Taurine,amino acid transmitters,and related molecules in the retina of the Australian lungfish Neoceratodus forsteri: a light-microscopic immunocytochemical and electron-microscopic study

The morphology of the retina of the Australian lungfish Neoceratodus forsteri was investigated by means of light- and electron microscopy, whilst immunocytochemical studies were performed to determine the cellular distributions of the major amino acid neurotransmitters and other amino acids. The distributions of glycine and GABA were similar to those previously described for teleost, amphibian and mammalian retinae. Labelling was abundant in amacrine cells, whilst GABA was also present in one layer of horizontal cells and some bipolar cells. Taurine was present in both rods and cones, but, unlike the mammalian or avian retina, was absent from other cellular structures, including glial elements. Unexpectedly, the photoreceptor terminals lacked an apparent content of the excitatory amino acid transmitter glutamate. The glutamate that was present in the rods and cones occupied a crescentic arc corresponding to the location of glycogen-rich paraboloids. Asparagine was also present in rods, albeit in the modified mitochondria that formed the elipsoids of the rod inner segments. Arginine, the precursor for formation of nitric oxide, was present in glial cells, and in the paraboloids of both rods and cones.     

The steroidogenic response to angiotensin II in the Australian lungfish,Neoceratodus forsteri

Corticosterone, aldosterone and cortisol were found to be present in lungfish plasma. Plasma levels of these hormones were measured in lungfish following separate single intramuscular injections of three forms of angiotensin II; [Asp¹, Ile⁵], [Asp¹, Val⁵] and [Asn¹, Val⁵]. Aldosterone levels were significantly elevated in response to [Asp¹, Ile⁵] AII and [Asn¹, Val⁵] AII injection. [Asp¹, Val⁵] AII increased plasma corticosterone levels. The difference between these data and the negative results previously reported by Blair-West et al. (1977) are discussed.Abbreviations AII angiotensin II - bw body weight - DOC deoxycorticosterone - RAS renin-angiotensin system - RIA radioimmuno assay     

Insulin-, glucagonand somatostatin-like immunoreactivity in the endocrine pancreas of the lungfish,Neoceratodus forsteri

Summary The endocrine pancreas of the Australian lungfish,Neoceratodus forsteri, was investigated immunocytochemically for the presence of polypeptide hormone-producing cells. Three cell types were identified, namely insulin-, glucagon-, and somatostatin-immunoreactive elements. The insulin cells are confined solely to the center of the islets. Glucagon and somatostatin cells are distributed peripherally around the central mass of the insulin cells. Isolated cells or clusters of glucagon and somatostatin cells are also dispersed within the exocrine parenchyma. The immunoreactive cell types are compared with those staining with standard histological procedures. The spatial relationships of the different cell populations are examined.     

Immunocytochemical localization and characterization of mammalian prolactin- and somatotropin-like material in the pituitary of the Australian lungfish,Neoceratodus forsteri

The aim of the present study was to define at the light-microscopic level expression of prolactin and somatotropin material in the pituitary gland of the Australian lungfish, Neoceratodus forsteri, by use of polyclonal antibodies against ovine prolactin (oPRL) and bovine somatotropin (bSTH). Substances immunologically related to mammalian oPRL as well as bSTH were detected in two morphologically different cell types in the distal lobe, corresponding to the acidophilic cells. The specificity of the antibodies was initially confirmed in a porcine tissue control system. First, our absorption studies confirm that in Neoceratodus the anti-oPRL identifies part of an oPRL-like molecule different from bSTH. Secondly, the anti-bSTH identifies both part of a bSTH-like molecule proper to bovine and Neoceratodus STH, and part of a bSTH-like molecule having antigenic determinants in common with both bSTH and oPRL. This part of the oPRL is, however, not shared with the Neoceratodus PRL as revealed by the anti-oPRL. Altogether these observations support the concepts: (1) that mammalian PRL and STH, or part of those, were established early in evolution, and (2) that dipnoans as living sarcopterygians have an ancestor in common with the early amphibians. The exact nature and physiological functions of the substances detected remain to be defined.     

