Localization of NO-Ergic Structures in Oral Lobes, Labia, and Esophagus of the Bivalve Mollusc Crenomytilus grayanus

Localization of NO-ergic elements in oral lobes, labia, and esophagus in the bivalve mollusc, mussel Crenomytilus grayanus was studied using histochemical technique [1] for detection of NADPH-diaphorase (EC 1.6.99.1). The NO-producing elements were revealed in all studied parts of the digestive system. NADPH-diaphorase was found in nerve and secretory cells as well as in nerve plexuses. Numerous NO-ergic nerve cells were observed in the basal part of epithelium of labia and of the initial part of esophagus as well as in the subepithelial area of these organs. In the middle and posterior parts of esophagus, only subepithelially located NO-ergic nerve cell are present. Basiepithelial NO-producing secretory cells are found in all the parts, but most of these cells are observed in labia and the initial part of esophagus. Subepithelial secretory cells labeled with diformazan granules are spread from the folded surface of oral lobes to the initial part of esophagus; no such cells were found on the smooth surface of the lobes. The deposit labeled basi- and subepithelial nerve plexuses in all studied organs except for oral lobes. These plexuses are the most developed in labia and the initial part of esophagus of the studied mollusc.     

NADPH-diaphorase activity in the digestive system of gastropod molluscs <Emphasis Type="Italic">Achatina fulica</Emphasis> and <Emphasis Type="Italic">Littorina littorea</Emphasis>

Distribution and morphological peculiarities of nitroxidergic elements throughout the entire length of digestive tract was studied for the first time in gastropod molluscs Littorina littorea (Prosobranchia) and Achatina fulica (Pulmonata) using histochemical detection of NADPH-diaphorase (NADPHd). NO-ergic cells and fibers were revealed in all parts of the mollusc digestive system beginning from esophagus. Intensive NADPHd activity is found in a great number of intraepithelial cells of the open type and their processes in the intraand subepithelial nerve plexuses, subepithelial neurons, granular connective tissue cells, and multiple nervous fibers distributed among muscular elements of digestive tract as well as those in nerves innervating the tract. NADPHd was also revealed in receptor cells in the oral area and in the A. fulica CNS ganglia innervating the digestive tract. A. fulica has a more complicated organization of A. fulica nitroxidergic system of the digestive tract. A system of glomerular structures formed by thin NADPHd-positive neural fibers coming from epithelium is found directly beneath the epithelium in esophagus, stomach, and midgut of the mollusc. More superficially under the main groups of muscular elements there are revealed small clusters of NADPHd-positive neurons that can be classified as primitive, non-structured microganglia. The distribution pattern and a possible functional role of nitroxidergic elements in digestive tract of molluscs as compared with other invertebrate and vertebrate animals are discussed.     

NO-Ergic Innervation of the Intestine of the Snow Sculpin Myoxocephalus brandti

Distribution, localization, and morphological peculiarities of NO-ergic nerve cells in the intestine of the snow sculpin Myoxocephalus brandti (Cottidae family) were studied using histochemical staining for NADPH-diaphorase ( NADPH-d). These cells were shown to be present in the pyloric appendages, middle and posterior parts of the intestine and in its rectal part. The NO-ergic cells are the most numerous in the myenteric plexus and circular muscle layer of all studied parts of the intestine. Single NO-ergic nerve cells are revealed in the submucosal plexus of pyloric appendages, middle and posterior parts of the intestine. No NO-ergic neural cells were found in subserosal and subepithelial plexuses, longitudinal layer of smooth muscle in all studied parts, and in the submucosal plexus of the rectal part of the intestine.     

Nitroxidergic Cells in the Intestine of the Bivalve Mollusk Modiolus kurilensis

The distribution of nitroxidergic (NO-ergic) cells in the direct and recurrent loops of the midgut and in the hindgut of the bivalve mollusk Modiolus kurilensis was studied using a NADPH-diaphorase (NADPH-d) histochemistry [13]. Intraepithelial NO-ergic cells were found in the intestinal groove, in the major typhlosole of the direct loop, in the recurrent loop of the midgut, as well as in the hindgut. The apical processes of these cells are directed to the intestinal lumen, whereas the basal processes connect to the basiepithelial NO-ergic plexus. The latter, in turn, have separate fibers that are in contactswith the subepithelial NO-ergic plexus. Both plexuses are well developed in the intestinal groove of the direct loop and in the recurrent loop of the midgut, as well as in the hindgut. In the major typhlosole and crystalline style sac of the direct loop, both basi- and subepithelial NO-ergic plexuses are less developed. In the minor typhlosole, the basiepithelial NO-ergic plexus is absent and only single fibers of the subepithelial plexus occur. In the connective tissue and muscular layers of the recurrent loop of the midgut, subepithelial NO-ergic nerve cells were found.     

