Intercellular junctions in the gill epithelium of the Atlantic hagfish,Myxine glutinosa

Summary The intramembrane organization of the occluding junctions in the gill epithelium of the Atlantic hagfish, Myxine glutinosa, was studied by means of freeze-fracture electron microscopy. Mitochondria-rich cells, characterized by assemblies of rod-shaped particles in the luminal plasma membrane and by an extensive intracellular amplification of the basolateral plasma membrane, are singly distributed between the pavement cells in the gill epithelium of this marine and stenohaline cyclostome. The occluding junctions between mitochondria-rich cells and pavement cells do not differ from those between adjacent pavement cells, concerning the number of superimposed strands (median 6, range 4–9) and their geometrical organization. These observations suggest that, in contrast to marine teleosts, the paracellular pathway plays a minor role in transepithelial ion movements in the hagfish gill epithelium. The findings are in agreement with the absence of hypoosmoregulatory mechanisms in hagfish, as have been evolved in various marine vertebrates. In addition, small communicating junctions are demonstrated between pavement cells; they possibly serve for a coordinated synthesis and secretion of mucus by the pavement cells.Dedicated to Prof. H. Leonhardt on the occasion of his 70th birthday     

Accumulation of exogenous catecholamines in the neural tube and non-neural tissues of the early fowl embryo

Summary Catecholamines (CA) were localized in stage 11–34 domestic fowl embryos by the formaldehyde-induced fluorescence (FIF) method after exposure in vivo or in vitro to CA (noradrenaline or -methylnoradrenaline), or the CA precursorl-DOPA. The effects of drugs known to alter CA metabolism in the adult were also investigated.Negligible FIF was observed in embryos which had not been exposed to CA. After CA loading, FIF could be seen in the neural tube and in non-neural tissues such as the notochord and gut mesenchyme and to a lesser degree in suprarenal area tissue, liver endothelium, sclerotome, and myotome. This FIF was inhibited by desmethylimipramine, a blocker of adult neuronal CA uptake (Uptake₁), but not by corticosterone, a blocker of adult extraneuronal CA uptake (Uptake₂). The notochord, dorsal pancreas and some blood cells were fluorescent afterl-DOPA loading, and this FIF could be greatly diminished by the DOPA decarboxylase inhibitor RO4-4602.The pattern of FIF in the axial structures (neural tube and notochord) correlated with axial flexure in both position and time, and the intensity of fluorescence was strongest cranially and caudally, where flexure is most pronounced. The FIF in gut mesenchyme cells was closely related to the movement of the intestinal protals during early gut tube formation, and to the regions of the developing intestine that undergo intense morphogenesis during their early formation.     

Conservative treatment in Scheuermann’s kyphosis: comparison between lateral curve and variation of the vertebral geometry

Background

Conservative treatment in the Scheuermann’s kyphosis obtain, during skeletal growth, remodelling of the deformed vertebras. In a previous paper on Scheuermann’s kyphosis, we have studied the geometry variations of all vertebrae included in the curve, before and after the treatment.

The purpose of this study was to confirm the effectiveness of conservative treatment in Scheuermann’s kyphosis and was to evaluate and compare the variation of the vertebral geometry with the curve trend in Cobb degrees, before and after conservative treatment.

Methods

From a consecutive series of patients, we selected 90 patients with thoracic Scheuermann’s kyphosis, treated using anti-gravity brace: 59 male, 31 female. The mean age at the beginning of the treatment was 14 years.

Radiographical measurements were performed on radiographs from a lateral projection, at the beginning (t1) and at the end of the treatment (t5). Vertebral geometry modifications at t1 and t5 were analysed according to the following parameters and evaluated by three independent observers: Anterior wedging angle (ALFA) of the apex vertebra and Posterior wall inclination (APOS) of the limiting lower vertebra. The curve was measured in Cobb degrees.

