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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., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to be disclosed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

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 .

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., (© K. Chiangkul et al., published by EDP Sciences, 2018.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.

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.

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., (Copyright © 2013 Wiley Periodicals, Inc.)

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.

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., (Copyright © 2013 Wiley Periodicals, Inc.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

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., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

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.

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.

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.

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., ((c) 2007 Wiley-Liss, Inc.)

Helicoids in the cocoon membrane of leeches Theromyzon tessulatum and Erpobdella punctata comprise a twisted superposition of layers, each containing a variable number of planes formed by unidirectional fibrils. Straight fibrils intersecting at different angles were displayed in tangential sections through the cocoon wall of each species. When the sectioning angle was below a certain value (i.e., the critical angle), bow-shaped lines apparent in oblique sections were replaced by a succession of layers containing straight fibrils, permitting a direct measurement of step-angle change between successive layers in a helicoid. By this methodology, we determined that no regularities exist in the succession of step-angles or in layer thicknesses within the cocoon membranes, but that the distribution of step-angles between layers was unique for each cocoon type.

During October 2004 to April 2005, when water temperature ranged between 18 and 22 degrees C, over than 500 specimens of leeches namely Alboglossiphonia polypompholyx, Batracobdelloides tricarinata, Helobdella conifera (Rhynchobdellida), Barbronia assiuti, and Salifa delicata (Arhynchobdellida) were collected from Al-Sont canal near Assiut city. About 80% of these specimens were infected with the peritrich ciliate Epistylis sp. The infection occurred in the field, both rhynchobdellid and arhynchobdellid leeches were infected, but Epistylis can infect rhynchobdellids more than arhynchobdellids. Both light and scanning electron microscopy showed the morphology of this parasite which attached to the surface of the leech body by a stalk. Histopathological studies revealed the damage of cuticle, epidermis, and dermis of the leech body wall at the area of attachment and the leech body seemed to be sloughy. Presence of this parasite in high numbers covering large areas of the body can impede gas exchange leading to leech suffocation and finally its death. Like the other sessile single-celled ciliates Epistylis spp. feed on bacteria and suspended organic debris, which are prevalent in nutrient-rich water, hence, Epistylis is a good indicator of poor water quality (pollution). The present study represents the first report of the effect of Epistylis on leeches collected from Egypt.

By the end of previtellogenesis, the oocytes of Glossiphonia heteroclita gradually protrude into the ovary cavity. As a result they lose contact with the ovary cord (which begins to degenerate) and float freely within the hemocoelomic fluid. The oocyte's ooplasm is rich in numerous well-developed Golgi complexes showing high secretory activity, normal and transforming mitochondria, cisternae of rER and vast amounts of ribosomes. The transforming mitochondria become small lipid droplets as vitellogenesis progresses. The oolemma forms microvilli, numerous coated pits and vesicles occur at the base of the microvilli, and the first yolk spheres appear in the peripheral ooplasm. A mixed mechanism of vitellogenesis is suggested. The eggs are covered by a thin vitelline envelope with microvilli projecting through it. The envelope is formed by the oocyte. The vitelline envelope is produced by exocytosis of vesicles containing two kinds of material, one of which is electron-dense and seems not to participate in envelope formation. The cortical ooplasm of fully grown oocytes contains many cytoskeletal elements (F-actin) and numerous membrane-bound vesicles filled with stratified content. Those vesicles probably are cortical granules. The follicle cells surrounding growing oocytes have the following features: (1) they do not lie on a basal lamina; (2) their plasma membrane folds deeply, forming invaginations which eventually seem to form channels throughout their cytoplasm; (3) the plasma membrane facing the ovary lumen is lined with a layer of dense material; and (4) the plasma membrane facing the oocyte forms thin projections which intermingle with the oocyte microvilli. In late oogenesis, the follicle cells detach from the oocytes and degenerate in the ovary lumen.

Protuberances on the cocoon surface of the leech, Theromyzon tessulatum, are roughly parallel rows of triangular prisms arranged equidistantly to each other on the outer surface of the cocoon membrane. The distance between neighboring protuberances is approximately 1.6 microm, the height approximately 0.5 microm and the semi-width approximately 0.3 microm. The fibrillar arrangement within the protuberance maintains some elements of the helicoids found within the cocoon membrane but a high proportion of large holes disrupt the symmetry of the protuberance ultrastructure. A procedure for 3D reconstruction of the protuberance using the complementarity between the paratangential and normal sections through the cocoon is presented. Our results demonstrate that the ultrastructure of protuberances show elements of a twisted fibrillar arrangement, but the demands of filling a narrow space ruled by acute angles appears to cause a high degree of ultrastructural disorganization.