Effects of vertebrate growth factors on digestive gland cells from the mollusc Pecten maximus L.: an in vitro study

In relation with the digestive cycle, the digestive gland cells of bivalve molluscs undergo a sequence of cytological changes which is controlled by external and internal effectors such as putative gastrointestinal hormones and growth differentiation factors. A tissue dissociation method was developed to investigate the in vitro effect of the vertebrate growth and differentiation factors: insulin, insulin growth factor I (IGF-I), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on the digestive gland cells of the scallop Pecten maximus. All these vertebrate peptides induced a dose-dependent increased incorporation of ³H-leucine and ¹⁴C-uridine in whole digestive gland cell suspensions. However, after Percoll density gradient purification of the digestive cells, only stem and undifferentiated enriched cell fractions were responsive to the different peptides. In addition, insulin and IGF-I, but not EGF and bFGF, stimulated ³H-leucine incorporation in control dispersed mantle edge cells. These results suggest that insulin-related peptides could work as general growth promoting factors in molluscs. On the other hand, EGF and bFGF, or at least their molluscan counterparts, may be efficient growth differentiation factors in the regenerative processes occurring in the digestive gland of molluscs. Accepted: 26 September 1997     

龟足消化系统形态和组织学特点

应用光学显微镜观察龟足（Capitulum mitella）消化系统的形态和组织结构。龟足的消化系统包括消化腺和消化道。消化腺一对,呈长囊状,含有分泌细胞（B细胞）、吸收细胞（R细胞）、储存细胞（F细胞）和胚细胞（E细胞）4种类型细胞。消化道呈U型,由口、食道、胃、肠、直肠和肛门组成,各部分的结构由内到外可分为黏膜层、黏膜下层、肌层和外膜4层。口器为咀嚼型,包括一片上唇、一对触须、一对大颚以及两对小颚。食道细短,具几丁质层但无基膜,管壁向腔内突起形成明显的纵褶突;食道前段的环肌特别发达,同时独有放射肌。胃略呈球袋状,肠较长;胃和肠的组织结构相似,没有几丁质层,上皮细胞都有发达的微绒毛。直肠细长,外膜分布有16组纵肌;直肠前段的组织结构与胃、肠相似,而直肠后段有几丁质层覆盖,黏膜层、黏膜下层、肌层和外膜渐退化,16组纵肌渐发达。肛门16组更加发达的纵肌挤入上皮细胞下方,在外膜外另出现一层明显的环肌。龟足消化道各部分的组织结构差异明显,反映了它们功能的差异。     

Nitric oxide-ergic organization of medullar cranial nuclei in teleost fishes

Traditional neuromorphological and NADPH-diaphorase methods were used to study and compare the topography and localization of medullar nuclei NO-ergic neurons in four teleost species: Pholis nebelosis, Hexagramus octogrammus, Carassius carassius and Pholidapus dybowskii. The peculiarity of medullar nuclei organization in teleost fishes consists in the weak topological disconnection of sensor and motor components with pronounced heteromorphism in their structure and the presence of subpopulations in their composition. In the fishes studied, NADPH-diaphorase was found in the trygeminal, octavo-lateral, facial, glossopharingeal and vagal nuclei. The number of NO-ergic neurons in the trygeminal nucleus was marginal and made up 9-14 % of the total cell number. About 40-60 % of NO-ergic cells were found in the octavo-lateral, facial, glossopharingeal and vagal nuclei.     

