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The catalase gene katA of the medicinal leech symblont Aeromonas veronii bv. sobria was cloned, sequenced, and functionally characterized. Southern hybridization, using an A. veronii katA-specific hybridization probe, suggested the presence of a single gene copy in many Aeromonas species. A. veronfi katA consisted of 1446 nt encoding a protein with a high degree of similarity to the small-subunit group III bacterial catalases. A catalase-null mutant (JG186) was constructed through gene-replacement mutagenesis. In the parent strain (HM21R), catalase activity was only detected in extracts of cells grown to early exponential phase following [H.sub.2][O.sub.2] induction, in which the ability to induce activity was inversely related to optical density. In contrast, induced JG186 cells were very sensitive to oxidative stress, with survival being affected even at low [H.sub.2][O.sub.2] concentrations. In contrast to the findings of previous reports of other symbiotic systems, the catalase mutant was not defective in its ability to competitively colonize or persist within its host, in both co-inoculation and sole-colonization assays. This body of evidence suggests either that oxidative stress, in the form of [H.sub.2][O.sub.2] exposure, is not encountered by the microbial partner under the examined symbiotic conditions or that compensatory mechanisms exist. The data suggest that although many colonization factors reoccur, each symbiotic system has also evolved specific mechanisms that affect symbiont-host dynamics.

Animals house a community of bacterial symbionts in their digestive tracts that contribute to their well being. The medicinal leech, Hirudo verbana, has a remarkably simple gut population carrying two extracellular microbes in the crop where the ingested blood is stored. This simplicity renders it attractive for studying colonization factors. Aeromonas veronii, one of the leech symbionts, can be genetically manipulated and is a pathogen of mammals. Screening transposon mutants of A. veronii for colonization defects in the leech, we found one mutant, JG752, with a transposon insertion in an ascU homolog, encoding an essential component of type III secretion systems (T3SS). Competing JG752 against the wild type revealed that JG752 was increasingly attenuated over time (10-fold at 18 h and >10,000-fold at 96 h). This colonization defect was linked to ascU by complementing JG752 with the operon containing ascU. Fluorescence in situ hybridization analysis revealed that at 42 h 38% of JG752 cells were phagocytosed by leech macrophage-like cells compared with Aeromonas | innate immunity | pathogenesis | symbiosis | Hirudo

The characterization of gut bacteria of the North American medicinal leech, Macrobdella decora, is presented. A new habitat is identified for Aeromonas jandae, suggesting that there is unexpected specificity between leeches and Aeromonas species.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.04.473 Byline: Sara J. Agee, Deirdre C. Lyons, David A. Weisblat Keywords: Leech; Annelid; Spiralian; nanos; D-quadrant specification; Maternal inheritants; Lophotrochozoa; Spiral cleavage; Micromeres Abstract: The gene nanos (nos) is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Drosophila. Expression of nos-related genes is associated with the germ line in a broad variety of other taxa, including the leech Helobdella robusta, where zygotically expressed Hro-nos appears to be associated with primordial germ cells. The function of maternally inherited Hro-nos transcripts remains to be determined, however. Here, the function of maternal Hro-nos is examined using an antisense morpholino (MO) knockdown strategy, as confirmed by immunostaining and western blot analysis. HRO-NOS knockdown embryos exhibit abnormalities in the distribution of micromeres during cleavage. Subsequently, their germinal bands are positioned abnormally with respect to the embryonic midline and the micromere cap, epiboly fails, and the HRO-NOS knockdown embryos die. This lethality can be rescued by injection of mRNA encoding an eGFP::HRO-NOS fusion protein. HRO-NOS knockdown embryos make their normal complements of mesodermal and ectodermal teloblasts, and the progeny of these teloblasts segregate into distinct mesodermal and ectodermal layers. These results suggest that maternal Hro-nos is required for embryonic development. However, contrary to previous suggestions, maternal inherited Hro-nos does not appear necessary for ectoderm specification. Author Affiliation: Department of Molecular and Cellular Biology, 385 LSA, University of California, Berkeley, CA 94720-3200, USA Article History: Received 4 January 2006; Revised 10 April 2006; Accepted 19 April 2006

The asymmetrical cell division that occurs during embryonic development of Helobdella robusta (leech) is studied.

