Fine structure of diffused pseudobranchial neurosecretory cells associated with carotid labyrinth in an air‐breathing catfish Clarias batrachus

Fish are known to have branchial chemoreceptors and even extrabranchial chemoreceptors to meet the challenges of aquatic environment. The pseudobranchial neurosecretory system associated with carotid labyrinth (CL) is one such example. CL – a chemosensory organ is well known in amphibians. The homologous structure also exists in fish. Clusters of neurosecretory cells, close to the CL and the first two efferent branchial arteries occur in catfish and a few other groups of teleosts. These cells belong to the pseudobranchial neurosecretory system (PNS). To reveal the ultrastructure of CL and the pseudobranchial neurosecretory cells (PNSCs), environmental scanning electron microscope (ESEM) and transmission electron microscope (TEM) investigations were made in an Asian air‐breathing catfish Clarias batrachus. Under ESEM, the PNS appeared as a mass of cells innervated by nerves and supplied by blood capillaries. The CL appeared to have a network of blood capillaries. The transmission electron microscopic investigations showed pear shaped PNSCs having different sizes of dense cored vesicles (DCVs), numerous mitochondria, nerve varicosities, indicating a secretory function of the cells. The CL shows a close association with PNSCs and smooth muscles. Although the exact function of the CL and associated PNSCs in the biology of fish is far from clear, their morphology suggests they are involved in a stress response such as to hypoxia.     

Mode of Association,Enzyme Producing Ability and Identification of Autochthonous Bacteria in the Gastrointestinal Tract of Two Indian Air-Breathing Fish,Murrel (<Emphasis Type="Italic">Channa punctatus</Emphasis>) and Stinging Catfish (<Emphasis Type="Italic">Heteropneustes fossilis</Emphasis>)

The mode of association of epithelium-associated bacteria in the gastrointestinal (GI) tract of two Indian air-breathing fish species, the murrel, Channa punctatus and the stinging catfish, Heteropneustes fossilis was demonstrated through scanning and transmission electron microscopy (SEM and TEM). The SEM examination revealed substantial numbers of rod shaped bacterial cells associated with the microvillus brush borders of enterocytes in proximal (PI) and distal regions (DI) of the GI tract of both the fish species. The TEM investigation indicated endocytosis and translocation of bacteria in the microvilli. The isolated bacterial strains (two each from the PI and DI of murrel and stinging catfish) were quantitatively evaluated for their extracellular amylase, cellulase and protease production. All the bacterial strains exhibited high cellulolytic activity than that of amylolytic and proteolytic enzymes. Only two strains, CPF1 and CPF2, isolated from the PI of murrel exhibited high proteolytic activity. Maximum amylase activity was exhibited by the strain, HFH5, isolated from the DI of stinging catfish. Totally six most promising enzyme-producing autochthonous bacterial strains were identified based on partial 16S rRNA gene sequence analytical results. All the strains showed close (92–99 %) similarity to Bacillus licheniformis.     

Ultrastructural evidence for the presence of secretory cells in the pineal parenchyma ofHeteropneustes fossilis

There is an extensive literature dealing with the study of indoles, especially serotonin and melatonin, but with considerably less emphasis on the cells and cell types involved in the synthetic process. In the present electron microscopical investigation of the pineal end vesicle ofHeteropneustes fossilis, pinealocytes have been revealed in the pineal parenchyma characterized with extensive synthetic apparatus viz., rough endoplasmic reticulum, free ribosomes, lipid droplets, mitochondria and Golgi bodies. Two sub-populations of the pinealocytes are easily distinguishable on the basis of electron opacity and the preponderance of one or other morphological profile: light cells and dark cells. Light cells represent the active phase of secretion while dark cells represent the storage and release phase of secretion. A neuroendocrine role for the pineal body inHeteropneustes fossilis is suggested which may be significant in view of the nocturnal habit of the fish. Dedicated to my father, Prof. C B L Srivastava     

Influence of continuous light and darkness on the secretory pinealocytes of<Emphasis Type="Italic">Heteropneustes fossilis</Emphasis>

