Structural changes in the zonulae occludentes of the chloride cells of young adult lampreys following acclimation to seawater

Summary Thin sections and freeze-fracture replicas have been used to study the structure of the zonulae occludentes of the branchial chloride cells in young adults of the anadromous lamprey Geotria australis, caught during their downstream migration to the sea and after acclimation to full-strength seawater (35). The chloride cells in the epithelium of the gill filaments of both freshwater- and seawater-acclimated animals form extensive multicellular complexes. In freshwater animals, the majority of chloride cells (64%) are covered by pavement cells and are thus not exposed to the external environment. Most of the other chloride cells are separated from each other by pavement cells or their processes. The zonulae occludentes between chloride cells and pavement cells and between adjacent chloride cells are extensive and characterised by a network of 4 (range 3–7) superimposed strands. In seawater-acclimated animals, the pavement cells cover only 30% of the chloride cells and their processes no longer occur between chloride cells. Whereas the zonulae occludentes between chloride cells and pavement cells are still extensive, those between chloride cells are shallow and comprise only a single strand or two parallel strands. The zonulae occludentes between the chloride cells of lampreys acclimated to seawater are similar to those in the gills of teleosts in seawater, and are thus considered to be leaky and to provide a low-resistance paracellular pathway for the passive transepithelial movement of Na⁺.     

The pronephros of the early ammocoete larva of lampreys (Cyclostomata,Petromyzontes): Fine structure of the renal tubules

Summary The renal tubules of the paired pronephros in early larvae (ammocoetes) of two lamprey species, Lampetra fluviatilis and Petromyzon marinus, were studied by use of light-, scanning- and transmission electron microscopy. They consist of (1) a variable number of pronephric tubules (3 to 6), and (2) an excretory duct. By fine-structural criteria, the renal tubules can be divided into 6 segments. Each pronephric tubule is divided into (1) the nephrostome and (2) the proximal tubule, the excretory duct consisting of (3) a common proximal tubule followed by (4) a short intermediate segment, and then by a pronephric duct composed of (5) a cranial and (6) a caudal section. The epithelium of the nephrostome displays bundles of cilia. The cells of the proximal tubule possess a brush border, many endocytotic organelles and a system of canaliculi (tubular invaginations of the basolateral plasmalemma). The same characteristics are encountered in the epithelium of the common proximal tubule; however, the number of these specific organelles decreases along the course of this segment in a posterior direction. In the intermediate segment, the epithelium appears structurally nonspecialized. The cells of the cranial pronephric duct lack a brush border; they have an extensive system of canaliculi and numerous mitochondria. The caudal pronephric duct is lined by an epithelium composed of light and dark cells; the latter are filled with mitochondria and the former contain mucus granules beneath the luminal plasmalemma. The tubular segments found in the pronephros are the same in structure and sequence as in the lamprey opisthonephroi. However, only the nephrostomes and proximal tubules occur serially in the pronephros, while the common proximal tubule, the intermediate segment and the cranial pronephric duct form portions of a single excretory duct.This paper is dedicated to the memory of Professor W. Bargmann, long-time editor of Cell and Tissue Research, the author of a splendid review on the structure of the vertebrate kidney and a master of German scientific writing.     

Intercellular junctions in the water-blood barrier of the gill lamella in the adult lamprey (Geotria australis,Lampetra fluviatilis)

Thin sections and freeze-fracture replicas of the water-blood barrier in the gill lamellae of adult lampreys (Geotria australis, Lampetra fluviatilis) demonstrate that the occluding junctions between epithelial pavement cells differ markedly from those between endothelial pillar cells in the structure and arrangement of their strands. The zonulae occludentes between pavement cells typically consist of complex networks of 4–6 strands, the mean number of which undergoes a small but significant decline when the animal is acclimated to seawater. In comparison, the occluding junctions between pillar cells are less elaborate and may represent maculae or fasciae, rather than zonulae occludentes. They do not apparently undergo a change when the animal enters saltwater. The results indicate that the main part of the paracellular diffusion barrier to proteins and ions is located in the epithelium rather than the endothelium. Communicating (gap) junctions are present between adjacent pavement cells, between pavement and basal cells and between pillar cells. These findings suggest that the epithelial cells and the pillar cells in the water-blood barrier of lampreys both form functional syncytia. The results are discussed in the context of ion-transporting epithelia in other aquatic vertebrates.This paper is dedicated to Professor H. Leonhardt on the occasion of his 75^th birthday     