Results

The results from our study showed that of the 90 patients with a thoracic curve mean value of Cobb degrees was 57.8 ± 6.0 SD at t1 and 41.3 ± 5.6 SD at t5. The differences between t1(angle at baseline) and t5 (end of treatment) were calculated for Cobb, ALFA and APOS angle and were respectively −16.4 ± 4.5, −6.4 ± 1.4 and −2.7 ± 1.2; tested with paired t-test were significative (p < 0.01). The results of the regression analysis to test the relationship between the three measures for the kyphosis (Cobb degree, ALFA and APOS) showed that the best association was between Cobb t5 and ALFA t5 (p < 0.01) and Cobb t1 and APOS t1 (p < 0.01). No significative association was found between the difference between ALFA and APOS.

Conclusion

We sustain that using new parameters to study vertebral remodelling allows us to reach a better comprehension of Scheuermann spine response to anti-gravity brace treatment. Furthermore, the evaluation of the ALFA angle of the apex vertebra confirms to be more reliable than Cobb’s angle because it cannot be affected by the radiological position.

     

Histochemical and ultrastructural study on the innervation of the byssus glands of Mytilus galloprovincialis

The aluminium-formaldehyde (ALFA) histofluorescence method reveals an extensive plexus of brilliant greenish monoaminergic elements in the glandular zones of the Mytilus foot, while only scanty nerve fibres are acetylcholinesterase-positive. By electron microscopy, bundles of nerve fibres can be seen i) in close connection with the intrinsic musculature located in the connective septa among the glands, and ii) near the cell bodies and necks of all the byssus glands. The nerve fibres show varicosities containing three types of vesicles: small clear (50-60 nm), small granular (80-90 nm), and large granular (160-200 nm). The regions of close apposition between nerve terminals and muscle or gland cells generally do not show typical pre- or postsynaptic specializations. Along the pedal groove, mainly in the proximal two thirds of the foot, peripheral bipolar neurons can be detected, both by fluorescence and electron microscopy.     

Recent developments in aldehyde-induced monoamine fluorescence: The aluminum-formaldehyde (ALFA) method applied to immature and adult central nervous tissue

Summary In this review, the new aluminum-formaldehyde (ALFA) histofluoresence method for the highly sensitive visualization of monoamine-containing neurones in adult and immature central nervous tissue is summarized. Animals are first perfused with a buffer containing high concentrations of aluminum ions and the brains are then freeze-dried, reacted with formaldehyde vapour and further processes according to the Falck-Hillarp fluorescence method.The ALFA technique applied to adult brains visualizes all known catecholamine neurone systems with a sensitivity comparable to, and for certain noradrenergic systems higher than, that of the previously published glyoxylic acid-Vibratome method. The catecholamine systems in immature brains are demonstrated with a sensitivity clearly superior to that of any other available method. If the ALFA method is combined with systemic injections of -methylnoradrenaline into young animals (less than one week old), there is a dramatic increase in the intensity and number of catecholaminergic fibres. Many catecholaminergic systems which have too low concentrations of transmitter to be visualized in the untreated animal even with the ALFA method, can be demonstrated after administration of -methylnoradrenaline.The use of freeze-dried, paraffin-embedded tissue in the ALFA method makes possible convenient storage and parallel processing of many specimens. This mode of processing also allowsen boc reaction, which is the only way by which consistent and reproducible fluorescence yields can be obtained throughout large series of sections and parallel-processed specimens. In animals pretreated withl-tryptophan and monoamine oxidase-inhibitor, the technique is also useful for studies on central indolamine-containing systems.     

Biochemical and cytogenetic biomarkers of pollutant exposure in marine fish (Aldrichetta forsteri Valenciennes and Sillago schomburgkii Peters) near industrial and metropolitan centres in South Australia

