Comparison of body wall histologic structure of two medicinal leeches Hirudo sulukii and Hirudo verbana (Hirudinida: Hirudinidae).

In this study, it was aimed to determine secretory cell types using histochemical properties of secretory cells and epidermis histology in the body wall of two medicinal leech species, Hirudo verbana and Hirudo sulukii. In addition, areas of epidermis epithelial cells, secretory cell types, and secretion areas of secretory cells stained with histochemical stains were statistically compared in both species. Epidermis is composed of single layer of cylindrical epithelium and secretory cells. The cuticle layer covers the epithelial layer. Some Type 1 cells within and close to the epidermis were determined as pear-shaped secretory cells. Type 2a and Type 2b secretory cells were found in large groups in the inner parts of the body wall, especially around muscles. While Type 1 cells were stained weakly with PAS and AB, Type 2b cells were stained darker than Type 2a cells. Statistical calculations showed that areas of epithelial and secretory cells were generally larger in H. sulukii than in H. verbana. Therefore, H. sulukii was thought to be a more resistant species compared to H. verbana. As secretion areas of secretory cells reacting with PAS and AB stains were generally larger in H. sulukii, it was concluded that mucus composition between the two species has different concentration.

An ultrastructural study of the ovary cord organization and oogenesis in the amphibian leech Batracobdella algira (Annelida, Clitellata, Hirudinida).

This study reveals the ovary micromorphology and the course of oogenesis in the leech Batracobdella algira (Glossiphoniidae). Using light, fluorescence, and electron microscopies, the paired ovaries were analyzed. At the beginning of the breeding season, the ovaries were small, but as oogenesis progressed, they increased in size significantly, broadened, and elongated. A single convoluted ovary cord was located inside each ovary. The ovary cord was composed of numerous germ cells gathered into syncytial groups, which are called germ-line cysts. During oogenesis, the clustering germ cells differentiated into two functional categories, i.e., nurse cells and oocytes, and therefore, this oogenesis was recognized as being meroistic. As a rule, each clustering germ cell had one connection in the form of a broad cytoplasmic channel (intercellular bridge) that connected it to the cytophore. There was a synchrony in the development of the clustering germ cells in the whole ovary cord. In the immature leeches, the ovary cords contained undifferentiated germ cells exclusively, from which, previtellogenic oocytes and nurse cells differentiated as the breeding season progressed. Only the oocytes grew considerably, gathered nutritive material, and protruded at the ovary cord surface. The vitellogenic oocytes subsequently detached from the cord and filled tightly the ovary sac, while the nurse cells and the cytophore degenerated. Ripe eggs were finally deposited into the cocoons. A comparison of the ovary structure and oogenesis revealed that almost all of the features that are described in the studied species were similar to those that are known from other representatives of Glossiphoniidae, which indicates their evolutionary conservatism within this family.

Ovary cord micromorphology in the blood-sucking haemadipsid leech Haemadipsa japonica (Hirudinida: Arhynchobdellida: Hirudiniformes).

This is the first report that describes histological and ultrastructural details of ovary organization in haemadipsid leeches. In Haemadipsa japonica, the female reproductive system is organized similar to that of other haemadipsids. Each of the paired and oval ovaries of H. japonica is comprised of the ovary wall (ovisac), which encloses two elongated, thread-like ovarian units termed ovary cords. Ovary cords are comprised of germ-line cells and associated somatic cells. Each cord is polarized and contains germ-line cells in the consecutive developmental stages that are sequentially located along the long cord axis. There were three zones in each cord: the club-shaped apical part, the thread-like middle part, and the basal-most end, which contains degenerating germ cells. Outside of the reproductive period, the middle part of the cord in leeches is smooth, and no growing oocytes are visible; alternatively, in mature specimens, several growing oocytes protrude from the cord, and several huge vitellogenic oocytes that are completely detached from the cord occur within the ovisac. Ovary cord organization and functioning in H. japonica are very similar to the 'Hirudo' type cords that were found in several hirudiniform leeches. This conclusion supports the view that all hirudiniform leeches have conservative ovary cord organization and a similar pattern of oogenesis. Germ-line cyst composition, architecture, and functioning were also found to be evolutionarily conservative characteristics when compared with all previously examined Clitellata. In the germ-line cysts found in H. japonica each cell is connected to the central and anuclear cytoplasmic mass (cytophore) via one intercellular bridge, and, as oogenesis progresses, the fate of interconnected cell diversifies: some of them (oocytes) grow and complete oogenesis, but the majority become nurse cells and finally degenerate. Thus, oogenesis in H. japonica, similar to other clitellates, can be considered meroistic.