In an earlier study onHeteropneustes fossilis, evidence of secretory activity in the pinealocytes had been demonstrated at the electron microscopic (EM) level and it was found to exist in two phases: a secretory phase (light cells) and a storage phase (dark cells). In the present investigation,H. fossilis was subjected to artificial photoperiods of continuous illumination and continuous darkness for a period of ten days and the effect on the secretory pinealocytes was studied at the EM level. Marked results were observed within the short period of ten days emphasizing the role of environmental photoperiod on the secretory activity of the pinealocytes. During continuous illuminated phase, both light and dark cells were observed: the light cells showed intense secretory activity and dark cells a storage one. During the dark phase both types of cells were present but in different metabolic states and neither of the cells demonstrated synthetic nor storage activity. Light cells were metabolically active but not secretory active and dark cells showed a necrotic condition. Phagocytotic activity of the dark cells was also seen. Intense neural activity was also observed during exposure to both the artificial photoperiods. The results highlight the role of light on the secretory activities of the pinealocytes of the catfish pineal organ.     

Telocytes in mice bone marrow: electron microscope evidence

Telocytes (TCs) are a novel type of interstitial cell of whom presence has been recently documented in many tissues and organs. However, whether TCs exists in bone marrow is still not reported. This study aims to find out TCs in mice bone marrow by using scanning electron microscope (SEM) and transmission electron microscope (TEM). SEM images showed that in mice bone marrow most of TCs have small spherical cell body (usually 4–6 μm diameter) with thin long telopodes (Tps; usually one to three). The longest Tp observed was about 70 μm, with an uneven calibre. Direct intercellular contacts exist between TCs. TEM shows mitochondria within dilations of Tps. Also, by TEM, we show the close spatial relations of Tps with blood vessels. In conclusion, this study provides ultrastructural evidence regarding the existence of TCs in mice bone marrow, in situ.     

Imaging of early conifer embryogenic tissues with the environmental scanning electron microscope

This article describes the usage of non-commercial environmental scanning electron microscope (ESEM) for the visualization of plant extracellular matrix in Abies alba and Abies numidica. Non sputter-coated samples free of using the common fixation technique observed at the relatively low humidity of the air environment with the pressure 550 Pa and the low temperature of the sample from −18 to −22°C give surprisingly very good results that show the natural structure of the tissues. This seems to be generally applicable. Moreover, a specially designed ionization detector of secondary electrons and a YAG:Ce³⁺ detector of backscattered electrons were used for better comparison.     

Recent developments in electron microscopical techniques for studying ion localization in plant cells

This paper reviews recent technical approaches to the study of element localization in plant cells. It is concerned with sample preparation; with the use of electron probe microanalysis in the low temperature scanning electron microscope; with the use of electron energy loss spectrocopy and electron spectroscopic imaging in the transmission electron microscope. Basic principles of instrumentation, special problems during cryopreparation of plant tissues, and the application of these techniques within selected fields of botanical interests are briefly discussed.Abbreviations EDXA Energy Dispersive X-Ray Analysis - EELS Electron Energy Loss Spectrometry - EPMA Electron Probe Microanalysis - ESEM Environmental Scanning Electron Microscope - ESI Electron Spectroscopic Imaging - HPF High Pressure Freezing - LTSEM Low Temperature Scanning Electron Microscope - SEM Scanning Electron Micrsocope - TEM Transmission Electron Microscope Botanisches Institut der Tierärztlichen Hochschule HannoverDedicated to Professor André Läuchli on the occasion of his 60th birthday     

Investigation of viability of plant tissue in the environmental scanning electron microscopy

The advantages of environmental scanning electron microscopy (ESEM) make it a suitable technique for studying plant tissue in its native state. There have been few studies on the effects of ESEM environment and beam damage on the viability of plant tissue. A simple plant tissue, Allium cepa (onion) upper epidermal tissue was taken as the model for study. The change of moisture content of samples was studied at different relative humidities. Working with the electron beam on, viability tests were conducted for samples after exposure in the ESEM under different operating conditions to investigate the effect of electron beam dose on the viability of samples. The results suggested that without the electron beam, the ESEM chamber itself can prevent the loss of initial moisture if its relative humidity is maintained above 90%. With the electron beam on, the viability of Allium cepa (onion) cells depends both on the beam accelerating voltage and the electron dose/unit area hitting the sample. The dose can be controlled by several of the ESEM instrumental parameters. The detailed process of beam damage on cuticle-down and cuticle-up samples was investigated and compared. The results indicate that cuticular adhesion to the cell wall is relatively weak, but highly resistant to electron beam damage. Systematic study on the effect of ESEM operation parameters has been done. Results qualitatively support the intuitive expectations, but demonstrate quantitatively that Allium cepa epidermal cells are able to be kept in a hydrated and viable state under relevant operation condition inside ESEM, providing a basis for further in situ experiments on plant tissues.     