Cocoons of the leech, Theromyzon tessulatum, are secreted underwater and sealed by two opercula (plugs) that are asymmetrically positioned on the upper aspect of the cocoon membrane. The opercula are protein-based, but their amino acid composition appears to differ from the surrounding cocoon membrane. Fluid deposited inside the cocoon by the parent leech contains several proteins ranging in size from ~40-100 kDa, most of which have pI's below 6.8. While inside the cocoon, late stage embryos display peristaltic contractions that may contribute to weakening of the opercula/membrane boundary. Juveniles break through the opercula after 2-3 wk and exit the cocoon over the course of several hours. The asymmetrical position of the opercula on the upper surface of the cocoon membrane may facilitate contact between the brooding parent leech and the emerging young, which attach to the venter of the parent and are brought to their first blood meal.

We investigated decision-making in the leech nervous system by stimulating identical sensory inputs that sometimes elicit crawling and other times swimming. Neuronal populations were monitored with voltage-sensitive dyes after each stimulus. By quantifying the discrimination time of each neuron, we found single neurons that discriminate before the two behaviors are evident. We used principal component analysis and linear discriminant analysis to find populations of neurons that discriminated earlier than any single neuron. The analysis highlighted the neuron cell 208. Hyperpolarizing cell 208 during a stimulus biases the leech to swim; depolarizing it biases the leech to crawl or to delay swimming., Understanding the mechanisms of behavioral choice would be a major step in bringing together neuroscience, psychology, and ethology (1). Research into decision-making has used several different strategies. One very productive [...]

Byline: S. S. Sergeeva (1), E. Zapryanova (2), O. S. Sotnikov (1), D. Deleva (2), A. Fil'chev (3) Keywords: anti-ganglioside antibodies; leech Retzius neurons; TTX-sensitive Na+ channels; TTX-resistant Na+ channels; spike activity; acclimation Abstract: The effects of anti-ganglioside antibodies on the functional states of two types of influx Na.sup.+ current channels were studied. Experiments used 20% anti-ganglioside antiserum prepared by standard methods by immunizing rabbits with total bovine brain gangliosides. These experiments showed that incubation of neurons in physiological saline containing antiserum induced discordance in the operation of the two types of influx current Na.sup.+ channels responsible for spike generation. This reaction was found to be associated with the slowed rate of activation of TTX-sensitive Na.sup.+ channels. Synaptic stimulation of cells in the presence of antiserum induced blockade of TTX-insensitive influx Na.sup.+ current channels. High-frequency synaptic activation of cells (10 Hz) showed that apart from blockade of TTX-insensitive Na.sup.+ channels, anti-ganglioside antibodies prevented plastic rearrangements in the gate system of TTX-sensitive Na.sup.+ channels. This resulted in impairment of the development of the acclimation process -- the response of the neuron to high-frequency stimulation seen in normal conditions. Author Affiliation: (1) I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, 6 Makarov Bank, 199034, St. Petersburg, Russia (2) Institute of Experimental Pathology and Anthropology, Bulgarian Academy of Sciences, Bulgaria (3) Institute of Experimental Pathology and Parasitology, Bulgarian Academy of Sciences, 25 G. Bonchev Street, 1000, Sofia, Bulgaria Article History: Registration Date: 18/10/2004