Oogenesis in the glossiphoniid leech Glossiphonia heteroclita (Hirudinea, Rhynchobdellida) is nutrimental, i.e., the growing oocyte is supported by specialized germline cells, the nurse cells. The main function of the nurse cells is to provide oocytes with cell organelles and RNAs (mainly rRNA). However, in studied leech species, irrespective of the nutrimental mode of oogenesis, the germinal vesicle (GV = oocyte nucleus) seems to be very active in rRNA production. As shown in the present study, during early previtellogenesis in the GV the meiotic chromosomes and prominent primary nucleoli occur. In late previtellogenesis the chromosomes condense and occupy a limited space of nucleoplasm in close vicinity to primary nucleolus, forming a karyosome. At the onset of vitellogenesis several prominent extrachromosomal DNA bodies appear in close association with the karyosome. At the same time, the primary nucleolus is no longer visible in the GV. As vitellogenesis proceeds the extrachromosomal DNA bodies undergo fragmentation and numerous spherical, RNA- and AgNOR-positive inclusions occur in the nucleoplasm. They are regarded as multiple nucleoli. Finally, in late oogenesis numerous accessory nuclei are formed in close proximity to the nuclear envelope. They usually contain one dense body, morphologically similar to multiple nucleoli. The amplification of rDNA genes, the occurrence of extrachromosomal DNA bodies, as well as the presence of multiple nucleoli and accessory nuclei are described for the first time in the phylum Annelida.

Placobdelloides bancrofti (Best, 1931) Sawyer, 1986 (Euhirudinea: Glossiphoniidae) is recorded for the first time from a location in North Queensland, Australia. Placobdelloides bancrofti was found feeding on the freshwater turtle Emydura krefftii and was collected from a new host species Elseya latisternum. Examination of live individuals enabled us to document external coloration and markings for the first time. Clarification of the original description of P. bancrofti is made, correcting the location of the anal pore, and then the taxonomic history of this species is discussed.

The cocoon of the leech Theromyzon trizonare consists of fibrils packed into an arrangement that produces both C- and S-like patterns of bow-shaped lines in sections oblique through the membrane. Sections normal to the cocoon membrane show layers containing cross-sections of fibrils (approximately 16 nm dia.) that are separated by a center-to-center distance of approximately 23 nm. In cross-section, each fibril presents a central hole approximately 5 nm in diameter. A structureless layer covers most of the exterior surface of the cocoon membrane, and short protuberances are apparent in some zones.

The uptake of horseradish peroxidase (HRP), applied as an extracellular tracer, is a classical method for studying endo/exocytosis of synaptic vesicles at the ultrastructural level. It is generally not considered that HRP may affect neuronal function. Reported here is the finding that extracellularly applied HRP (0.1%) perturbs dense core vesicles in the synaptic processes of leech neurons. The strength of the effect varies with neuronal class. In sensory afferents, the number of dense core vesicles increases 5-fold, while there is only a 2-fold increase in central neurons.

We have used electron microscopy to measure quantitatively the morphology of electrical synapses in a circuit that has been proposed to account for the positional discrimination of the leech. Injection of a presynaptic nociceptive sensory neuron and the postsynaptic anterior pagoda neuron with HRP showed gap junctions in the neuropil. After double labeling, La(3+)-treated ganglia revealed labeled gap junctions from 2.0 to 3.5 nm wide. Between the labeled axon terminals, there were innexons with diameters of 8 to 10 nm. The innexon's central pore diameter was 2 nm, and the mean of the center-to-center distance between two innexons was 30 nm. Except for the gap junction areas of nociceptive sensory neuron axon terminals, the other ultrastructural parameters measured by freeze fracture were similar to those of samples labeled with HRP and filled with La(3+). These data suggested that the gap width, innexon diameter, and its central pore do not on their own account for the mechanism of positional discrimination, which may depend rather on the differences in distribution and number of gap junctions.

