of approximately 50-150 microm and extends over approximately 15 microm around the mean penetration depth, which is adjustable between 0 and 50 microm. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches. For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery. When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

Proteome modifications of the medicinal leech nervous system under bacterial challenge.

Once considered as lacking intrinsic immune mechanisms, the CNS of vertebrates is now known to be capable of mounting its own innate immune response. Interestingly, while invertebrates have been very useful in the interpretation of general vertebrate innate immunity mechanisms, only scarce data are available on the immune response of nervous tissue within this group. This study provides new data on the innate immune response of medicinal leech Hirudo medicinalis CNS. We identified several spots in 2-D gels of leech CNS proteins that showed specific changes following bacterial challenge, thus demonstrating the ability of the leech nervous system to mount a response to an immune stress. Protein identifications were based on comparison of sequence data with publicly available databases and a recently established leech ESTs database. The broad nature of the identified proteins suggests a clear involvement of cytoskeletal rearrangements, endoplasmic reticulum stress, modulation of synaptic activity and calcium mobilization, all during the first 24 hours of this response. Moreover, several of these proteins are specifically expressed in glial cells, suggesting an important role for glial cells in the immune response of the leech nervous system, similar to what has been observed in vertebrates.

[Study on oxygen consumption rate and suffocation point of Whitmania pigra].

OBJECTIVE: To study the oxygen consumption rate and suffocation point of Whitmania pigra. METHOD: Using the method of respiratory chamber, the oxygen consumption rate, oxygen consumption volume and suffocation point were obtained. RESULT: The oxygen consumption rate(OCR) ranged from 0.049 to 0.094 mg x g(-1) x h(-1), the oxygen consumption volume(OCV) from 0.44 to 0.67 mg x p(-1) x h(-1), and suffocation point(SP) from 0.9 to 1.51 mg x L(-1), when the average weight of W. pigra was 10 g and the water temperature varied from 15 to 35 degrees C. OCR ranged from 0.044 to 0.058 mg x g(-1) x h(-1), OCV from 0.19 to 0.77 mg x p(-1) x h(-1) and SP from 1.4 to 1.57 mg x l(-1) when water temperature was 20 degrees C. CONCLUSION: OCR rose when the water temperature and weight increased, but SP was justly opposite. The weight was negatively correlated with OCR and positively with OCV. OCR had little difference from day to night.

A cephalic projection neuron involved in locomotion is dye coupled to the dopaminergic neural network in the medicinal leech.

It is widely appreciated that the selection and modulation of locomotor circuits are dependent on the actions of higher-order projection neurons. In the leech, Hirudo medicinalis, locomotion is modulated by a number of cephalic projection neurons that descend from the subesophageal ganglion in the head. Specifically, descending brain interneuron Tr2 functions as a command-like neuron that can terminate or sometimes trigger fictive swimming. In this study, we demonstrate that Tr2 is dye coupled to the dopaminergic neural network distributed in the head brain. These findings represent the first anatomical evidence in support of dopamine (DA) playing a role in the modulation of locomotion in the leech. In addition, we have determined that bath application of DA to the brain and entire nerve cord reliably and rapidly terminates swimming in all preparations exhibiting fictive swimming. By contrast, DA application to nerve cords expressing ongoing fictive crawling does not inhibit this motor rhythm. Furthermore, we show that Tr2 receives rhythmic feedback from the crawl central pattern generator. For example, Tr2 receives inhibitory post-synaptic potentials during the elongation phase of each crawl cycle. When crawling is not expressed, spontaneous inhibitory post-synaptic potentials in Tr2 correlate in time with spontaneous excitatory post-synaptic potentials in the CV motor neuron, a circular muscle excitor that bursts during the elongation phase of crawling. Our data are consistent with the idea that DA biases the nervous system to produce locomotion in the form of crawling.

Distribution and development of dopamine- and octopamine-synthesizing neurons in the medicinal leech.