Detailed ultrastructure of the Hirudo (Annelida: Hirudinea) salivary gland.

Medical leeches have been widely used in medical applications and treatments for millennia. Studies on the salivary glands of blood-sucking leeches have focused on their bioactive secretions and mechanisms of action, with little attention to ultrastructure. In this study, we examined dissected embryonic and adult Hirudo verbana salivary glands by scanning electron microscopy (SEM). Gland cells of embryos were physically separated while adults displayed highly developed cell bunches in which each cell was connected to others by fine channels. Channels from each bunch combined to form a larger canal that opened to the jaw. Secreted material from these glands prevent blood from clotting and allow the adult to feed while sucking blood.

A new species of Branchellion Savigny, 1822 (Hirudinida: Piscicolidae), a marine leech parasitic on the giant electric ray Narcine entemedor Jordan & Starks (Batoidea: Narcinidae) off Oaxaca, Mexico.

Branchellion spindolaorum n. sp. (Hirudinida: Piscicolidae) is described based on specimens found parasitising the giant electric ray Narcine entemedor Jordan & Starks off the coast of Oaxaca, Mexico. The new species can be clearly distinguished from the other species of Branchellion Savigny, 1822 by the presence of 30 pairs of lateral branchiae and 10 pairs of pulsatile vesicles. The definition of the genus Branchellion is expanded to include species with either 30, 31 or 33 pairs of foliaceous (plate-like) lateral branchiae in the urosome. In addition, we provide for the first time for the genus, scanning electron micrographs of the secondary suckers located on the ventral surface of the posterior sucker. Additionally, partial DNA sequences of the mitochondrial cytochrome c oxidase subunit 1 (cox1) were generated and compared with homologous sequences of other species of the genus. Branchellion spindolaorum n. sp. represents the fourth species of the genus known in the Eastern Pacific and the first record of a leech parasitising N. entemedor.

Phylogenetic Position and Description of a New Species of Medicinal Leech from the Eastern United States.

A new species of medicinal leech, Macrobdella mimicus n. sp., is described from specimens collected in Maryland; this is the first description of a North American macrobdellid since 1975. Superficially, the new species resembles the well-known Macrobdella decora, as both species possess 4 accessory pores arranged symmetrically on the ventral surface, yet the new species is distinguished from M. decora in possessing 4-4½ annuli (rather than 3½) between the gonopores and 4 annuli (rather than 5 annuli) between the female gonopore and the first pair of accessory pores. Phylogenetic analyses, based on 2 mitochondrial and 2 nuclear loci for a set of closely related taxa, confirms the placement of the new species within the family Macrobdellidae and places it as the sister taxon to M. decora and M. diplotertia.

Redescription of the Siamese shield leech Placobdelloides siamensis with new host species and geographic range.

The Siamese shield leech Placobdelloides siamensis (Oka, 1917) Sawyer, 1986 (Euhirudinea: Glossiphoniidae) was collected from five new host species, Southeastern Asian Box Turtle (Cuora amboinensis), Yellow-headed Temple Turtle (Heosemys annandalii), Malayan Snail-eating Turtle (Malayemys macrocephala), Mekong Snail-eating Turtle (M. subtrijuga), and Khorat Snail-eating Turtle (M. khoratensis) and was found for the first time in Udon Thani, Thailand. Examination of live leeches provided, for the first time, data on coloration and the combination of parental care behavior, both carrying cocoons and attaching cocoons to the substrate. This species was separated from its congeners based on the following characters: one pair of eyes; spines at proboscis subterminal; mouth terminal on oral sucker; absent plaque in neck region; gonopores located in furrow and separated by two annuli; distinctly triannulated mid-body segments; crop with seven pairs and branched caeca; caudal sucker slightly over half of maximum body width; and strongly dorsal papillae. Phylogenetic relationships based on the COI and ND1 genes were clarified and demonstrated that the species is distinct from others. The original description was amended and the taxonomic history is discussed.