This project aimed to measure biochemical and cytogenetic biomarkers in marine fish (Aldrichetta forsteri and Sillago schomburgkii) associated with industrial and urban centres in South Australia. These sites were Port Pirie (affected by metal-contaminated outflows), Barker Inlet (adjacent to Metropolitan Adelaide), and Wills Creek (reference site). The biochemical biomarkers included sorbitol dehydrogenase (SDH) and alanine aminotransferase (ALAT) in serum, adenylate levels (ATP, ADP and AMP) and adenylate energy charge (AEC) in gill and liver, and sodium/potassium ATPase (Na⁺, K⁺-ATPase) in gill. Erythrocyte micronucleus frequency was a marker of cytogenetic effect. Serum enzyme levels were generally higher in fish from Port Pirie and Barker Inlet than in those from Wills Creek, with SDH demonstrating the clearest site-associated differences. Tissue adenylates were consistently lower at Port Pirie than elsewhere, suggesting a greater metabolic strain in fish at this site. AEC in gill and liver were consistently lower at Port Pirie than at Wills Creek, with Barker Inlet generally between these two. The reversed rank order was observed with erythrocyte micronucleus frequencies. Seasonal variations in the biomarkers may be attributed either to seasonal physiological changes in fish or changes in pollutant input levels or compositions. Na⁺, K⁺-ATPase did not differ between sites nor seasons in this study. This work shows that biochemical and cytogenetic differences occur in marine fish at specific locations in South Australia. It also shows that of these tests, serum SDH and erythrocyte micronuclei are potentially the most sensitive and reliable biomarkers of pollutants effects on marine fish. The results also suggest that these data may be used as a baseline against which future changes in marine water quality, and their consequent biological effects, can be compared.     

High toxicity of the novel bloom-forming species Chattonella ovata (Raphidophyceae) to cultured fish

A toxicological study of an axenic cell line of novel species Chattonella ovata Y. Hara et Chihara (Raphidophyceae) revealed that cultured species of sea bream (Pagrus major), horse mackerel (Trachurus japonicus), and yellowtail (Seriola quinqueradiata) were killed by 4.1–6.8 × 10³, 5.4 × 10³, and 2.8 × 10³ cells/mL, respectively. The sensitivity of the gill lamellae to C. ovata differed among the fish species tested. This finding revealed that C. ovata was highly toxic to the cultured fish. Histological examination showed that edema and hyperplasia of the secondary gill lamellae of red sea bream and horse mackerel occurred when exposed to, or killed by C. ovata, whereas severe damage in the gill lamellae was not observed in yellowtail. Chattonella produced high amounts of superoxide anion radicals and hydrogen peroxide, possibly responsible for the fish death observed. Based on the results of this study and occurrence of a red tide by this organism in China in 2001, we consider this organism to be one of the harmful algae in coastal waters. This is the first report demonstrating that C. ovata is highly toxic to fish, and that it produces superoxide and hydrogen peroxide.     

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.     

Non-infectious gill disorders of marine salmonid fish

Gill disorders present a significant challenge in salmon (Salmo salar and Oncorhynchus sp.) farming regions throughout the world. This review of gill disorders and diseases of marine fish is focused on the non-infectious causes of gill disease in marine stage salmonids and these are grouped into harmful algae, such as Karenia mikimotoi, harmful zooplankton, such as Pelagia noctiluca, other environmental challenges, such as pollutants, as well as nutritional and genetic or congenital causes. The present level of understanding of these gill disorders is reviewed with regard to risk factors, potential impacting factors, methods of best practice to mitigate non-infectious gill disease and disorders, as well as knowledge gaps and avenues for future research.     

A new species of deepwater flathead,Bembras adenensis (Scorpaeniformes: Bembridae) from the western Indian Ocean

A new bembrid,Bembras adenensis, is described on the basis of specimens collected from the Gulf of Aden, western Indian Ocean. This species is easily distinguished fromB. japonica, the only known congener of the genus, by the following combination of characters: larger orbital diameter, longer head length, pectoral-fin with a small black blotch near tip of upper rays, 3 gill rakers on upper gill arch, 19 pectoral-fin rays, and 15 anal-fin rays (vs. smaller orbital diameter, shorter head length, pectoral-fin with several small spots forming irregular bands, 2 gill rakers on upper gill arch, usually 17 pectoral-fin rays, and 14 anal-fin rays inB. japonica).     