Distribution of lamprey gonadotropin-releasing hormone-III (GnRH-III) in brains of larval lampreys (Petromyzon marinus)

Two immunoreactive forms of gonadotropinreleasing hormone (GnRH), lamprey GnRH-I and lamprey GnRH-III, were found in neurons in larval sea lampreys (Petromyzon marinus). Using antisera preferentially directed against either lamprey GnRH-I or-III, dense reaction product was seen in cell bodies in the rostral hypothalamus and preoptic area. Reaction product was also dense in fibers to and within the neurohypophysis, in addition to numerous fibers which projected caudally, beyond the neurohypophysis through the mesencephalon. The majority of immunoreactive GnRH was lamprey GnRH-III, and when lamprey GnRH-I was seen, it was in cells that appeared to contain both forms of GnRH. A small number of cells found in the caudal hypothalamus contained only immunoreactive lamprey GnRH-III, and these may constitute a functional subgroup within the population of GnRH neurons. In animals undergoing metamorphosis there was a large increase in reaction product in all GnRH-containing cells and fibers. A striking change within the distribution of GnRH cells was localized to a distinct group of GnRH-immunoreactive cells (GnRH-I and-III) in the ventral anterior hypothalamic area. These cells were minimally detectable in larvae, but during metamorphosis became densely filled with immunoreactive product in perikarya and distal processes. The results are consistent with the hypothesis that lamprey GnRH-III is an important form of GnRH during the maturation of GnRH cells and fibers, and further indicates that these cells have attained their normal positions in the preoptic area and hypothalamus before metamorphosis.     

Communicating (gap) junctions between chloride cells in the gill epithelium of the lamprey,Geotria australis

Summary Freeze-fracture replicas show that communicating (gap) junctions are present between chloride cells in the gill epithelium of young adults of the Southern Hemisphere lamprey, Geotria australis, acclimated to full-strength sea water. The junctions, which were already present when these lampreys were migrating downstream, may help coordinate the secretory activities of the chloride cells during the marine phase of the lamprey life cycle.     

Structural organization of ovarian follicle cells in the cotton bug (Dysdercus intermedius) and the honeybee (Apis mellifera)

Summary The somatic epithelia of Dysdercus and Apis follicles were analyzed by electron microscopy, and the patterns of F-actin and microtubules were studied by fluorescence microscopy. The epithelia in both species differ considerably in shape and in the organization of the cytoskeleton. During previtellogenic stages, the epithelium consists of columnar-shaped cells with small (Dysdercus) or no (Apis) lateral intercellular spaces. During vitellogenesis, the follicle cells round up; the intercellular spaces increase in size in Dysdercus follicles, whereas in Apis follicles they remain small. Along the basal surface of the follicle cells, there are conspicuous parallel bundles of microfilaments perpendicular to the anteroposterior axis of the follicles. In the honeybee, these microfilament bundles are present in long filopodia, most of which are embedded in thickenings of the basement membrane and extend over the surfaces of neighbouring cells. In the cotton bug, the basal surface of the follicle cells is thrown into parallel folds. The microfilament bundles are located just underneath the cell membrane where the folds contact the basement membrane. In the polar regions of the Dysdercus follicle, the epithelial cells become flat and adhere to each other without forming intercellular spaces. The basement membrane is particularly thick in the polar areas; this has also been observed in Apis follicles around the intercellular bridge connecting oocyte and nurse cells.     