In Retzius neurones of the medicinal leech, Hirudo medicinals, kainate activates ionotropic glutamate receptors classified as AMPA/kainate receptors. Activation of the AMPA/kainate receptor-coupled cation channels evokes a marked depolarization, intracellular acidification, and increases in the intracellular concentrations of [Na.sup.+] ([[[Na.sup.+].sub.i]) and [Ca.sup.2+]. Qualitatively similar changes are observed upon the application of carbachol, an activator of acetylcholine receptor-coupled cation channels. Using multibarrelled ion-selective microelectrodes it was demonstrated that kainate, but not carbachol, caused additional increases in the intracellular free [Mg.sup.2+] concentration ([[[Mg.sup.2+].sub.i]. Experiments were designed to investigate whether this kainate-induced [[[Mg.sup.2+].sub.i] increase was due to a direct [Mg.sup.2+] influx through the AMPA/kainate receptor-coupled cation channels or a secondary effect due to the depolarization or the ionic changes. It was found that: (a) Similar [[[Mg.sup.2+].sub.i] increases were evoked by the application of glutamate or aspartate. (b) All kainate-induced effects were inhibited by the glutamatergic antagonist DNQX. (c) The magnitude of the [[[Mg.sup.2+].sub.i] increases depended on the extracellular [Mg.sub.2+] concentration. (d) A reduction of the extracellular [Ca.sub.2+] concentration increased kainate-induced [[[Mg.sup.2+].sub.i] increases, excluding possible [Ca.sup.2+] interference at the [Mg.sup.2+]-selective microelectrode or at intracellular buffer sites. (e) Neither depolarizations evoked by the application of 30 mM [K.sup.+], nor [[[Na.sup.+].sub.i] increases induced by the inhibition of the [Na.sup.+]/[K.sup.+] ATPase caused comparable [[[Mg.sup.2+].sub.i] increases. (f) Inhibitors of voltage-dependent [Ca.sup.2+] channels did not affect the kainate-induced [[[Mg.sup.2+].sub.i] increases. Moreover, previous experiments had already shown that intracellular acidification evoked by the application of 20 mM propionate did not cause changes in [[[Mg.sup.2+].sub.i]. The results indicate that kainate-induced [[[Mg.sup.2+].sub.i] increases in leech Retzius neurones are due to an influx of extracellular [Mg.sup.2+] through the AMPA/kainate receptor-coupled cation channel. [Mg.sup.2+] may thus act as an intracellular signal to distinguish between glutamatergic and cholinergic activation of leech Retzius neurones. KEY WORDS: magnesium * ion-selective microelectrode * glutamate receptor * ion channels * divalent cations

The voltage sensitivity of hemicyanine dyes ANNINE-6 and ANNINE-5 with anellated benzene rings and without free CC single and double bonds is studied in Retzius neurons from Hirudo medicinalis. The Annine dyes are the most efficient fluorescent probes of neuronal activity on the basis of a well-defined physical mechanism.

Byline: N. I. Chalisova (1), I. P. Baskova (2), L. L. Zavalova (3), V. A. Pennijainen (1) Keywords: nerve tissue culture; medicinal leech; protease inhibitors Abstract: The effects of components from medicinal leech (Hirudo medicinalis) salivary gland secretions and the therapeutic agent Piyavit on the growth of chick embryo neurites in organotypic culture were studied. Native destabilase and bdellin A at concentrations of 0.01, 0.02, 0.05, and 0.1 ng/ml, bdellin B at a concentration of 0.05 ng/ml, and eglin at a concentration of 0.1 ng/ml had neurite-stimulating activity, evident on the third day of organotypic culture of spinal ganglia. The stimulatory activity of destabilase was lost after revere-phase chromatography. The neurite-stimulating activity of the extract of the therapeutic agent Piyavit (200 ng/ml) in organotypic ganglion culture appeared to result from the neurite-stimulating salivary gland components within this agent, suggesting that Piyavit could be used for the treatment of neurodegenerative disorders. Author Affiliation: (1) Laboratory for Excitable Membrane Physiology, I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, 6 Makarov Bank, 199034, St. Petersburg, Russia (2) Faculty of Biology, M. V, Lomonosov Moscow State University, Vorob'ev Heights, 119899, Moscow, Russia (3) Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117871, Moscow, Russia Article History: Registration Date: 12/10/2004

The nanos-class gene of the leech Helobdella robusta (Hro-nos) is present as a maternal transcript whose levels decay during cleavage; HRO-NOS protein is more abundant in the D quadrant cells relative to the A, B, and C quadrants; and HRO-NOS is more abundant in the ectodermal precursor cell (DNOPQ) than in its sister mesodermal precursor (DM) (Pilon and Weisblat, 1997). Here, using in situ hybridization, we show that Hro-nos mRNA is broadly distributed throughout the zygote, is concentrated in both animal and vegetal teloplasm during stage I and is at higher levels in DNOPQ than in DM at stage 4b. Hro-nos expression increases after stage 7, as judged by in situ hybridization, developmental RT-PCR, and western blots; this increase must therefore represent later zygotic expression. Of particular interest, during stages 9 and 10, each of 11 mid-body segments (M8-M18) has a pair of Hro-nos positive 'spots' comprising of one or two large cells each. These spots later disappear in an anteroposterior progression. We find that these Hro-nos-expressing cells are of mesodermal origin, arising in a segmentally iterated manner from the M lineage, and correspond to cells previously proposed as primordial germ cells (PGCs; Burger, 1891; Weisblat and Shankland, 1985). These results support the proposal that nanos-class genes functioned in the specification of germline cells in the ancestral bilaterian and possibly in a separate process related to embryonic polarity in the ancestral protostome. Key Words: annelid; leech; Helobdella; germline; nanos.