New fluorophore-forming reactions for histochemical visualization of N-acetylated and tertiary indolamines using glyoxylic acid,aluminum-formaldehyde and trifluoroacetic acid anhydride as reagents

Summary N-acetylated and tertiary indolamines, some of which are possible neurotransmitter candidates in the CNS, cannot be visualized with the standard Falck-Hillarp histofluorescence method and very little is known about their cellular localization. The present investigation demonstrates that glyoxylic acid (GA), formaldehyde (FA) in combination with aluminum ions (the ALFA method) and trifluoroacetic acid anhydride (TFAA) are capable of forming fluorescent compounds from N-acetylated (e.g. melatonin and N-acetyl-5-hydroxytryptamine) and tertiary (e.g. bufotenin) indolamines in histochemical protein models. With GA and FA-aluminum more vigorous reaction conditions were required for demonstration of these compounds compared to those needed for optimal visualization of primary catecholand indolamines (prolonged reaction time and higher concentration of GA and FA and aluminum ions). The fluorophore formation from N-acetylated and tertiary indolamines, which represents a new reaction principle in amine fluorescence histochemistry, is proposed to proceed as follows. In the first step, the indole reacts in 2-position with the reagent. The intermediate formed is dehydrated in the second step, yielding a strongly fluorescent 2-methylene derivative, which either per se or as the corresponding autoxidized dimer constitutes the main fluorophore. TFAA and related anhydrides represent new and potent reagents for histochemical visualization of N-acetylated indolamines such as melatonin. In contrast to the GA and ALFA reactions the optimal formation of fluorphores with TFAA required only mild reaction conditions (2–10 min at 0–20° C). The main fluorophore formed from melatonin has been identified and the reaction with TFAA is proposed to proceed as follows. An unstable intermediate, the isoimidinium carboxylate, is formed in the first step and this compound is then cyclized to form the fluorophore, 6-methoxy-1-methyl-3,4-dihydro--carboline. The GA and ALFA methods are already widely used for visualization of catecholamine systems. The fluorescence microscopical and microspectrofluorometric analysis did not, however, veveal any specific structures containing N-acetylated or tertiary indolamines in the rat CNS. The TFAA reaction was highly specific for N-acetylated indolamines when applied to protein models. However, in tissue a disturbing background fluorescence appeared, which under all reaction conditions tested, developed concomitantly with the specific fluorescence from melatonin. The problem with this background reaction has to be solved before the TFAA reaction can be applied for demonstration of N-acetylated indolamines in tissue.     

5-HT nerve terminals in the fourth ventricle of the rat brain: Their identification and distribution studied by fluorescence histochemistry and electron microscopy

Summary The occurrence and distribution of supra-ependymal nerve terminals storing serotonin (5-HT) are described for the fourth ventricle of the rat brain. The nerve terminals were identified as monoaminergic 1) fluorescence-histochemically, by the presence of a varicose, formaldehyde-induced fluorescence (FIF) on the free surface of the ependyma, 2) electron microscopically, by the presence of electron dense (chromaffin) cores in small (50 nm) and large (100 nm) vesicles found within the varicose regions of supra-ependymal nerve fibres, and 3) by the absence of both the FIF and chromaffin dense cores after treatment with reserpine. Moreover, the serotonergic nature of these nerve fibres could be concluded from 1) the yellow colour of the FIF, 2) the increased FIF after treatment with nialamide or reserpine+nialamide, 3) the diminished FIF and absence of chromaffin dense cores after treatment with p-CPA, and finally 4) the persistence of the FIF and chromaffin dense cores after treatment with -MPT.A high density of 5-HT nerve terminals occurred throughout the floor of the fourth ventricle and on the floor and roof of the lateral recess. Few 5-HT nerve terminals occurred only on the roof of the fourth ventricle (velum medullare, lamina epithelialis of the tela chorioidea), and the surface of the choroid plexus epithelia was devoid of such nerves. Virtually all nerve terminals in the fourth ventricle appear to be serotonergic.Results presented in part at the Autumn Meeting of the British and Italian Pharmacological Societies, Bristol, 1974 (Lorez et al., 1974). The skilful assistance of Mr. R. Wybrecht, Mr. R. Reese and Mrs. M. Gschwind is gratefully acknowledged.     

The effect of input pressure and flow on the pattern and resistance to flow in the isolated perfused gill of a teleost fish

Summary When an isolated gill arch of the marine teleost,Ophiodon elongatus, was perfused under conditions which mimic those present in the intact animal, only two thirds of the gill lamellae were perfused. An increase in either input (afferent) pressure and flow or input pulse pressure caused an increase in the number of lamellae perfused as well as altering the distribution of the efferent outflow between the efferent artery and the venolymphatic drainage of the gill. The gill is compliant and an increase in efferent pressure reduced gill resistance to flow without altering the number of lamellae perfused. In these experiments there was no simple relationship between the number of lamellae perfused and gill resistance.These observations are of importance in the interpretation of results from pharmacological and ion exchange studies of isolated gills as well as indicating how cardiovascular changes could bring about alterations in gill blood flow in the intact fish.     