Differences in carbohydrate signaling control sequential steps in synaptic growth of sensory afferents in the leech. The relevant glycans are constitutive and developmentally regulated modifications of leechCAM and Tractin (family members of NCAM and L1) that are specific to the surface of sensory afferents. A mannosidic glycosylation mediates the dynamic growth of early afferents as they explore their target region through sprouting sensory arbors rich with synaptic vesicles. Later emerging galactosidic glycosylations serve as markers for subsets of the same sensory afferents that correlate with different sensory modalities. These developmentally regulated galactose markers now oppose the function of the constitutive mannose marker. Sensory afferents gain cell-cell contact with central neurons and self-similar afferents, but lose filopodia and synaptic vesicles. Extant vesicles are confined to sites of en passant synapse formation. The transformation of sensory afferent growth, progressing from mannose- to galactose-specific recognition, is consistent with a change from cell-matrix to cell-cell contact. While the constitutive mannosidic glycosylation promotes dynamic growth, developmentally regulated galactosidic glycosylations of the same cell adhesion molecules promote tissue stability. The persistence of both types of neutral glycans beyond embryonic age allows their function in synaptic plasticity during habituation and learning.

Intracellular rickettsia-like structures were found in the tissues of a glossiphoniid leech, Torix tagoi, by transmission electron microscopy. Diagnostic PCR analysis using specific primers suggested that of the nine glossiphoniid species examined, two species, T. tagoi and Hemicrepsis marginata, harbored bacteria of the genus Rickettsia. A 1.5-kb eubacterial 16S rRNA gene segment obtained from each of these species was amplified by PCR, cloned, and sequenced. Phylogenetic analysis of the 16S rRNA gene demonstrated that the Rickettsia species found in the leeches constituted a novel clade that is distinct from the clade of arthropod-associated Rickettsia species. In natural populations, 97.7% (43 of 44) of T. tagoi leeches and 100% (9 of 9) of H. marginata leeches carried Rickettsia, suggesting that infection with Rickettsia is prevalent in these leeches. This is the first report of Rickettsia found in annelids.

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 micrometer/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., (Copyright 2000 Academic Press.)

Sensory afferents in the leech are labeled with both constitutive and developmentally regulated glycosylations (markers) of their cell adhesion molecules (CAMs). Their constitutive mannose marker, recognized by Lan3-2 monoclonal antibody (mAb), mediates the formation of their diffuse central arbors. We show that, at the ultrastructural level, these arbors consist of large, loosely organized axons rich with filopodia and synaptic vesicles. Perturbing the mannose-specific adhesion of this first targeting step leads to a gain in cell-cell contact but a loss of filopodia and synaptic vesicles. During the second targeting step, galactose markers divide afferents into different subsets. We focus on the subset labeled by the marker recognized by Laz2-369 mAb. Initially, the galactose marker appears where afferents contact central neurons. Subsequently it spreads proximally and distally, covering the entire afferent surface. Afferents now gain cell-cell contact, with central neurons and self-similar afferents, but lose filopodia and synaptic vesicles. Extant synaptic vesicles prevail where afferents are apposed to central neurons. These neurons develop postsynaptic densities and en passant synapses are forming. Perturbing the galactose-specific adhesion of this second targeting step causes a loss of cell-cell contact but a gain in filopodia and synaptic vesicles, essentially returning afferents to the first targeting step. The transformation of afferent growth, progressing from mannose- to galactose-specific adhesion, is consistent with a change from cell-matrix to cell-cell adhesion. By performing opposing functions in a temporal sequence, constitutive and developmentally regulated glycosylations of CAMs collaborate in the synaptogenesis of afferents and the consolidation of self-similar afferents., (Copyright 1999 Academic Press.)

Glossiphoniids are iteroparous hirudineans that brood their offspring. The young are attached by the posterior sucker to the parent's ventral side until development is complete. The weight loss of the brooding adults is commonly attributed to their reduced possibility of feeding and to the increased metabolic expense for movements needed to ventilate the offspring. The present study showed that there is a passage of nutritive substrates between the parental body wall and the young's sucker. A possible correlation between this passage and the reabsorption of some muscle fibers that occurs in the adult's body wall is also analyzed.