The membrane-attack complex of the complement system in blood seems to prevent many bacteria from colonizing the digestive tract of the medicinal leech Hirudo medicinalis. This could explain why Aeromonas veronii bv. sobria is the only bacterium found in the leech's digestive tract.

The population coding in a small network underlying the leech local bend is reported. A complete set of sensory inputs to the network were monitored and manipulated. The population vector was found to correlate with bend direction, linking a proposed algorithm for neural population coding with synaptic and network mechanisms.

The homeobox gene Lox3 is expressed in a segmentally iterated pattern within the endoderm of the leech Helobdella. We use that expression here to study endoderm differentiation following experimental ablations of mesoderm. Lox3 RNA was first detected by in situ hybridization at the stage when a definitive cellular endoderm is formed from its syncytial precursor and was never observed in derivatives of other germ layers. Expression is initially distributed throughout the endoderm, but rapidly disappears from specific regions of the nascent gut wall so as to produce a pattern of segmental stripes. The stripe pattern differs markedly between midgut organs, with thin stripes of Lox3 expression in the intercaecal constrictions of the crop and wide stripes of Lox3 expression marking the caecal bulges of the intestine. Lox3 expression in the rectum is not obviously segmental. Ablation of segmental mesoderm in the early Helobdella embryo prevents the formation of definitive endoderm and the expression of Lox3 RNA and leads to abnormalities in the morphogenesis of the gut tube. These endodermal deficits are precisely coextensive with the zone of mesodermal deficiency, suggesting that the mesoderm normally acts to promote the formation of the endodermal cell layer via local cell interactions. The segmental pattern of Lox3 expression is largely unaffected in portions of the endoderm surrounding such deficits, suggesting that endodermal segmentation is not established by lateral interactions within that tissue layer. Rather, we propose that the segmental organization of the endoderm is imprinted by vertical interactions with the segmental mesoderm.

Molecular developmental studies of fly and mouse embryos have shown that the identity of individual body segments is controlled by a suite of homeobox-containing genes called the Hox cluster. To examine the conservation of this patterning mechanism in other segmented phyla, we here describe four Hox gene homologs isolated from glossiphoniid leeches of the genus Helobdella. Based on sequence similarity and phylogenetic analysis, the leech genes Lox7, Lox6, Lox20, and Lox5 are deemed to be orthologs of the Drosophila genes 1ab, Dfd, Scr, and Antp, respectively. Sequence similarities between Lox5 and Antp outside the homeodomain and phylogenetic reconstructions suggest that the Antennapedia family of Hox genes (as defined by Burglin, 1994) had already expanded to include at least two discrete Antp and Ubx/abdA precursors prior to the annelid/arthropod divergence. In situ hybridization reveals that the four Lox genes described in this study are all expressed at high levels within the segmented portion of the central nervous system (CNS), with variable levels of expression in the segmental mesoderm. Little or no expression was seen in peripheral ectoderm or endoderm, or in the unsegmented head region (prostomium). Each Lox gene has a distinct anterior expression boundary within one of the four rostral segments, and the anterior-posterior (AP) order of these expression boundaries is identical to that reported for the orthologous Hox gene products in fly and mouse. This finding supports the idea that the process of AP axis differentiation is conserved among the higher metazoan phyla with respect to the regional expression of individual Hox genes along that axis. One unusual feature of leech Hox genes is the observation that some genes are only expressed during later development - beginning at the time of terminal cell differentiation - whereas others begin expression at a much earlier stage, and their RNA ceases to be detectable shortly after the onset of expression of the 'late' Hox genes. The functional significance of this temporal disparity is unknown, but it is noteworthy that only the two 'early' Hox genes display high levels of mesodermal expression.