Surface ultrastructure and internal properties of the Erpobdella testacea (Hirudinae, Arynchobdellidae) cocoon membrane.

The Erpobdella testacea cocoon membrane is studied for the first time by transmission and scanning electron microscopy. It has an ovoid form, displays a cambered dorsal side in which various micro-organisms are attached and a flattened ventral side. Symmetrically positioned, 2 opercula occur at the distal ends of the cocoon. The internal ultrastructure reveals fibrils (17.5nm) packed in layers forming C, S, bow shaped, parallel and stippling patterns lines, interrupted by Polygon-shaped cavities (1.8µm). Transverse sections show that each fibril presents an external dark part (6nm) and a central hole approximately 5.06nm in diameter. These features are discussed with bibliographic data signalized for other species. Phylogenetic and functional significations of the cocoon wall structure in leeches are suggested.

Prevalence, Abundance, and Intensity of Implanted Spermatophores in the Leech Haementeria officinalis (Glossiphoniidae: Hirudinida) from Guanajuato, Mexico.

Fertilization through hypodermic implantation of spermatophores has been recorded in at least 4 groups of leeches: Glossiphoniidae, Piscicolidae, Ozobranchidae, and Erpobdelliformes. In Piscicola respirans (Piscicolidae), vector tissue responsible for sperm transfer from a specialized region of the body to the ovaries has led to the non-random attachment of spermatophores on the body surface of the recipient leech. It has been suggested that in glossiphoniid leeches, spermatophores are implanted in any part of the body surface of the recipient leech without a clear pattern or preference for region. In order to determine if the donor leech implants its spermatophores in a specific area of the conspecific recipient's body, we surveyed 81 specimens of Haementeria officinalis (Clitellata: Glossiphoniidae) from a wild population in Guanajuato, Mexico, and recorded the distribution of the spermatophores over the recipient's body surface. We describe for the first time a spermatophore of H. officinalis using scanning electron and light microscopy. Spermatophores were found attached dorsally between somites XVII and XXI 59.57% of the time, and the rest were found in other parts of the body, including on the ventral surface. The non-specific attachment for spermatophores does not support the presence of specialized tissue responsible for sperm transfer and instead attributes the placement of implantation to mechanical characteristics of the copulation process.

Ultrastructural analysis of apoptosis and autophagy in the midgut epithelium of Piscicola geometra (Annelida, Hirudinida) after blood feeding.

Cell death in the endodermal region of the digestive tract of the blood-feeding leech Piscicola geometra was analyzed using light and transmission electron microscopes and the fluorescence method. Sexually mature specimens of P. geometra were bred under laboratory conditions and fed on Danio rerio. After copulation, the specimens laid cocoons. The material for our studies were non-feeding juveniles collected just after hatching, non-feeding adult specimens, and leeches that had been fed with fish blood (D. rerio) only once ad libitum. The fed leeches were prepared for our studies during feeding and after 1, 3, 7, and 14 days (not sexually mature specimens) and some weeks after feeding (the sexually mature). Autophagy in all regions of the endodermal part of the digestive system, including the esophagus, the crop, the posterior crop caecum (PCC), and the intestine was observed in the adult non-feeding and feeding specimens. In fed specimens, autophagy occurred at very high levels--in 80 to 90 % of epithelial cells in all four regions. In contrast, in adult specimens that did not feed, this process occurred at much lower levels--about 10 % (esophagus and intestine) and about 30 % (crop and PCC) of the midgut epithelial cells. Apoptosis occurred in the feeding adult specimens but only in the crop and PCC. However, it was absent in the non-feeding adult specimens and the specimens that were collected during feeding. Moreover, neither autophagy nor apoptosis were observed in the juvenile, non-feeding specimens. The appearance of autophagy and apoptosis was connected with feeding on toxic blood. We concluded that autophagy played the role of a survival factor and was involved in the protection of the epithelium against the products of blood digestion. Quantitative analysis was prepared to determine the number of autophagic and apoptotic cells.