Molecular Characterization of a Deep-Sea Methanotrophic Mussel Symbiont that Carries a RuBisCO Gene

In our previous investigation on the genes of 1,5-ribulose bisphosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39) in deep-sea chemoautotrophic and methanotrophic endosymbioses, the gene encoding the large subunit of RuBisCO form I (cbbL) had been detected in the gill of a mussel belonging to the genus Bathymodiolus from a western Pacific back-arc hydrothermal vent. This study further examined the symbiont source of the RuBisCO cbbL gene along with the genes of 16S ribosomal RNA (16S rDNA) and particulate methane monooxygenase (EC 1.14.13.25; pmoA) and probed for the presence of the ATP sulfurylase gene (EC 2.7.7.4; sopT). The 16S rDNA sequence analysis indicated that the mussel harbors a monospecific methanotrophic Gammaproteobacterium. This was confirmed by amplification and sequencing of the methanotrophic pmoA, while thiotrophic sopT was not amplified from the same symbiotic genome DNA. Fluorescence in situ hybridization demonstrated simultaneous occurrence of the symbiont-specific 16S rDNA, cbbL and pmoA, but not sopT, in the mussel gill. This is the first molecular and visual evidence for a methanotrophic bacterial endosymbiont that bears the RuBisCO cbbL gene relevant to autotrophic CO₂ fixation.     

Flagellate Cryptobia branchialis (Bodonida: Kinetoplastida), ectoparasite of tilapia from the Salton Sea

An infestation of young tilapia, Oreochromis mossambicus Peters, by the flagellate Cryptobia branchialiswas observed at the Salton Sea, California, in September, 1997. This is the first report of C. branchialis in a highly saline water-body (43 g l^–1). Ultrastructure of C. branchialis as well as its effect on the gills of tilapia were studied using the scanning and transmission electron microscopy. No direct effect of C. branchialis on the epithelial cells of fish gills was observed. However, alterations of gill general structure, such as deposition of copious mucus on the gill surface, swelling of filaments, reduction of respiratory lamellae and their transformation into short club-shaped structures were found in infected fish. This suggests mortality of young tilapia may arise from decreased gill function in response to Cryptobia infestation.     

Application of the aluminum-formaldehyde (ALFA) histofluorescence method for demonstration of peripheral stores of catecholamines and indolamines in freeze-dried paraffin-embedded tissue,cryostat sections and whole-mounts

Summary This paper describes new procedures for highly sensitive visualization of monoamine stores in peripheral tissues, taking advantage of the recently introduced aluminum-catalysed formaldehyde (ALFA) reaction. The tissues are exposed to an aluminum sulphate solution (with or without formaldehyde fixation) in a perfusion and/or immersion step, followed by formaldehyde vapour treatment. Procedures are described for freeze-dried, paraffin embedded tissue, cryostat sections and whole mount preparations. For all these tissue preparations the ALFA method gives a highly sensitive and precise demonstration of catecholamine-containing neurons and 5-HT-containing cells in a variety of peripheral tissues. For freeze-dried tissue and cryostat sections the ALFA method represents an improvement in comparison with other available methods. This is particularly noticeable for the very delicate adrenergic nerves in such organs as the thyroid, ovary, pancreas and the gastrointestinal tract.     

Distribution and abundance of a nemertean egg predator ( Carcinonemertes sp.) on a leucosiid crab,Randallia ornata

The purple globe crab, Randallia ornata (Randall, 1839) (Decapoda: Leucosiidae) is a common crustacean found on sandy beaches from Northern California to Baja California, Mexico. An undescribed species of Carcinonemertes, which shares biological features with a partially described worm from Tasmania was recovered from R. ornata. The worm reaches 10 mm in length and secretes a sturdy tube with a distinctive spiral shape. The total prevalence of the worm on R. ornatawas 70% (85% for ovigerous female crabs, 67% for post-ovigerous female crabs, and 45% for male crabs). Intensities reached 32 worms per crab. Adult worms were found in crab egg masses, regressed adults were recovered from under the abdomen of post-ovigerous females, and encysted juveniles were found on the gill lamellae of non-ovigerous female crabs and occasionally from the gill lamellae of male crabs.     