Immunohistochemical studies on peptide- and amine-containing endocrine cells and nerves in the gut and the rectal gland of the ratfish Chimaera monstrosa

Summary The occurrence and distribution of endocrine cells and nerves were immunohistochemically demonstrated in the gut and rectal gland of the ratfish Chimaera monstrosa (Holocephala). The epithelium of the gut mucosa revealed open-type endocrine cells exhibiting immunoreactivity for serotonin (5HT), gastrin/cholecystokinin (CCK), pancreatic polypeptide (PP)/FMRFamide, somatostatin, glucagon, substance P or gastrin-releasing peptide (GRP). The rectum contained a large number of closed-type endocrine cells in the basal layer of its stratified epithelium; the majority contained 5HT- and GRP-like immunoreactivity in the same cytoplasm, whereas others were immunoreactive for substance P. The rectal gland revealed closed-type endocrine cells located in the collecting duct epithelium. Most of these contained substance P-like immunoreactivity, although some reacted either to antibody against somatostatin or against 5HT. Four types of nerves were identified in the gut and the rectal gland. The nerve cells and fibers that were immunoreactive for vasoactive intestinal peptide (VIP) and GRP formed dense plexuses in the lamina propria, submucosa and muscular layer of the gut and rectal gland. A sparse network of gastrin- and 5HT-immunoreactive nerve fibers was found in the mucosa and the muscular layer of the gut. The present study demonstrated for the first time the occurrence of the closed-type endocrine cells in the mucosa of the rectum and rectal gland of the ratfish. These abundant cells presumably secrete 5HT and/or peptides in response to mechanical stimuli in the gut and the rectal gland. The peptide-containing nerves may be involved in the regulation of secretion by the rectal gland.     

Avian enteric nerve plexuses

Summary The enteric nerve plexuses of the domestic fowl (Gallus domesticus) were investigated in sections and stretch preparations by means of the cholinesterase and glyoxylic acid fluorescence histochemical techniques. Cholinesterase-positive and varicose and non-varicose fluorescent nerve fibres were distributed at all levels of the gut in myenteric, submucosal, muscle and mucosal plexuses, and in a perivascular plexus. The density of the innervation and the detailed distribution of the nerves varied in different parts of the intestinal tract. All nerve plexuses appeared to be best developed in the rectum. Whereas the circular muscle coat contained a substantial number of nerves at all levels of the gut, the longitudinal coat was well innervated only in the rectum. The major portion of the mucosal plexus appeared to be associated with the intestinal glands. The nerve cell bodies were restricted to the myenteric and submucosal plexuses and were mainly cholinesterase-positive. Fluorescent ganglion cells were not observed. Pretreatment of stretch preparations with NADH: Nitro BT to stain ganglion cells showed that the majority of the cells were surrounded by a meshwork of fluorescent varicose fibres, although none of the fibres appeared to be associated with individual cells. The perivascular plexus was mainly associated with the arteries. The functional significance of the innervation is discussed.We would like to thank the British Council for financial support for Mr. H.A. Ali     

Effect of copper ions on spatial density of NO synthase-positive cells in intestine of the mussel <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> (mollusca: bivalvia: mytilidae). A histochemical study

By the histochemical method of detection of NADPH-diaphorase (NADPH-d) (EC1.6.99.1) [1] the state of nitroxidergic enteric nervous system of the mussel Crenomytilus grayanus was studied under conditions of an increased copper concentration in water. Under the action of copper ions the density of distribution of NADPH-d-positive cells has been established to be changed as compared with control throughout 28 days. A sharp rise of proportion of the labeled cells and their enzyme activity was noted after one day of the experiment. The labeled bipolar cells were of dark blue color and were located within the epithelium. There were revealed numerous nerve fibers penetrating the intestinal epithelium throughout its entire length as well as bipolar nerve cells in epithelium of the minor typhlosole and of crystalline style sac; in control molluscs the NADPH-d-positive cells in these parts were absent. After 7 days the difference between control and experimental decreased and remained at this level after 14 days, while after 21 days of exposition the proportion of labeled cells in the experimental mussels was lower than in control, but increased again after 28 days. It is suggested that nitric oxide is an important protective factor of the intestinal epithelium of the mussel C. grayanus and participates in adaptation of this mollusc to action of the elevated concentration of copper ions in water.     