Barbronia weberi (Clitellata, Hirudinida, Salifidae) has ovary cords of the Erpobdella type.

The organization of the ovaries in representative of the Salifidae (Hirudinida, Erpobdelliformes) was studied at the ultrastructural level for the first time. Like in other leeches, the ovaries of Barbronia weberi are composed of an outer envelope (i.e., an ovisac made up of two coelomic epithelia, muscle cells, and connective tissue) and several internal units, which are broadly similar to the ovary cords found in representatives of the Erpobdellidae. There are usually 6-8 ovary cords that are twisted or cambered with a narrow apical part and a broader, irregularly shaped distal end in each ovisac of B. weberi. Each ovary cord is built from somatic and germ-line cells and the latter tend to form multicellular cysts that are equipped with a central cytoplasmic core (cytophore). There are two morphologically different subpopulations of germ-line cells: oocytes and more numerous nurse cells. Growing oocytes form protuberances on the ovary cord surface and eventually detach from the cord and float freely in the ovisac lumen, whereas the other components of germ-line cysts (i.e., nurse cells and cytophore) degenerate. It should be pointed out that there is a prominent gradient of germ-cell development along the long axis of the cord. The somatic cells form the ovary cord envelope (the so-called spongiosa cells) and also penetrate the spaces between germ-line cells. Both kinds of the somatic cells, that is, those forming the cord envelope and the somatic cells that are associated with oocytes (follicular cells) have a well-developed system of intercellular channels. Additionally, one prominent somatic cell, the apical cell, was found at the apical tip of each ovary cord. Because the aforementioned features of ovary cords found in B. weberi are very similar (with a few minor exceptions) to the ovary cords that have been described in Erpobdella octoculata and E. johanssoni, we propose the term "ovary cords of the Erpobdella type" for them. Our results support a close phylogenetic relationship between Salifidae and Erpobdellidae.

May salivary gland secretory proteins from hematophagous leeches (Hirudo verbana) reach pharmacologically relevant concentrations in the vertebrate host?

Saliva of hematophagous leeches (Hirudo sp.) contains bioactive proteins which allow the leech proper feeding and storage of ingested blood, but may also exert effects in the host. Leech therapy is used to treat many different ailments in humans, although only a small fraction of salivary proteins are characterized yet. Moreover, we do not know whether complete transfer of salivary proteins stored in the unicellular salivary glands in a leech to the host during feeding may generate concentrations that are sufficiently high to affect physiological processes in the host. Our 3D reconstruction of a portion of internal leech tissue from histological sections revealed that one leech contains approx. 37,000 salivary gland cells. Using tissue slices from pig liver and mouse skeletal muscle for reference, we obtained data for protein densities in leech salivary gland cells. As individual salivary cells are voluminous (67,000 µm(3)) and the stored proteins are densely packed (approx. 500 µg/mm(3)), we extrapolated that a single leech may contain up to 1.2 mg of salivary proteins. Analyzing protein extracts of unfed or fed leeches by 2D electrophoresis, we calculated the relative molar amounts of individual salivary proteins in the mass range of 17-60 kDa which may be released from a single leech during feeding. Distribution of these salivary proteins in the host (assumed plasma volume of 5 l) may result in concentrations of individual compounds between 3 and 236 pmol/l. Such concentrations seem sufficiently high to exert biochemical interactions with target molecules in the host.

Operculum ultrastructure in leech cocoons.