The oblique muscle organizer (Comb- or C-cell) in the embryonic medicinal leech, Hirudo medicinalis, provides an amenable situation to examine growth cone navigation in vivo. Each of the segmentally iterated C-cells extends an array of growth cones through the body wall along oblique trajectories. C-cell growth cones undergo an early, relatively slow period of extension followed by later, protracted and rapid directed outgrowth. During such transitions in extension, guidance might be mediated by a number of factors, including intrinsic constraints on polarity, spatially and temporally regulated cell and matrix interactions, physical constraints imposed by the environment, or guidance along particular cells in advance of the growth cones. Growth cones and their environment were examined by transmission electron microscopy to define those factors that might play a significant role in migration and guidance in this system. The ultrastructural examination has made the possibility very unlikely that simple, physical constraints play a prominent role in guiding C-cell growth cones. No anatomically defined paths or obliquely aligned channels were found in advance of these growth cones, and there were no identifiable physical boundaries, which might constrain young growth cones to a particular location in the body wall before rapid extension. There were diverse associations with many matrices and basement membranes located above, below, and within the layer in which growth cones appear to extend at the light level. Additionally, a preliminary examination of myocyte assembly upon processes proximal to the growth cones further implicates a role for matrix-associated interactions in muscle histogenesis as well as process outgrowth during embryonic development.

The stereotyped distribution of identified neurons and glial cells in the leech nervous system is the product of stereotyped cell migrations and rearrangements during embryogenesis. To examine the dependence of long-distance cell migrations on positional cues provided by other tissues, embryos of Theromyzon rude were examined for the effects of selective ablation of various embryonic cell lines on the migration and final distribution of neural and glial precursor cells descended from the bilaterally paired ectodermal cell lines designated q bandlets. The results suggest that neither the commitment of q-bandlet cells to migrate nor the general lateral-to-medial direction of their migration depend on interactions with any other cell line. However, the ability of the migrating cells to follow their normal pathways and to find their normal destinations does depend on interactions with cells of the mesodermal cell line, which appears to provide positional cues that specify the migration pathways.

The giant anterior salivary gland cells from the large mammalian blood-sucking, glossiphoniid leech, Haementeria ghilianii, can be subdivided into three morphologically and functionally distinct regions: 1) a soma, responsible for the synthesis and storage of secretory products; 2) a long cell process, responsible for the storage and intracellular transport of the secretory vesicles; and 3) the site of exocytosis at the process terminal. The giant somata are densely packed with secretory vesicles. Deep plasmalemmal invaginations invade the soma and form an extensive system of extracellular lacunae. The rough endoplasmic reticulum (ER) and the Golgi apparatus are organized in the cell periphery, near the highly branched nucleus, and along the lacunae. The somata taper into long processes extending over several centimeters to the proboscis tip. These contain secretory vesicles through their whole length. In the process periphery, the vesicles are completely ensheathed by a concentric subplasmalemmal smooth ER cisterna. This originates deeply within the soma and extends through the whole cell process to its terminal. The ER provides support for up to several hundred longitudinally oriented microtubules. Secretion occurs at the very tip of the cell processes, each of which terminates at the proboscis tip at the base of a cuticular pore. We found synapses close to the sites of exocytosis, providing morphological evidence for neuronal control of secretion.

In embryos of the glossiphoniid leech, Helobdella triserialis, as in many annelids, cytoplasmic reorganization prior to first cleavage generates distinct animal and vegetal domains of yolk-deficient cytoplasm, called teloplasm. Both domains are sequestered to the D' macromere, progenitor of the definitive segmental tissues, during the first three rounds of cell division. And it has been believed that during the fourth round of cell division, the obliquely equatorial cleavage of macromere D' cleanly segregates animal teloplasm into an ectodermal precursor, cell DNOPQ, and vegetal teloplasm into a mesodermal precursor, cell DM. But here we report a hitherto unobserved cytoplasmic rearrangement between the second and the fourth divisions that seems to mix the animal and vegetal domains of teloplasm. The newly observed rearrangement consists of the movement of vegetal teloplasm toward the animal pole of cell D' between the second and the fourth cell divisions. Animal and vegetal teloplasms form a single pool of teloplasm in cell D' which is then divided between DM and DNOPQ at the fourth division. The movement of teloplasm was inferred by examination of embryos fixed and sectioned between the second and the fourth rounds of cleavage and was confirmed in living embryos microinjected with rhodamine 123, a fluorescent mitochondrial stain.