Monoamine- and peptide-containing elements in the nemertine digestive tract

Detailed comparative-histological data on localization and morphological peculiarities of catecholamine-, serotonin-, neurotensin-and FMRFamide-containing elements in the nemertine digestive system are presented for the first time. Using fluorescent histochemistry and immunohistochemistry, pharynx, esophagus, and midgut were studied in five species from three genera of the White Sea nemertines. Described in various nemertine species are intra-and subepithelial cells of the open and closed type, containing biologically active substances. Their processes are distributed in basi-and subepithelial nerve plexuses and can make contact with lumen of the digestive tract. Species-specificity in localization of the cells containing certain biologically active substances is noticed both along the length of the tract and with respect to its epithelial layer. Peculiarities and common regularities in distribution are discussed, as well as a possible function of monoamine-and peptide-containing elements in the digestive tract of the studied nemertines and other invertebrates.     

Monoamine- and peptide-containing elements in the nemertine digestive tract

Detailed comparative-histological data on localization and morphological peculiarities of catecholamine-, serotonin-, neurotensin- and FMRFamide-containing elements in the nemertine digestive system are presented for the first time. Using fluorescent histochemistry and immunohistochemistry, pharynx, esophagus, and midgut were studied in five species from three genera of the White Sea nemertines. Described in various nemertine species are intra- and subepithelial cells of the open and closed type, containing biologically active substances. Their processes are distributed in basi- and subepithelial nerve plexuses and can make contact with lumen of the digestive tract. Species-specificity in localization of the cells containing certain biologically active substances is noticed both along the length of the tract and with respect to its epithelial layer. Peculiarities and common regularities in distribution are discussed, as well as a possible function of monoamine- and peptide-containing elements in the digestive tract of the studied nemertines and other invertebrates.     

Endosymbiotic and Host Proteases in the Digestive Tract of the Invasive Snail Pomacea canaliculata: Diversity,Origin and Characterization

Digestive proteases of the digestive tract of the apple snail Pomacea canaliculata were studied. Luminal protease activity was found in the crop, the style sac and the coiled gut and was significantly higher in the coiled gut. Several protease bands and their apparent molecular weights were identified in both tissue extracts and luminal contents by gel zymography: (1) a 125 kDa protease in salivary gland extracts and in the crop content; (2) a 30 kDa protease throughout all studied luminal contents and in extracts of the midgut gland and of the endosymbionts isolated from this gland; (3) two proteases of 145 and 198 kDa in the coiled gut content. All these proteases were inhibited by aprotinin, a serine-protease inhibitor, and showed maximum activity between 30°C and 35°C and pH between 8.5 and 9.5. Tissue L-alanine-N-aminopeptidase activity was determined in the wall of the crop, the style sac and the coiled gut and was significantly higher in the coiled gut. Our findings show that protein digestion in P. canaliculata is carried out through a battery of diverse proteases originated from the salivary glands and the endosymbionts lodged in the midgut gland and by proteases of uncertain origin that occur in the coiled gut lumen.     

Location of immunoreactive leucine enkephalin in the digestive gland of the Mollusca Mizuhopecten yessoensis (Jay)

Distribution of leu-enkephalin-like peptides in the digestive gland of the Japanese scallop has been investigated by means of the indirect immunohistochemical method. The immunoreactive leu-enkephalin is revealed in supranuclear vacuoles of separate large secretory cells. These cells are situated in epithelium of the digestive tubules along the gland periphery. Their number is from 1-3 up to 5-6 per tubue section. The basal part of the cell and small apical vacuoles do not contain any products of the reaction. It is possible that in the bivalve molluscs, as in vertebrates, the leu-enkephalin-immunoreactive elements participate in regulation of digestion.     

云南盘鳇消化系统解剖学、组织学及消化酶活性研究

采用形态学、组织学及酶学方法对云南盘逗(Discogobio yunnanensis)成体消化系统进行研究。结果表明, 云南盘逗消化系统有以下特征: 口下位, 口腔上皮分布有较多味蕾及杯状细胞, 食道粗大, 含有大量黏液细胞, 无胃, 肠道较长, 盘旋于体腔中, 成鱼盘旋10回, 肠道系数为5.06±0.61, 肠分为前中后三段, 肠腔中密布肠绒毛。消化腺为肝胰脏, 肝脏分为左右两叶, 胰脏弥散分布在肝脏中。消化系统不同部位消化酶活性大小不同, 脂肪酶活性: 肝胰脏>前肠>中肠>后肠, 胰蛋白酶、淀粉酶、碱性磷酸酶活性: 前肠>中肠>肝胰脏>后肠。云南盘逗口下位, 食道粗短, 肠道细长, 肠绒毛丰富, 肠道含较高的胰蛋白酶和淀粉酶活性, 消化系统所具有的这些特征与其以固着藻类为食有关。     