Gill chemoreceptors and cardio-respiratory reflexes in the neotropical teleost pacu, <Emphasis Type="Italic">Piaractus mesopotamicus</Emphasis>

This study examined the location and distribution of O₂ chemoreceptors involved in cardio-respiratory responses to hypoxia in the neotropical teleost, the pacu (Piaractus mesopotamicus). Intact fish and fish experiencing progressive gill denervation by selective transection of cranial nerves IX and X were exposed to gradual hypoxia and submitted to intrabuccal and intravenous injections of NaCN while their heart rate, ventilation rate and ventilation amplitude were measured. The chemoreceptors producing reflex bradycardia were confined to, but distributed along all gill arches, and were sensitive to O₂ levels in the water and the blood. Ventilatory responses to all stimuli, though modified, continued following gill denervation, however, indicating the presence of internally and externally oriented receptors along all gill arches and either in the pseudobranch or at extra-branchial sites. Chemoreceptors located on the first pair of gill arches and innervated by the glossopharyngeal nerve appeared to attenuate the cardiac and respiratory responses to hypoxia. The data indicate that the location and distribution of cardio-respiratory O₂ receptors are not identical to those in tambaqui (Colossoma macropomum) despite their similar habitats and close phylogenetic lineage, although the differences between the two species could reduce to nothing more than the presence or absence of the pseudobranch.     

The effect of ageing on the SIF-cells in the hypogastric (main pelvic) ganglion of the rat

Summary The effect of ageing on SIF-cells was studied by the formaldehyde-induced fluorescence (FIF) method and by electron microscopy (EM). Microspectrofluorimetry was used to record emission spectra in FIF preparations. In newborn and in young adult (8–12 weeks) rats a single type of SIF-cell emitting greenish-yellow FIF was found while in aged rats a second type of SIF-cell emitting yellowish-brown granular FIF was also present. The intensity of the yellowish-brown FIF was lower than that of the greenish-yellow FIF. Also a few bright cytoplasmic fluorescent areas were occasionally found in some SIF-cells. The distribution of the SIF-cells through the ganglion did not change remarkably with age.In EM after glutaraldehyde-fixation in newborn and in young adult rats two types of small granule-containing (SGC) cells were distinguished according to the size of the dense cored vesicles, 1) 50–150 nm and 2) 50–250 nm. In aged rats, a third type of SGC-cells containing 50×250 nm elongated dense core vesicles could also be distinguished. After KMnO₄-fixation in newborn and in young adult rats the classification was identical with glutaraldehyde-fixation. In aged rats three types of storage granules were found after KMnO₄, 1) 100–300 nm empty vesicles and 2) 100–300 nm vesicles with small dense core, 3) 100–500 nm irregular in shape and filled with electron opaque material with a more dense core.     

A comparison of the major lipid classes and fatty acid composition of marine unicellular cyanobacteria with freshwater species

The fatty acid composition of two motile (strains WH 8113 and WH 8103) and one nonmotile (strain WH 7803) marine cyanobacteria has been determined and compared with two freshwater unicellular Synechocystis species (strain PCC 6308 and PCC 6803). The fatty acid composition of lipid extracts of isolated membranes from Synechocystis PCC 6803 was found to be identical to that of whole cells. All the marine strains contained myristic acid (14:0) as the major fatty acid, with only traces of polyunsaturated fatty acids. This composition is similar to Synechocystis PCC 6308. The major lipid classes of the nonmotile marine strain were identified as digalactosyl diacylglycerol, monogalactosyl diacylglycerol, phosphatidylglycerol, and sulfoquinovosyl diacylglycerol, identical to those found in other cyanobacteria.Abbreviations DGDG Digalactosyl diacylglycerol - MGDG Monogalactosyldiacylglycerol - PG Phosphatidylglycerol - SGDG sulfoquinovosyl diacylglycerol - gc gas chromatography - ms mass spectrometry