Two ultrastructural approaches were used in photoreceptor cells of the leech, Hirudo medicinalis, to (a) investigate the intracellular topography of the smooth endoplasmic reticulum (SER) and (b) identify among the various subregions of the SER those which might function as Ca-sequestering sites. When the cells are prefixed with CaCl2-containing glutaraldehyde and postfixed with osmium tetroxide-ferricyanide (OsFeCN), only a part of the total SER is specifically stained. The stained SER cisternae include the submicrovillar cisternae (SMC), subsurface cisternae (SSC), the nuclear envelope, Golgi-associated SER, paracrystalline SER, and SER associated with glycogen areas. An extensive tubular SER cisternal system always remains unstained. When the cells are permeabilized by saponin and subsequently incubated with Ca2+, MgATP, and oxalate, the SMC (Walz, 1979, Eur. J. Cell Biol. 20:83-91), the SSC and the nuclear envelope contain electron-opaque Ca-oxalate precipitates indicating their ability to function as an effective Ca2+ sink. The results show that the very elaborate SER in this photoreceptor cell includes many functionally heterogeneous subregions. Of special physiological significance are those components (SMC and SSC) which are effective in Ca2+-buffering in the immediate vicinity of the plasma membrane.

In the present contribution the distributive pattern of phosphatases and non-specific esterase amongst the constituents of the leech eye has been revealed and the significance of the enzymatic locales in relation to the visual processes has been discussed. All the phosphatases studied demonstrate absence of the activity in the lens. Activity of the phosphatases, however, is restricted to the plasma membrane and associated cytoplasm to varying degrees. The significance of the phosphatases in the nerve impulses and transmission has been highlightened. The observation of considerable significance relates the demonstration of a high degree of non-specific esterase activity in the lens, which has defied all the phosphatases. Another observation relates to the positive activity of 5-nucleotidase in the group of axons constituting optic nerve. The significance of this observation has also been discussed.

The position, structure and function of the valves within the lateral sinus of the medical leech, Hirudo medicinalis, are described on the basis of vital, light- and electron microscopy. In this species the valvular apparatus consists of multiple elongated fir cone-shaped fibrous villi surrounding the orifices of the latero-lateral and latero-dorsal vessel like a tentacular crest. Each villus is covered by a thin sheet of a continuous endothelium. The valves prevent the backflow of hemolymph during systolic contraction of the lateral sinus. The endothelium contains many small mitochondria and polyribosomes in the perinuclear cytoplasm and it develops deep projections into the underlying connective tissue. Each of these consists of a multilayered system of closely interwoven thin endothelial membranes. The endothelium is anchored to its basement membrane by means of a great number of poorly defined hemidesmosomes. The fibrous tissue of the villi consists mainly of a homogeneous vitreous matrix in which few cellular components and very fine filaments are dispersed. Close to the endothelium this matrix appears to be condensed to form a multilayered framework made out of a basement membrane-like material. Though the valves themselves are devoid of muscle cells, those situated at their base and thus belonging to the vascular wall proper, display some specific morphological features: in particular the nuclei of these cells show a distinct fibrous lamina. Moreover, these muscle cells seem to be innervated only by one type of axon, containing both small, lucent synaptic vesicles as well as some of the dense-core variety. These findings are compared with data from earlier works and are discussed in relation to the hemodynamic functions of this valvular apparatus.

Microvillar (receptive) and external (non-receptive) portions of the plasmalemma of photoreceptor cells of Hirudo were compared electron microscopically in thin sections and freeze-fracture replicas. A morphometric approximation showed that the surface area of the microvillar membrane is about 19 times larger than that of the external membrane. The microvillar membrane most probably undergoes extensive membrane turnover. In both segments of the membrane the particles associated with the P- and the E-fracture faces are randomly distributed except at some specific sites. The particles adhere predominantly to the P-faces. The particle densities on the fracture faces of the microvillar membrane differ from those of the external membrane. The P-face particles of the external membrane appear to be larger than those of the microvillar membrane. It is suggested that the P-face particles of the microvillar membrane represent sites where the photopigment is incorporated into the membrane. The distinguishing structural features correspond to the functional differences postulated for both portions of the plasma membrane.