Sublethal effects of experimental exposure to mercury in European flat oyster Ostrea edulis: Cell alterations and quantitative analysis of metal

Oysters display a diversity of uptake mechanisms for metallic elements and distribution in the target organs, namely gills and the digestive gland. Various tissues of the flat oyster, Ostrea edulis, were studied following experimental exposure to 0.025 m (5 g l) of mercury, for up to 34 days. All animals survived the treatment. Data indicate Hg accumulation in gill tissue with a maximum concentration of 38.76 g g dry weight after 25 days of exposure. Hg levels were lower in remaining tissues, in which the maximum concentration (18.47 mg g-1 dry weight) was reached after 18 days of exposure. After these times, concentration in both tissues decreased. Results show that oysters can accumulate Hg from the environment, without their survival being affected during the experimental period. Structural alteration of epithelial tissues of gill and digestive gland of flat oyster was comparable with effects described for other metallic elements in bivalve molluscs. Interstitial tissue was disorganized in the digestive gland, and ultrastructural changes in intracellular endomembranes were detected in epithelial cells of the digestive gland after 18 days of treatment. After 25 days, absorptive epithelial cells of gills showed highly dilated, swollen microvilli. These intracellular alterations are parameters of the incipient response to the accumulation of mercury.     

A study on early tolerance of Mactra chinensis philippi to salinity

In order to evaluate the early tolerance of Mactra chinensis to salinity, the treatments of salinity gradients and salinity gradual changes were set in this study, and the post growth and development of juveniles were analyzed in recovery experiment, respectively. The result showed that the optimum hatching of zygotes was found at a salinity from 24 to 32, which is narrower than that of larvae (20–32); a slight of low salinity (16–32) will benefit the early growth and development of M. chinensis; at planktonic and creeping stages, low salinity stress (20) was conducive to promoting the growth of juvenile M. chinensis philippi; 4, 48 was the ultimate salinity of M. chinensis; The range of early tolerance of larvae M. chinensis philippi to salinity can be widened through a short period of salinity acclimation.     

Histopathological effects of larval digenea on the digestive epithelia of the marine prosobranch Cerithidea californica: Fine structural changes in the digestive gland

The effects of natural infections by rediae of the heterophyid Euhaplorchis californiensis on the digestive gland cells of Cerithidea californica (Gastropoda: Prosobranchiata) were studied through the use of light and electron microscopy. Pathological changes in secretory and digestive cells, including the disruption of digestive vacuoles and cell-cell junctions, the deformation of lumenal microvilli, the lysis of distal plasma membranes, and nuclear polymorphism, usually accompany the migration of larval trematodes into the digestive gland. Degenerating secretory cells are further characterized by Golgi complexes which assume an inflated appearance and a reduction of rough endoplasmic reticulum in the cell's perinuclear region. Finally, an increase in excretory and autophagic activities in the digestive gland cells of infected snails may be considered possible mechanisms by which the host is able to compensate for adverse metabolic or nutritional stresses of trematode parasitism.     

Localization of NADPH-diaphorase activity in the pineal gland of the domestic pig

The study showed the presence of NADPH-diaphorase containing structures in the pineal gland of the domestic pig. NADPH-diaphorase activity was found in the nerve fibers and in the endothelial cells of the vasculature. The nerve fibers were localized in the capsule, around the blood vessels as well as in the parenchyma. The positive staining was not observed in the pig pinealocytes.     

Nitroxidergic Innervation of the Digestive Tract of the Shishamo Smelt Hypomesus japonicus (Teleostei: Salmoniformes)

The distribution and morphology of nitroxidergic elements in the esophagus, stomach, and intestine of the shishamo smelt Hypomesus japonicus (Teleostei: Salmoniformes) were studied. Nitroxidergic cells and fibers were found in all examined parts of the digestive tract, occurring most frequently in the stomach and rectal portion of the intestine. The spatial density of the nerve cells and fibers was maximum in the myenteric plexus and circular muscle layer and decreased in the longitudinal muscle layer and in the submucous plexus.