Scanning and transmission electron microscopy of the subfornical organ of the grass frog (Rana pipiens)

Summary The ventricular surface of the subfornical organ of the frog is made up of ependymal cells with numerous apical microvilli, occasional cytoplasmic protrusions and many vacuoles projecting into the lumen of the third ventricle. Between these cells dendrites of cerebrospinal fluid-contacting neurons reach the ventricle to terminate in bulbous enlargements. In addition, flask-shaped encephalo-chromaffin cells, containing granulated vesicles and aggregates of filaments in their cytoplasm, project into the cerebrospinal fluid. Surrounding the centrally located capillaries are enlarged dendrites and axons of heterogeneous morphology, some of which appear to originate within the subfornical organ, intermingled with dendrites and axons of normal structure. The glial cells in this region, especially the microglial cells, often contain large lipofuscin inclusions, suggestive of degeneration and subsequent phagocytosis of some of the enlarged dendrites and axons. The normally scarce neurosecretory peptidergic axons become more evident and form typical Herring bodies in stalk-transected animals. Neuronal perikarya of varying morphology are predominantly located peripheral to the region of enlarged dendrites and axons. Supraependymal macrophages are particularly numerous on the subfornical organ.Abbreviations used CSF cerebrospinal fluid - SEM scanning electron microscope, scanning electron microscopy - SFO subfornical organ - TEM transmission electron microscope, transmission electron microscopy Supported, in part, by NIH grant NB 07492The skillful technical assistance of J.G. Linner and the secretarial assistance of Ann Gerdom are gratefully acknowledged. The SEM studies were made possible through a grant from the Graduate College of Iowa State University and the use of the SEM facility in the Department of Botany     

Fine structure of pars neuro-intermedia of the loach,Misgurnus fossilis L.

Summary Two types of glandular cells are present in the pars intermedia of the loach, Misgurnus fossilis. Basophils are characterized by the presence in their cytoplasm of numerous secretory granules containing electron-dense and homogeneous material and by scarce endoplasmic reticulum. Weak acidophils contain in their cytoplasm abundant endoplasmic reticulum and numerous granules of different electron densities.The distal part of the neurohypophysis is composed of several types of neurosecretory axons, strongly branched pituicytes, numerous capillaries, and connective tissue elements. The axon terminals form the neuroglandular, neurovascular and neurointerstitial contacts. Some axon terminals are closely apposed to the basement membrane separating neurohypophysis from meta-adenohypophysis. At points of absence of continuity of this membrane, some neurosecretory axons become directly contiguous with cytoplasmic membranes of the intermedia cells.The investigation was partly supported by a research grant from the Zoological Committee of the Polish Academy of Sciences.     

Sublethal malathion induced changes in the ovary of an air-breathing fish,Heteropneustes fossilis: a histological study

This paper describes effects of a sublethal (1.2 mg 1^–1) organophosphate, malathion, on the ovary of an air breathing catfish, Heteropneustes fossilis. The study focuses on microscopic changes that occur on ovigerous lamellae, oocytes at different stages of development and the nucleus of the immature oocyte. Also, change in estrogen levels in blood serum is investigated. Clumping of cytoplasm appears after 24 h of exposure to malathion. Clumping intensified after 48 h. Degeneration in the follicular cells was also observed. After 72 h exposure the number of nucleoli increased, nuclear materials shrunk, oocytes became adhered. With 96 h of exposure, nuclear materials of all the oocytes shrunk to a smaller clump. The oocytes fused together, and follicular epithelium became loose and ruptured. A few atretic oocytes were visible. Radioimmunoassay of the estrogen level in blood serum after 72 h of exposure of malathion showed a reduction in the level. This study showed that the histopathological condition of the gonad is reflected in malfunctioning of the endocrine system and hormonal disbalance.     