A study conducted on the release of neurotransmitter serotonin from neurons of the leech reveals that properties such as charge, amplitude, kinetics are due to exocytosis of small clear vesicles (SSV) and large dense-core vesicles (LDCV) present in the cells. Carbon fibers were used as electrochemical detectors. Two types of signals were observed, corresponding to the two types of vesicles. Exocytosis of SSV is faster than LDVC.

A solid-phase chemical synthetic method can be used to prepare analogs of the biologically active N-terminal region of hirudin, although it has a relatively long polypeptide chain with three cross-linking disulfide bridges. The Y3W analog of N-terminal fragment 1-47 was prepared by replacing Tyr3 with typtophan. Hirudin, a polypeptide with 64/66 amino acids residues, inhibits thrombin, a serine protease that plays an important role in thrombotic diseases. The analog has the same properties of folding and stability as the natural fragment, but has five times greater ability to inhibit thrombin.

Fab antibody fragments against a mannose-containing antigen on developing axons in embryonic leech can inhibit axonal defasciculation and arborization and this process follows first-order kinetics. This indicates that the developing axons extend independently of each other.

A superanumerary equal division induction of embryonic stem cells (O/P teleoblasts) suggests that the developmental fates of the progeny of three ipsilateral O/P teleoblasts are decided by their early mitotic patterns or by the phenotypes of their progeny. Ipsilateral sister O/P teleoblasts show equipotency but not equivalency. Bandlets obtained from duplicate O/P teleoblasts form progeny of the same fate in the presence of three O/P derived bandlets.

The midbody segments of Hirudo medicinalis have two types of sensory cilia that project from the skin into the surrounding water. These are the proposed mechanosensory S cilia and the putative chemosensory G cilia. The cells bearing these cilia are clustered together into structures known as sensilla. The skin of each midbody segment is externally divided into five annuli. Fourteen large sensilla carrying S and G cilia are found on the central annulus of each segment. Small sensilla carrying only G cilia are found on all five annuli. In this paper we show a positive correlation between the size of a neural sensillum and the number of S and G cilia it contains. Any one leech contains a range of sizes of neural sensilla, but the average area increases with the weight of the leech. In contrast, the annular sensilla show only a modest increase in size with leech weight.

The antibody A2B5 identifies a glycolipid in leech embryo and can be used as a neuron marker for its ganglioside identifying ability. Leech glucolipid resembles vertebrate gangliosides as both require the same conditions for extraction and can be retained on a silicic acid column. Both are resorcinol positive and contain epitopes that can be detected by A2B5. A2B5, which is expressed in a spatially controlled manner on anon tracts in a leech, reveals a spatial and temporal correspondence between C-cell growth cone labelling and its entry into a rapid phase of development.

In response to a moderate mechanical stimulus, the leech withdraws by forming a U-shaped local bend beneath the stimulus. Interneurons in the local bending reflex receive multiple sensory inputs, and have outputs to many motor neurons, suggesting that stimulus location on the body is represented in a distributed fashion. However, it is possible that as yet unidentified interneurons dedicated to the detection of specific spatial locations are necessary for the full range of the local bending motor output. Using the backpropagation algorithm to optimize connections in a model of the reflex, we showed that the distributed processing model of sensorimotor integration was consistent with our knowledge of the connection strengths between identified neurons in the reflex. The model further showed that the spatial and temporal constraints on the linkage between sensory representations and motor output can be satisfied by the appropriate connectivity between layers of interneurons and motor neurons in the leech, as well as in other systems with similar circuitry.