Clitellate annelids (i.e., oligochaetes including leeches) secrete cocoons as part of their normal reproductive cycle. Typically, the cocoon sheath is passed over the head of the leech and sealed at both ends by opercula (i.e., glue-like material secreted by the clitellum). Both the fibrous cocoon wall (CW) and opercula are chemically-related biomaterials that share unusual physiochemical properties, including thermal and chemical resiliency. To explore the underlying morphology of the operculum, we examined cocoons from four leech species (i.e., Myzobdella lugubris, Theromyzon tessulatum, Erpobdella obscura, and Erpobdella punctata) by transmission (TEM) and scanning electron microscopy (SEM). Transmission electron micrographs of all opercula revealed a common, ultrastructural pattern comprising an electron-dense mosaic of ordered polygons that surrounded interspersed cavities. The long axes of cavities were often oriented directionally, suggesting that operculum material is pliable prior to solidification and distorted as a consequence of cocoon deposition. Concomitantly, the operculum permeates jagged edges of the cocoon sheath sealing the cocoon, which provides a mechanically strong CW/operculum boundary. SEM of leech opercula revealed globular nanoparticles comparable to that observed in bioadhesives from disparate animal phyla (e.g., mussel, barnacle, sea star), suggesting a convergent mechanism of bioadhesion among animals.

An ultrastructural study of the ovary cord organization and oogenesis in Erpobdella johanssoni (Annelida, Clitellata: Hirudinida).

The aims of the present study were to analyze the ovary cord structure and oogenesis in Erpobdella johanssoni under light, fluorescent and transmission electron microscopy and to compare the obtained results with other clitellate annelids, especially with other arhynchobdellid leeches. Each of the paired ovaries is composed of the ovary wall (ovisac) and several (7-8) short, cone-shaped ovary cords. The ovary cords are of the "Erpobdella" type, i.e. they are short and polarized and five zones containing germ cells at consecutives stages of their development can be distinguished along their long axis. One, huge somatic cell (the apical cell), oogonia and premeiotic germ cells occur at the tip of the apical part of the ovary cord - zone I. Below, in zone II germ cells enter meiosis, whereas in zone III only a few cells continue meiosis and gather nutrients (oocytes), while the rest become nurse cells. In zone IV, huge vitellogenic oocytes form protuberances on the surface of the cord, and degenerating germ cells were observed at the base of the ovary cord (zones IV and V). The germline cells form syncytial cysts in zones I-III. The germline cysts have broadly the same architecture as in the ovaries of all of the clitellate annelids that have been described to date. Each germ cell in a cyst has only one cytoplasmic bridge connecting it to the common cytoplasmic mass - the cytophore. The cytophore is poorly developed, and it has the form of thin, long cytoplasmic strands. The presence of two categories of germ cells suggests a meroistic mode of oogenesis. The germline cysts are closely associated with somatic, follicular cells. There are two subpopulations of follicular cells: one envelops the growing oocytes, while the second is distributed between other germ cells. The entire ovary cord is additionally enveloped by a layer of somatic cells with a spongy appearance - the spongiosa cells. A characteristic feature of vitellogenic oocytes is the condensation of the chromosomes into a karyosome. Fully grown oocytes are excluded from the ovary cords and float freely in the ovisac lumen.

Affiliations between bacteria and marine fish leeches (Piscicolidae), with emphasis on a deep-sea species from Monterey Canyon, CA.

Leeches within the Piscicolidae are of great numerical and taxonomic importance, yet little is known about bacteria that associate with this diverse group of blood-feeding marine parasites of fish and elasmobranchs. We focused primarily on the bacteria from a deep-sea leech species of unknown identity, collected at ∼ 600 m depth in Monterey Canyon, CA, along with two shallow-living leech genera, Austrobdella and Branchellion, from Los Angeles Harbor, CA. Molecular analysis of all five leech species revealed a dominance of gammaproteobacteria, which were distinct from each other and from previously reported freshwater leech symbionts. Bacteria related to members of the genus Psychromonas (99% similarity in 16S rRNA) were dominant in the deep-sea leech species (80-94% of recovered ribotypes) collected over 19 months from two different locations. Psychromonas was not detected in cocoons or 2-16 week-old juveniles, suggesting that acquisition is via the environment at a later stage. Transmission electron microscopy did, however, reveal abundant bacteria-like cells near areas of thinning of the juvenile epithelial surface, as well as Psychromonas sparsely distributed internally. Electron and fluorescence in situ microscopy of adults also showed Psychromonas-like bacteria concentrated within the crop. Despite the apparent non-transient nature of the association between Psychromonas and the deep-sea leech, their functional role, if any, is not known. The prevalence, however, of an abundant bacterial genus in one piscicolid leech species, as well as the presence of a dominant bacterial species in singular observations of four additional marine species, suggests that members of the Piscicolidae, possibly basal within the class Hirudinea, form specific alliances with microbes.