Ultrastructure of cell types of the olfactory epithelium in a catfish,Heteropneustesfossilis (Bloch)

Transmission electron microscopical study of olfactory epithelium of a mud-dwelling catfish,Heteropneustes fossilis (Bloch) shows receptor, supporting, goblet and basal cells. The receptor cells are of ciliated and microvillous type. Both ciliated and microvillous receptor cells are provided with olfactory knob. The dendrite of all the receptor cells bears many longitudinally arranged microtubules. Occurrence of the rod cell and its function is quite debatable. Specialized juctional complexes between the receptor and adjacent cells are clearly noted. The supporting cells are both ciliated and nonciliated. The ciliated supporting cells are responsible for water ventilation in the olfactory chamber as well as in the inter-lamellar spaces. This facilitates better perception of odours by the receptor cells. In addition to providing mechanical support to other cells, the nonciliated supporting cells also have a secretory function which is evident from the present study. The different stages of maturity of goblet cells are well documented. The presence of white cells in the olfactory epithelium is a very rare finding.     

Surface ultrastructure of the olfactory rosette of an air-breathing catfish,Heteropneustes fossilis (Bloch)

The surface architecture of the olfactory rosette ofHeteropneustes fossilis (Bloch) has been studied by scanning electron microscopy. The olfactory rosette is an oval structure composed of a number of lamellae arranged pinnately on a median raphe. The raphe is invested with epithelial cells and pits which represent goblet cell openings. On the basis of cellular characteristics and their distribution the lateral surface of each olfactory lamella is identified as sensory, ciliated non-sensory and non-ciliated non-sensory epithelium. The sensory epithelium is provided with receptor and supporting cells. The ciliated non-sensory epithelium is covered with dense cilia obscuring the presence of other cell types. The non-ciliated non-sensory epithelium is with many polygonal areas containing cells.     

Modulation of redox regulatory molecules and electron transport chain activity in muscle of air breathing fish Heteropneustes fossilis under air exposure stress

Responses of redox regulatory system to long-term survival (>18 h) of the catfish Heteropneustes fossilis in air are not yet understood. Lipid and protein oxidation level, oxidant (H₂O₂) generation, antioxidative status (levels of superoxide dismutase, catalase, glutathione peroxidase and reductase, ascorbic acid and non-protein sulfhydryl) and activities of respiratory complexes (I, II, III and IV) in mitochondria were investigated in muscle of H. fossilis under air exposure condition (0, 3, 6, 12 and 18 h at 25 °C). The increased levels of both H₂O₂ and tissue oxidation were observed due to the decreased activities of antioxidant enzymes in muscle under water deprivation condition. However, ascorbic acid and non-protein thiol groups were the highest at 18 h air exposure time. A linear increase in complex II activity with air exposure time and an increase up to 12 h followed by a decrease in activity of complex I at 18 h were observed. Negative correlation was observed for complex III and V activity with exposure time. Critical time to modulate the above parameters was found to be 3 h air exposure. Dehydration induced oxidative stress due to modulation of electron transport chain and redox metabolizing enzymes in muscle of H. fossilis was clearly observed. Possible contribution of redox regulatory system in muscle tissue of the fish for long-term survival in air is elucidated. Results of the present study may be useful to understand the redox metabolism in muscle of fishes those are exposed to air in general and air breathing fishes in particular.     

A study of the budding ofSaccharomyces uvarum Beijerinck with the scanning electron microscope

Vegetative cells ofSaccharomyces uvarum Beijerinck in the exponential growth phase were examined with the scanning electron microscope. The existence of two types of scars — birth scars and bud scars — was confirmed. Birth scars had larger diameters than bud scars; both remained visible on old cells. The distribution of the buds on the mother cell did not appear to be a random one: there seemed to be a more or less emphasized cell polarity. The author wishes to thank Mr. Bert for technical assistance in the use of the scanning electron microscope.     