Although the medicinal leech is a well-studied system in which many neurons and circuits have been identified with precision, descriptions of the distributions of some of the major biogenic amines, such as dopamine (DA) and octopamine (OA), have yet to be completed. In the European medicinal leech Hirudo medicinalis and the American medicinal leech Macrobdella decora,we have presented the first immunohistochemical study of DA neurons in the entire central nervous system, and of OA-immunoreactive (ir) neurons in the head and tail brains. Dopaminergic neurons were identified using the glyoxylic acid method and antisera to DA and its rate-limiting synthetic enzyme tyrosine hydroxylase (TH). Octopaminergic neurons were recognized using a highly specific antiserum raised against OA. An antibody raised against DA-beta-hydroxylase (DbetaH), the mammalian enzyme that converts DA to norepinephrine (NE), was found to immunostain OA-ir neurons. This antibody appears to cross-react with the closely related invertebrate enzyme tyramine-beta-hydroxylase, which converts tyramine to OA, suggesting that the OA-ir cells are indeed octopaminergic, capable of synthesizing OA. Because the DbetaH antiserum selectively immunostained the OA-ir neurons, but not the DA-synthesizing cells, our results also indicate that the DA-ir neurons synthesize DA and not NE as their end product. The expression of TH immunoreactivity was found to emerge relatively early in development, on embryonic day 9 (47-48% of development). In contrast, OA expression remained absent as late as embryonic day 20. Higher order processes of some of the dopaminergic and octopaminergic neurons in the adult brain were observed to project to a region previously described as a neurohemal complex. Several TH-ir processes were also seen in the stomatogastric nerve ring, suggesting that DA may play a role in the regulation of biting behavior. By mapping the distributions and developmental expression pattern of DA and OA neurons in the leech, we aim to gain a better understanding of the functional roles of aminergic neurons and how they influence behavior.

Proteinase inhibitors from the medicinal leech Hirudo medicinalis.

The medicinal leech Hirudo medicinalis produces various types of proteinase inhibitors: bdellins (inhibitors of trypsin, plasmin, and acrosin), hirustasin (inhibitor of tissue kallikrein, trypsin, alpha-chymotrypsin, and granulocyte cathepsin G), tryptase inhibitor, eglins (inhibitors of alpha-chymotrypsin, subtilisin, and chymasin and the granulocyte proteinases elastase and cathepsin G), inhibitor of factor Xa, hirudin (thrombin inhibitor), inhibitor of carboxypeptidase, and inhibitor of complement component C1s. This review summarizes data on their primary and tertiary structures, action mechanisms, and biological activities.

Chemical and thermal stimuli have short-lived effects on the retzius cell in the medicinal leech.

During the appetitive phase of feeding, hungry leeches detect a prey by the integration of signals perceived by different sensory systems. Earlier reports suggested that chemical or thermal sensory stimulation of the lip was associated with increased afferent activity in cephalic nerves connecting the lip to the central nervous system. These authors further suggested that this activity was relayed to Retzius cells in segmental ganglia, which then released serotonin to initiate and control all aspects of feeding behavior. In this study, we show that chemosensory or thermal activation of the lip lasting for at least 5 min produces a distinct signal in the cephalic nerves consisting of action potentials of low amplitude. These small amplitude signals are clearly distinguishable from the large action potentials evoked by mechanosensory stimuli applied to the same area of the lip. Both types of sensory stimuli also evoke an increase in the firing frequency of the Retzius cells in segmental ganglia. However, the response recorded in the nerves and the Retzius cells during a maintained stimulus is not constant but decreases with an exponential time course. These results agree with our earlier observations on a semi-intact feeding preparation in which we showed that the firing frequency of the Retzius cell decreased as soon as the leech began to ingest its meal. Therefore, our data provide further evidence suggesting that it is unlikely that heat or chemical cues maintain the Retzius cell in an active state throughout the consummatory phase of feeding.