Peroxisomes in intestinal and gallbladder epithelial cells of the stickleback,Gasterosteus aculeatus L. (Teleostei)

Summary The occurrence of microbodies in the epithelial cells of the intestine and gallbladder of the stickleback, Gasterosteus aculeatus L., is described. In the intestine the organelles are predominantly located in the apical and perinuclear zone of the cells and may contain small crystalline cores. In gallbladder epithelial cells the microbodies are distributed randomly. The latter organelles are characterized by the presence of large crystalloids. Cytochemical and biochemical experiments show that catalase and D-amino acid oxidase are main matrix components of the microbodies in both the intestinal and gallbladder epithelia. These organelles therefore are considered peroxisomes. In addition, in intestinal mucosa but not in gallbladder epithelium a low activity of palmitoyl CoA oxidase was detected biochemically. Urate oxidase and L- hydroxy acid oxidase activities could not be demonstrated.     

Coexistence of NADPH-diaphorase and vasoactive intestinal polypeptide in the enteric nervous system of the Atlantic cod (Gadus morhua) and the spiny dogfish (Squalus acanthias)

The distribution of NADPH (nicotinamide adenine dinucleotide phosphate)-diaphorase in nerve cells in the gastrointestinal tract has been investigated and compared in three fish species representing different evolutionary branches. In mammals, NADPH-diphorase is identical to nitric oxide synthase (NOS) and can, in the presence of NADPH, reduce the dye nitroblue tetrazolium, resulting in a blue product. Using this method, we have found numerous NADPH-diaphorase-containing nerve cells in the myenteric plexus of the Atlantic cod (Gadus morhua) and the spiny dogfish (Squalus acanthias) but none in the hagfish (Myxine glutinosa). In the cod, nerve fibres were sparsely stained, whereas in the dogfish, they formed a dense pattern of fibre bundles. Double-staining for NADPH-diaphorase and the neuropolypeptides VIP (vasoactive intestinal polypeptide) and PACAP (pituitary adenylate cyclase activating peptide) revealed three separate populations designated VIP/NADPH, VIP/- and NADPH/-. The majority but not all of the NADPH-diaphorase-positive cells also showed VIP or PACAP immunoreactivity and vice versa. The presence of NADPH-diaphorase in neurons and the distribution of these neurons in the gastrointestinal tract of the two species indicate a physiological role for nitric oxide in the control of gut motility.     

Ultrastructure of the presumed ion-transporting cells in the gills of ammocoete lampreys,Lampetra fluviatilis (L.) and Lampetra planeri (Bloch)

Summary Mitochondria-rich cells were located in the interplatelet area of gill filaments from ammocoete Lampetra fluviatilis and L. planeri. The ultrastructure of this cell type differs from typical teleost chloride cells by the absence of a tubular, smooth endoplasmic reticulum (SER). This difference is discussed in relation to the presumed functions of the cell and to the evolutionary histories of lampreys and teleosts. It is concluded that the mitochondria-rich cell is responsible for the active uptake of ions by the ammocoete gill.     

Taxonomic and functional implications of stigma morphology in species ofCassia,Chamaecrista, andSenna (Leguminosae:Caesalpinioideae)

Two stigma forms occur inChamaecrista andSenna, but only one inCassia. In the common chambered form, a stigma pore is positioned on the reflexed style tip and is the entrance to a tapering chamber. The pore rim is fringed by hairs which vary in number, size, distribution and shape. In the alternative form the stigma is situated at the apex of the curved style and is crateriform. The crater rim is fringed by hairs of variable number and shape. The stigmatic hairs are predominantly unicellular and cutinized. Stigma and hair differences aid in the taxonomy of the genera. Their functions in pollination biology are discussed.     