Australian lungfish (Neoceratodus forsteri: Dipnoi) have low genetic variation at allozyme and mitochondrial DNA loci: a conservation alert?

Genetic variation at allozyme and mitochondrialDNA loci was investigated in the Australianlungfish, Neoceratodus forsteri Krefft1870. Tissue samples for genetic analysis weretaken non-lethally from 278 individualsrepresenting two spatially distinct endemicpopulations (Mary and Burnett rivers), as wellas one population thought to be derived from ananthropogenic translocation in the 1890's(Brisbane river). Two of 24 allozyme lociresolved from muscle tissue were polymorphic.Mitochondrial DNA nucleotide sequence diversityestimated across 2,235 base pairs in each of 40individuals ranged between 0.000423 and0.001470 per river. Low genetic variation atallozyme and mitochondrial loci could beattributed to population bottlenecks, possiblyinduced by Pleistocene aridity. Limited geneticdifferentiation was detected among rivers usingnuclear and mitochondrial markers suggestingthat admixture may have occurred between theendemic Mary and Burnett populations duringperiods of low sea level when the drainages mayhave converged before reaching the ocean.Genetic data was consistent with theexplanation that lungfish were introduced tothe Brisbane river from the Mary river. Furtherresearch using more variable genetic loci isneeded before the conservation status ofpopulations can be determined, particularly asanthropogenic demands on lungfish habitat areincreasing. In the interim we recommend amanagement strategy aimed at conservingexisting genetic variation within and betweenrivers.     

Endothelial nitric oxide synthase in the amphibian, Xenopus tropicalis

Nitric oxide (NO) is generated by NO synthase (NOS) of which there are three isoforms: neuronal NOS (nNOS, nos1), inducible NOS (iNOS, nos2), and endothelial NOS (eNOS, nos3). This study utilised the genome of Xenopus tropicalis to sequence a nos3 cDNA and determine if eNOS protein is expressed in blood vessels. A nos3 cDNA was sequenced that encoded a 1177 amino acid protein called XteNOS, which showed closest sequence identity to mammalian eNOS protein. The X. tropicalis nos3 gene and eNOS protein were determined to be an orthologue of mammalian nos3 and eNOS using gene synteny and phylogenetic analyses, respectively. In X. tropicalis, nos3 mRNA expression was highest in lung and skeletal muscle and lower in the liver, gut, kidney, heart and brain. Western analysis of kidney protein using an affinity-purified anti-XteNOS produced a single band at 140kDa. Immunohistochemistry showed XteNOS immunoreactivity in the proximal tubule of the kidney and endocardium of the heart, but not in the endothelium of blood vessels. Thus, X. tropicalis has a nos3 gene that appears not to be expressed in the vascular endothelium.     

The histology of tooth formation in the Australian lungfish, Neoceratodus forsteri Krefft

The histology of developing toothplates of Neoceratodusforsteri from the time of first appearance of the tooth primordia to the adult condition has been investigated. The dentition develops by the formation of a shell of primary epithelial and mesenchymal matrices. Within the shell, secondary mesenchymal matrix and central material, both containing columns of tertiary matrix, are laid down. Primary epidielial matrix appears to contain collagen and is closely associated with the epithelium of the mouth. All other tooth tissues as well as the supporting bone develop in association with mesenchyme. Primary, secondary and tertiary mesenchymal matrices appear to contain collagen. Central dentine contains some fibres, possibly of reticulin or collagen, within a matrix of unknown composition.
The tooth is attached to the underlying bone by a pedestal of bone and this grows with the tooth material.
New tooth tissues are formed in the pulp cavity in layers below the older material, causing the toothplate to grow in every dimension as the animal grows.
An evolutionary pathway is suggested for lungfish with a dentition of cusps arranged in radiating ridges.     