Heartbeat in the medicinal leech is paced by a neural oscillator comprising two elemental oscillators whose activity is coordinated by intersegmental coordinating fibers. The elemental oscillators each consist of a bilateral pair of heart interneurons linked by reciprocal inhibitory synapses. The activity cycle of each elemental oscillator consists of alternating bursts of action potentials (plateau/burst phase) and periods of inhibition (inactive phase). Oscillation ensues in the reciprocally inhibitory pairs because each neuron is able to escape from the inhibition of its contralateral partner and thus move on to the plateau/burst phase. We have identified and described membrane currents that contribute to oscillation and studied graded synaptic transmission between the neurons, using discontinuous current clamp and switching single electrode voltage clamp techniques. A hyperpolarization-activated inward current, I(sub h), plays a major role in escape from inhibition, and Ca2+ currents produce the plateau potentials that support burst formation and mediate graded synaptic transmission. To consolidate our knowledge and guide future research, we have constructed a first generation computer model of a neural oscillator based on reciprocal inhibition, using Hodgkin-Huxley equations and a synaptic transfer model, derived from our biophysical studies, with Nodus software (De Schutter, 1989). This model has confirmed an important role for I(sub h) in sustaining oscillation and has implicated a similarly important role for outward currents (particularly I(sub A)), which remain to be studies. Neural oscillators based on reciprocal inhibition appear to be ubiquitous, and our studies, biophysical and computational, provide insights into how they may operate.

The primary structure of two hirudins from Hirudinaria manillensis was analyzed and its anticoagulant activity determined and compared with similar hirudins extracted from Hirudinaria medicinalis. Results indicated differences in amino acid sequencing especially at the C-terminus of the different hirudins. Variations in the carbohydrate moiety were also noted although glycosylation occurs at a single site. They all showed comparable antithrombin activity and their structure showed similarity to human blood group H determinants.

THE SPECIFICATION OF ECTODERMAL and mesodermal fate occurs early in the development of most animals and is achieved by a variety of mechanisms, including the segregation of localized determinants during [...]

Analysis of 16S rRNA gene clone libraries confirmed the existence of Aeromonas veronii and revealed a second symbiont, clone, PW3, a novel member of the Rikenellaceae, within the crop, a large compartment where ingested blood is stored before digestion. The existence of a two-member bacterial community in the crop gives a unique opportunity for investigating both symbiont-symbiont and symbiont-host interactions in a natural model of digestive-tract associations.

The organization of the cytoskeleton in the early first interphase zygote and its involvement in organelle redistribution were studied in the glossiphoniid leech Theromyzon trizonare by confocal and electron microscopy, immunofluorescence, and time-lapse video imaging after microinjection of labeled tubulin and/or actin and loading with a mitotracker. The cytoskeleton consists of an inner or endoplasmic and an outer or ectoplasmic domain. The inner domain consists of a monaster whose fibers retract from the zygote periphery by the end of the early first interphase. The outer domain is built upon a network of microtubules and microfilaments cytasters. Short pulses of microinjected labeled actin or tubulin and Taxol treatment demonstrate that cytasters are centers of microtubule and microfilament nucleation. Immunostaining with anti-centrophilin, anti-BX-63, and anti-AH-6 indicates that the network of cytasters includes centrosomal antigens. Cytasters move in an orderly fashion at speeds of 0.5-2/[micro]m/min, in an energy-dependent process retarded and finally blocked by the ATP analogue AMP-PNP and high concentrations of Taxol. Colliding cytasters fuse and form larger cytoskeletal nucleation centers. The leech zygote is a highly compartmentalized cell whose cytasters function as articulated components of a very dynamic cytoskeletal system engaged in bulk transportation of organelles during ooplasmic segregation. [C] 2000 Academic Press Key Words: cytasters; leech zygote; mitochondria transport; zygote cytoskeleton; cytoskeleton assembly; cytoskeleton dynamics; cytoskeleton organization centers; ooplasmic segregation.

The turtle-blood feeding leeches, Placobdella ornata and Placobdella parasitica, consumed the soft tissue of Physella virgata and Stagnicola elodes gastropods in the laboratory. Placobdella ornata also damaged Helisoma anceps in apparent feeding attempts. The leeches generally ate low numbers of gastropods and approximately 3 days were required for the leeches to locate and consume the gastropods. This is the first documented report of P. ornata and P. parasitica consuming gastropods under laboratory conditions.

Cell-Cell Interactions in the Guidance of Late-Developing Neurons in Caenorhabditis elegans CUES PROVIDED BY NEURONS (1, 2), glia, other associated cells (1, 3), and components of the extracellular matrix (4) [...]