Structural and functional organization of the mitochondrial respiratory chain: A dynamic super-assembly

The structural organization of the mitochondrial oxidative phosphorylation (OXPHOS) system has received large attention in the past and most investigations led to the conclusion that the respiratory enzymatic complexes are randomly dispersed in the lipid bilayer of the inner membrane and functionally connected by fast diffusion of smaller redox components, Coenzyme Q and cytochrome c. More recent investigations by native gel electrophoresis, however, have shown the existence of supramolecular associations of the respiratory complexes, confirmed by electron microscopy analysis and single particle image processing. Flux control analysis has demonstrated that Complexes I and III in mammalian mitochondria and Complexes I, III, and IV in plant mitochondria kinetically behave as single units with control coefficients approaching unity for each single component, suggesting the existence of substrate channelling within the supercomplexes. The reasons why the presence of substrate channelling for Coenzyme Q and cytochrome c was overlooked in the past are analytically discussed. The review also discusses the forces and the conditions responsible for the formation of the supramolecular units. The function of the supercomplexes appears not to be restricted to kinetic advantages in electron transfer: we discuss evidence on their role in the stability and assembly of the individual complexes and in preventing excess oxygen radical formation. Finally, there is increasing evidence that disruption of the supercomplex organization leads to functional derangements responsible for pathological changes.     

Cytoskeletal organization and cell organelle transport in basal epithelial cells of the freshwater sponge Spongilla lacustris

Summary Different antibodies against actin, tubulin and cytokeratin were utilized to demonstrate the spatial organization of the cytoskeleton in basal epithelial cells of the freshwater sponge Spongilla lacustris. Accordingly, actin is localized in a cortical layer beneath the plasma membrane and in distinct fibers within the cytoplasmic matrix. Microtubules exhibit a different distributional pattern by radiating from a perinuclear sheath and terminating at, the cell periphery; in contrast, intermediate filaments are lacking. Cytoplasmic streaming activity was studied by in-vivo staining of mitochondria and endoplasmic reticulum by means of fluorescent dyes. Single-frame analysis of such specimens revealed a regular shuttle movement of mitochondria and other small particles between the cell nucleus and the plasma membrane, which can be stopped in a reversible manner with the use of colcemid or colchicine but not with cytochalasin D. The results point to the microtubular system as a candidate for cell organelle transport, whereas the actomyosin system rather serves for changes in cellular shape and motility.     

FMRFamide-like immunoreactivity in the brain of the Pacific hagfish,Eptatretus stouti (Myxinoidea)

Summary The distribution of FMRFamide-like immunoreactivity was investigated in the brain of a myxinoid, the Pacific hagfish,Eptatretus stouti, by means of immunocytochemistry. In the forebrain, labelled cell bodies occurred in the infundibular nucleus of the hypothalamus and some closely adjacent nuclei. Labelled fibers formed a diffuse network in the forebrain, but there was no evidence for the presence of intracerebral ganglionic cells of the terminal nerve or a central projection of the terminal nerve. In the hindbrain, a group of labelled cells was found in the trigeminal sensory nucleus. A distinet terminal arborization occurred in the ventrally adjacent nucleus A of Kusunoki and around the nuclei of the branchial motor column. These findings suggest that FMRFamide may play a role in the central control of branchiomotor activity.     

Structural and functional characteristics of primary cell cultures derived from the adrenal gland tissue of neonatal mallard ducklings (Anas platyrhynchos)

Summary Primary cell cultures were prepared from the adrenal glands of one-day-old mallard ducklings (Anas platyrhynchos). The cells attached equally well to uncoated plastic and glass surfaces and on surfaces that had been coated with collagen. The phase of logarithmic growth occurred between the second and the fourth day, and the cells became confluent between the fifth and the sixth day. Staining with Sudan black B and toluidine blue and viewing fixed preparations by transmission electron microscopy indicated that the cultures consisted mostly of steroidogenic cells. A smaller population of chromaffin cells was also present. Scanning electron microscopy showed that most of the cells had long filopodia, and some cells had numerous surface blebs that were interpreted as exocytotic vesicles. When incubated in Krebs-Henseleit buffer containing 1–24 ACTH the cultured cells released three corticosteroids, namely, corticosterone, aldosterone and deoxycorticosterone. These responses occurred within 15 min of exposure to medium containing 1–24 ACTH and continued throughout a 60-min period of continuous stimulation. The minimally effective concentration of 1–24 ACTH was 0.078 ng per ml (0.0234 nM) and, as the concentration was increased up to 10 ng per ml (2.99 nM), the total output of each hormone during the 60-min incubation period increased significantly according to the following semi-logarithmic relationship: Y=a+b log X, where Y=the total output of hormone, X=the concentration of 1–24 ACTH in the medium, and a=the total output of hormone when the medium contained 1.0 ng of 1–24 ACTH per ml. The total outputs of each hormone in the presence of a maximally effective concentration of 1–24 ACTH, however, were low compared to the responses of similarly stimulated tissue slices taken from the neonatal duckling. It is concluded that most of the cells comprising the confluent cultures were derived from steroidogenic cells in the neonatal adrenal. These cells appeared to retain corticotropin receptors during the course of developing into confluent monolayers, but their diminished steroidogenic capacity to respond when stimulated maximally suggests that some generational changes may have occurred.This work was supported by grants to James Cronshaw and W.N. Holmes from the University of California Committee on Research and the National Science Foundation (DIR-8820923), Washington, DC, USA     