Morphology and ultrastructure of the midgut in Piscicola geometra (Annelida, Hirudinea).

This paper presents information on the organization of the midgut and its epithelium ultrastructure in juvenile and adult specimens of Piscicola geometra (Annelida, Hirudinea), a species which is a widespread ectoparasite found on the body and gills and in the mouth of many types of fish. The analysis of juvenile nonfeeding specimens helped in the explanation of all alterations in the midgut epithelium which are connected with digestion. The endodermal portion (midgut) of the digestive system is composed of four regions: the esophagus, the crop, the posterior crop caecum, and the intestine. Their epithelia are formed by flat, cuboidal, or columnar digestive cells; however, single small cells which do not contact the midgut lumen were also observed. The ultrastructure of all of the regions of the midgut are described and discussed with a special emphasis on their functions in the digestion of blood. In P. geometra, the part of the midgut that is devoid of microvilli is responsible for the accumulation of blood, while the epithelium of the remaining part of the midgut, which has a distinct regionalization in the distribution of organelles, plays a role in its absorption and secretion. Glycogen granules in the intestinal epithelium indicate its role in the accumulation of sugar. The comparison of the ultrastructure of midgut epithelium in juvenile and adult specimens suggests that electron-dense granules observed in the apical cytoplasm of digestive cells take part in enzyme accumulation. Numerous microorganisms were observed in the mycetome, which is composed of two large oval diverticles that connect with the esophagus via thin ducts. Similar microorganisms also occurred in the cytoplasm of the epithelium in the esophagus, the crop, the intestine, and in their lumen. Microorganisms were observed both in fed adult and unfed juvenile specimens of P. geometra, which strongly suggests that vertical transmission occurs from parent to offspring.

Stratified bacterial community in the bladder of the medicinal leech, Hirudo verbana.

Most animals harbour symbiotic microorganisms inside their body, where intimate interactions occur between the partners. The medicinal leech, Hirudo verbana, possesses 17 pairs of excretory bladders that harbour a large number of intracellular and extracellular symbiotic bacteria. In this study, we characterized the bladder symbionts using molecular phylogenetic analyses, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH). Restriction fragment length polymorphism (RFLP) and sequence analyses of 16S rRNA gene clone libraries suggested that six bacterial species co-colonize the leech bladders. Phylogenetic analyses revealed that these species belong to the alpha-Proteobacteria (Ochrobactrum symbiont), beta-Proteobacteria (Beta-1 and Beta-2 symbionts), delta-Proteobacteria (Bdellovibrio symbiont) and Bacteroidetes (Niabella and Sphingobacterium symbionts). Species-specific PCR detection and FISH confirmed the localization of the symbiotic bacteria in the bladders. The Ochrobactrum, Beta-1, Bdellovibrio and Sphingobacterium symbionts were consistently detected in 13 leeches from two populations, while infection rate of the other symbionts ranged between 20% and 100% in the two leech populations. Transmission electron microscopy observations of the bladders revealed epithelial cells harbouring a number of intracellular bacilli and an additional type of extracellular, rod-shaped bacteria in the luminal region. Fluorescence in situ hybridization with group-specific oligonucleotide probes revealed the spatial organization of the bacterial species in the bladder: the Ochrobactrum symbiont was located intracellularly inside epithelial cells; the Bacteroidetes were localized close to the epithelium in the lumen of the bladder; and the Bacteroidetes layer was covered with dense beta-proteobacterial cells. These results clearly demonstrate that a simple but organized microbial community exists in the bladder of the medicinal leech.