The microvilli of leech photoreceptors have diameters in the range of 60-100 nm. Each contains a bundle of microfilaments extending into the photoreceptor soma as a rootlet (Walz 1979 b). Apparent thicknesses of individual filaments are estimated to be 4-5 nm, consonant with those of identified actin filaments in the basement membranes of blowflies (Blest and De Couet 1983). Frozen sections of leech photoreceptors labelled with antibodies against scallop actin exhibited strong binding to the rootlet region but not to the microvilli, which are destroyed by the severe saponin or acetone extraction needed to permeabilise the preparation. NBD-phallacidin binds strongly but non-specifically to the photoreceptors and does not allow positive identification of F-actin. The cytoskeletons of the microvilli and rootlets are adequately preserved by conventional routines of fixation, and similar results were obtained when retinae were pretreated with either 0.5 mM Ca2+, 10 mM EGTA, 2 mM Ep-475 (a specific inhibitor of thiol proteases) or 2 mM Ep-475 combined with 0.5 mM Ca2+. Unlike the axial cytoskeletons of arthropod photoreceptor microvilli, those of the leech are stable to cellular insult.

The electron microscopic investigation has revealed in the body of the leech two types of vessels, that together with common signs possess a number of peculiarities in composition of endothelial cells. These vessels have different level of organization: the vessels of the first type, concerning the parenchyma of the leech, are nutritive, and the vessels of the second type combine capacitance and transport functions and are the place of hemolymph renovation at the expense of an active metabolic and synthetic activity of the endothelial cells. A fact is stated concerning a specific position of mitochondria in the endotheliocytes of the second type vessels: from the vessel's lumen they are not covered with plasmolemma along their whole extent and come into an immediate contact with hemolymph. Therefore, a hypothesis is suggested on functional activity of the endothelial cell mitochondria in the second type vessels. Presumptive differentiation of the two major vessels of the leech body makes it possible to suppose that there exist functional predecessors of the vessels of branchial and pulmonary type, that is vessels of lesser and greater circulation.

Sensilla that line the upper edge of the lip in the leech Hirudo medicinalis and that contain chemoreceptors required for feeding were examined in the scanning and transmission electron microscopes. The sensilla include two size-classes of ciliated button-like mounds--one about 35 microns in diameter and another about 10 microns in diameter. The larger sensilla are at the center of unpigmented patches of skin which are visible in the light microscope, while the smaller sensilla have not been previously described as distinct structures. Electron microscopy, though not light microscopy, shows that the lip sensilla differ markedly from the segmental sensilla of the leech, which have been shown to mediate mechanoreception and photoreception. In particular, the chemosensory lip sensilla contain multiciliated cells with cilia of a uniform length, whereas the segmental sensilla contain uniciliated cells with long, whip-like cilia, as well as multiciliated cells with short, stiff cilia. Thus, the two types of sensilla differ morphologically as well as functionally. In addition to the ciliated sensilla along the upper lip, structures consisting of a short, club-like process surrounded by granular material were observed inside the mouth. These structures may also be chemosensory organs.

Microphotometric measurements are used to investigate the functional properties of Ca2+-sequestering smooth endoplasmic reticulum (SER) in leech photoreceptors. 10-30 intact cells are mounted in a perfusion chamber, placed between crossed polarizers in a microphotometer, and permeabilized by saponin treatment. Subsequent perfusion with solutions containing Ca2+, MgATP, and oxalate leads to Ca uptake by SER. When the solubility product of Ca-oxalate is exceeded in the SER, birefringent Ca-oxalate precipitates form in the cisternae, leading to a large increase in the optical signal recorded from the preparation. The rate of increase in light intensity is used to measure the rate of Ca uptake. Ca uptake rate is linear with time over much of its course, can be switched on/off by the addition/withdrawal of Ca2+, ATP, or oxalate to/from the medium, and is inhibited by mersalyl and tetracaine. The Ca uptake mechanism has a high specificity for MgATP (KM,MgATP is approximately 0.8 mM). Uptake rates observed with dATP, GTP, UTP, ITP, and CTP are only 20-30% of the rate measured in ATP. The Ca pump has a high affinity for Ca2+ ions: the threshold for activation of the pump is approximately 5 x 10(-8) M, the apparent KM,Ca is approximately 4 x 10(-7) M. When Na+ or Li+ is substituted for K+, Ca uptake rate is decreased by 40-50%. The results show that the Ca2+-sequestering SER in leech photoreceptors shares some basic properties with skeletal muscle sarcoplasmic reticulum and supports the idea that certain subregions of the SER in invertebrate photoreceptors function as effective Ca2+ sinks/buffers close to the plasmalemma.