The antennae of neopseustid moths: Morphology and phylogenetic implications, with special reference to the sensilla (Insecta, Lepidoptera, Neopseustidae)

The antennae of Lepidoptera Neopseustidae were examined with the scanning electron microscope. The studied species, Nematocentropus cfr. omeiensis, Neopseustis meyricki, Synempora andesae, Apoplania valdiviana and Apoplania penai possess nine types of antennal flagellum sensilla: multiporous large sensilla basiconica, multiporous thin sensilla basiconica, multiporous small sensilla basiconica, multiporous sensilla trichodea, multiporous sensilla coeloconica; uniporous sensilla chaetica; aporous sensilla chaetica, aporous stylus-shaped sensilla chaetica, aporous sensilla styloconica.The multiporous sensillum type here termed “multiporous large sensillum basiconicum” is unknown from other Lepidoptera and probably constitutes an autapomorphy of the family Neopseustidae. This sensillum type is remarkable by having a single base in female Apoplania and Synempora while in male Apoplania it has a bifid or trifid base, and in male Synempora it is composed of two or three incompletely separated hairs. This may be the first recorded example of a sexually dimorphic lepidopteran sensillum type. The stylus-shaped sensillum chaeticum is a primitive type which occurs only in some lower Lepidoptera.     

Ultrastructure of the aortic diverticula of the adult dragonfly Sympetrum danae (Odonata: anisoptera)

Summary The aorta of Sympetrum danae possesses two dorsal diverticula: one in the mesothorax and one in the metathorax. They are very similar in form and position. Each diverticulum has a dorsal valve through which blood is pumped from the wings down into the aorta. The wall of the aortic diverticula consists of two simple cell layers: an outer epidermis-like layer and an inner muscle layer. The nuclei of the muscle cells are situated close to the lumen of the diverticula. The mitochondria are evenly dispersed between the myofibrils and are often paired up on either side of the Z-band. The Z-bands are thick and fragmented. The length of the sarcomeres varies from 3.3 to 6.1 . The A-band length is about 3 . The myofibrils consist of thick (250 Å) and thin (85 Å) filaments. Each thick filament is surrounded by 9–12 thin filaments. The sarcoplasmic reticulum is well developed and separates the myofibrils with one or two layers. The T-tubules are flattened and branch irregularly like a two-dimensional tree between the lamellar myofibrils. Intercalated discs are observed.The peculiarities of the muscle of aortic diverticula in S. danae are discussed in relation to various muscles of other insects and arthropods.     