A novel Salifa-Saprolegnia association.

During the period from December 2006 to March 2007, about 1000 freshwater leeches, Salifa delicata, were collected from Al-sont canal, adjacent to Assiut city, Egypt. In the laboratory, 96% of Salifa delicata showed signs of oomycotal infection (cotton-wool like appearance radiating out in whorled pattern) and died within 3 days. Direct microscopy and culture proved Saprolegnia hypogyna to be the pathogen. Histopathological studies showed necrotic lesions, destruction of cuticle, epidermis, dermis, muscle layers, botryoidal tissue and even the gut with the oomycete hyphae penetrating the damaged tissues. To the best of the authors' knowledge, this represents the first record of a novel association between the leech, Salifa delicata, and the oomycete, Saprolegnia hypogyna, but the second report on the histopathology of saprolegniasis within leeches.

Cathepsin L and cystatin B gene expression discriminates immune coelomic cells in the leech Theromyzon tessulatum.

Previous studies evidenced that cystatin B-like gene is specifically expressed and induced in large circulating coelomic cells following bacterial challenge in the leech Theromyzon tessulatum. In order to understand the role of that cysteine proteinase inhibitor during immune response, we investigated the existence of members of cathepsin family. We cloned a cathepsin L-like gene and studied its tissue distribution. Immunohistochemical studies using anti-cathepsin L and anti-cystatin B antibodies and ultrastructural results demonstrated the presence of three distinct coelomic cell populations: (1) the chloragocytes, which were initially defined as large coelomocytes, (2) the granular amoebocytes and (3) small coelomic cells. Among those cells, while chloragocytes contain cystatin B and cathepsin L, granular amoebocytes contain only cathepsin L and the third cell population contains neither cathepsin nor inhibitor. Finally, results evidenced that cathepsin L immunopositive granular amoebocytes are chemoattracted to the site of injury and phagocyte bacteria.

Oogenesis in four species of Piscicola (Hirudinea, Rhynchobdellida).

Piscicola has a pair of elongated sac-shaped ovaries. Inside the ovaries are numerous small somatic cells and regularly spherical egg follicles. Each follicle is composed of three types of cells: many (average 30) germ cells (cystocytes) interconnected by intercellular bridges in clones (cysts), one intermediate cell, and three to five outer follicle cells (envelope cells). Each germ cell in a clone has one intercellular bridge connecting it to the central anucleate cytoplasmic mass, the cytophore. Each cluster of germ cells is completely embedded inside a single huge somatic follicle cell, the intermediate (interstitial) cell. The most spectacular feature of the intermediate cell is its development of a system of intracytoplasmic canals apparently formed of invaginations of its cell membrane. Initially the complex of germ cell cluster + intermediate cell is enclosed within an envelope composed of squamous cells. As oogenesis progresses the envelope cells gradually degenerate. All the germ cells that have terminated their mitotic divisions are of similar size and enter meiotic prophase, but one of the cystocytes promptly starts to grow faster and differentiates into the oocyte, whereas the remaining cystocytes withdraw from meiosis and become nurse cells (trophocytes). Numerous mitochondria, ER, and a vast amount of ribosomes are transferred from the trophocytes via the cytophore toward the oocyte. Eventually the oocyte ingests all the content of the cytophore, and the trophocytes degenerate. Little vitellogenesis takes place; the oocyte gathers nutrients in the form of small lipid droplets. At the end of oogenesis, an electron-dense fibrous vitelline envelope appears around the oocyte, among short microvilli. At the same time, electron-dense cortical granules occur in the oocyte cortical cytoplasm; at the end of oogenesis they are numerous, but after fertilization they disappear from the ooplasm. In the present article we point out many differences in the course of oogenesis in two related families of rhynchobdellids: piscicolids and glossiphoniids.