Focused ion beam (FIB)/scanning electron microscopy (SEM) in tissue structural research

The focused ion beam (FIB) and scanning electron microscope (SEM) are commonly used in material sciences for imaging and analysis of materials. Over the last decade, the combined FIB/SEM system has proven to be also applicable in the life sciences. We have examined the potential of the focused ion beam/scanning electron microscope system for the investigation of biological tissues of the model organism Porcellio scaber (Crustacea: Isopoda). Tissue from digestive glands was prepared as for conventional SEM or as for transmission electron microscopy (TEM). The samples were transferred into FIB/SEM for FIB milling and an imaging operation. FIB-milled regions were secondary electron imaged, back-scattered electron imaged, or energy dispersive X-ray (EDX) analyzed. Our results demonstrated that FIB/SEM enables simultaneous investigation of sample gross morphology, cell surface characteristics, and subsurface structures. The same FIB-exposed regions were analyzed by EDX to provide basic compositional data. When samples were prepared as for TEM, the information obtained with FIB/SEM is comparable, though at limited magnification, to that obtained from TEM. A combination of imaging, micro-manipulation, and compositional analysis appears of particular interest in the investigation of epithelial tissues, which are subjected to various endogenous and exogenous conditions affecting their structure and function. The FIB/SEM is a promising tool for an overall examination of epithelial tissue under normal, stressed, or pathological conditions.     

Responses of the genital system and the urinogenital papilla of the female catfish,Heteropneustes fossilis (Bloch) to sex hormones

Summary Intraperitoneal injections of diethylstilbestrol and testosterone propionate into the catfish,Heteropneustes fossilis (Bloch) elicited pronounced changes in the female reproductive system and the urinogenital papilla. Considerable gonadal and oviducal hypotrophy occurred in the hormone recipients. Marked histological alterations were also encountered in the genital system of the experimental females. Another interesting response was the inhibition of the urinogenital papilla of the treated fishes. It is suggested that the changes in the ovary and the oviduct were induced by the sex hormones throught the inhibition of the gonadotrophic activity of the anterior hypophysis. The changes in the urinogenital papilla proved that this structure in the female catfish is a true secondary sexual character and is under the estrogenic control of the ovary.

---

Zusammenfassung Intraperitoneale Injektionen von Diäthylstilböstrol und von Testosteronpropionat haben bei Heteropneustes fossilis ausgesprochene Veränderungen an den weiblichen Geschlechtsorganen und an der Urogenitalpapille zur Folge. Bei den Hormonempfängern kommt es zu einer beträchtlichen Hypotrophie der Keimdrüse wie des Ovidukts. Im Genitalsystem der Versuchstiere sind auch deutliche histologische Veränderungen nachzuweisen. Eine bemerkenswerte Folgeerscheinung ist die Hemmung der Urogenitalpapille der behandelten Fische. Es ist anzunehmen, daß die Veränderungen im Eierstock und im Eileiter durch die Geschlechtshormone über eine Hemmung der gonadotropen Aktivität der Hypophyse veranlaßt werden. Die Veränderungen an der Urogenitalpapille beweisen, daß dieses Organ beim weiblichen Heteropneustes den Charakter eines wahren sekundären Geschlechtsmerkmales besitzt und unter der östrogenen Kontrolle des Eierstocks steht.

     

Identification of adenohypophysiotropic neurohormone producing neurosecretory cells in Rana temporaria

Summary Electron microscopic investigation of neural isolated and normal pars ventralis of the tuber cinereum showed the presence, in Rana temporaria, of a tubero-hypophysial neurosecretory system. A striking structural resemblance between this parvicellular, aldehydefuchsin negative, tubero-hypophysial neurosecretory system and the magnocellular, aldehydefuchsin positive, hypothalamo-hypophysial neurosecretory system was observed. Six different neurosecretory cell types are described, characterized by different shape and size of their respective secretory granules. The nature of the secretory product of these cells is briefly discussed.Of the results of this investigation, a preliminary note has been published (Dierickx, 1971).The authors wish to thank Prof. Dr. H. Steyaert and Dr. W. Moerdijk of the Laboratory for Mathematical Statistics, Ghent University, for invaluable advice and assistance with their computers. They thank Dr. G. De Waele for his help with the scanning electron microscopy.     

A scanning electron microscope study of the gills of the air-breathing catfish, Clarias batrachus L.

Using the scanning electron microscope, the gills of the air-breathing catfish, Clarias batrachus , have been studied. The overall morphology of the gills are similar to other teleosts. In contrast to water-breathing species, however, microridges are absent from the surfaces of the secondary lamellae and only short microvilli are present. Long, convoluted microridges are present on the epithelial cells of the gill filaments. The possible roles of these structures in relation to water flow are discussed.