Ontogenetic development of neuropeptide Y-like-immunoreactive cells in the gastroenteropancreatic endocrine system of the dogfish

This immunocytochemical study was carried out to elucidate the ontogenetic development of neuropeptide Y-like-immunoreactive cells in the gastroenteropancreatic endocrine system of the cloudy dogfish, Scyliorhinus torazame. Immunostained cells first appeared in the pancreas of the embryo at the 15-mm stage, and were also detected in the vitellointestinal duct of the yolk stalk at the 20-mm stage. These cells were polymorphic, with occasional processes that were sometimes directed toward the vascular wall or into the cavity of the vitellointestinal duct. At the 34-mm stage, immunostained cells could also be found in the proximal part of the spiral intestine and, by the 74-mm stage, immunopositive cells were present in the gastric mucosa. In the gut and pancreas, the cells gradually increased in number with development, whereas in the vitellointestinal duct and internal yolk sac, they decreased and seemed to disappear following hatching. Thus, in juveniles, the distribution of the neuropeptide Y-like-immunoreactive cells in the gastroenteropancreatic endocrine system had attained that of adults. Electron-microscopic immunocytochemistry demonstrated that, in the labeled cells of the vitellointestinal duct, the neuropeptide Y-like antigen was located in cytoplasmic granules, as in the cells of the gut and pancreas.This paper is dedicated to Professor Yoshiharu Honma, on the occasion of his retirement and inauguration as Emeritus Professor     

Effects of ammonia,chloroquine, and monensin on the vacuolar apparatus of an absorptive epithelium

Summary The distribution of two major immunoreactive forms of somatostatin, somatostatin-14 and somatostatin-34, within the brain, pancreas and intestine of adult lampreys, Petromyzon marinus, was identified using antisera raised against these peptides. Immunostaining of the brain is similar in juveniles and upstream migrants, and somatostatin-14 is the major somatostatin form demonstrated. A few somatostatin-34-containing cells are localized within the olfactory bulbs, thalamus and hypothalamus, but cells immunoreactive to anti-somatostatin-34 in the hypothalamus and thalamus do not co-localize somatostatin-14. Immunostaining of pinealocytes within the pineal pellucida with anti-somatostatin-14 may infer a novel function for this structure. Somatostatin-14 and somatostatin-34 are co-localized within D-cells of the cranial pancreas and caudal pancreas of juveniles and upstream migrants. Numerous somatostatin-34-immunoreactive cells are distributed within the epithelial mucosa of the anterior intestine but not all of these cells cross-react with anti-somatostatin-14. It appears that somatostatin-34 is the major somatostatin in the pancreo-gastrointestinal system of adult lampreys.     

Mast cell types in the lymph nodes of the opossum Didelphis albiventris (Marsupialia,Didelphidae)

Summary Using histological and histochemical techniques, we have found a unique population of mast cells in the lymphatic sinuses of lymph nodes from different anatomical regions of the opossum. The lymphatic-sinus mast cells of the medullary sinuses were numerous, and could be easily distinguished from the connective-tissue mast cells of the dermis and lymph node capsule by their larger size and their enlarged cytoplasmic granules that were also more heterogeneous in shape and staining properties.     

Effects of entomopathogenic bacterium Photorhabdus temperata infection on the intestinal microbiota of the sugarcane stalk borer Diatraea saccharalis (Lepidoptera: Crambidae)

Photorhabdus temperata is an entomopathogenic bacterium that is associated with nematodes of the Heterorhabditidae family in a symbiotic relationship. This study investigated the effects of P. temperata infection on the intestinal microbiota of the sugarcane stalk borer Diatraea saccharalis. Histopathology of the infection was also investigated using scanning electron microscopy. Groups of 20 larvae were infected by injection of approximately 50 bacterial cells directly into the hemocoel. After different periods of infection, larvae were dissected and different tissues were used for bacterial cell quantification. P. temperata was highly virulent with an LD₅₀ of 16.2 bacterial cells at 48 h post-infection. Infected larvae started dying as soon as 30 h post-infection with a LT₅₀ value of 33.8 h (confidence limits 32.2–35.6) and an LT₉₀ value of 44.8 h (CL 40.8–51.4). Following death of the larvae, bacteria from the midgut did not invade the hemocoel. In the midgut epithelium, P. temperata occupied the space underneath the basal lamina. The cultivable intestinal bacterial populations decreased as soon as 1 h post-infection and at 48 h post-infection, 90% of the gut microbiota had died. The role of P. temperata in control of the midgut microbiota was discussed.