Vascularization of the pituitary of the Australian lungfish and Neoceratodus forsteri

The vascularization of the brain and the pituitary region of the Australian lungfish, Neoceratodus forsteri is described from serial section reconstruction. The distal lobe has no direct arterial blood supply and receives blood solely from a pituitary portal system basically similar to that of other sarcopterygians. The primary capillary plexus of the median eminence receives its arterial blood from the infundibular arteries, which on their way distribute some small branches to the prechiasmatic region. The primary plexus also receives capillaries from the adjacent pial hypothalamic plexus. The primary capillary plexus of the median eminence comprises a rostral 'uncovered' and caudal 'covered' part which are not sharply delineated. Distinct portal vessels connect the 'uncovered' rostral part of the primary plexus with the secondary capillary plexus supplying the rostral subdivision of the pars distalis. The 'covered' caudal part of the primary plexus merges into the proximal subdivision of the pars distalis, apparently without formation of distinct portal vessels. The primary plexus has some connections with the plexus intermedius via a hypophysial stem capillary plexus. The plexus intermedius has a substantial arterial supply and gives off capillaries to the parenchyma of the pars intermedia. The adenohypophysis is drained into an unpaired hypophysial vein. The significance of the vascular pathways is discussed from comparative, functional, and evolutionary viewpoints.     

Ultrastructure of developing tooth plates in the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi)

While the lungfish dentition is partially understood as far as morphology and light microscopic structure is concerned, the ultrastructure is not. Each tooth plate is associated with a dental lamina that develops from the inner layer of endodermal cells that form the oral epithelium. Dentines, bone and cartilage of the jaws differentiate from mesenchyme cells aggregating beneath the oral endothelium. Enamel, in the developing and in the mature form, has similarities to that of other early vertebrates, but unusual characters appear as development proceeds. Ameloblasts are capable of secreting enamel, and, with mononuclear osteoclasts, of remodelling the bone below the tooth plate. The forms of dentine, all based largely on an extracellular matrix of collagen and mineralised with biological apatite, differ from each other and from the underlying bone in the ultrastructure of associated cells and in the mineralised extracellular matrices produced. Cell processes emerging from the odontoblasts and from the osteoblasts vary in length, degree of branching and of anastomoses between the processes, although all of the cell types have large amounts of rough endoplasmic reticulum. Mineralisation of the extracellular matrices varies among the enamel, dentines and bone in the tooth plate. In addition, the development of the hard tissues of the tooth plates indicates that many of the similarities in fine structure of the dentition in lungfish, to tissues in other fish and amphibia, apparent early in development, disappear as the dentition matures.     

Cranial nerves in the Australian lungfish,Neoceratodus forsteri,and in fossil relatives (Osteichthyes: Dipnoi)

Three systems, two sensory and one protective, are present in the skin of the living Australian lungfish, Neoceratodus forsteri, and in fossil lungfish, and the arrangement and innervation of the sense organs is peculiar to lungfish. Peripheral branches of nerves that innervate the sense organs are slender and unprotected, and form before any skeletal structures appear. When the olfactory capsule develops, it traps some of the anterior branches of cranial nerve V, which emerged from the chondrocranium from the lateral sphenotic foramen. Cranial nerve I innervates the olfactory organ enclosed within the olfactory capsule and cranial nerve II innervates the eye. Cranial nerve V innervates the sense organs of the snout and upper lip, and, in conjunction with nerve IX and X, the sense organs of the posterior and lateral head. Cranial nerve VII is primarily a motor nerve, and a single branch innervates sense organs in the mandible. There are no connections between nerves V and VII, although both emerge from the brain close to each other. The third associated system consists of lymphatic vessels covered by an extracellular matrix of collagen, mineralised as tubules in fossils. Innervation of the sensory organs is separate from the lymphatic system and from the tubule system of fossil lungfish.     

Skin structure in the snout of the Australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

Many fossil lungfish have a system of mineralised tubules in the dermis of the snout, branching extensively and radiating towards the epidermis. The tubules anastomose in the superficial layer of the dermis, forming a plexus consisting of two layers of vessels, with branches that expand into pore canals and flask organs, flanked by cosmine nodules where these are present. Traces of this system are found in the Australian lungfish, Neoceratodus forsteri, consisting of branching tubules in the dermis, a double plexus below the epidermis and dermal papillae entering the epidermis without reaching the surface. In N. forsteri, the tubules, the plexus and the dermal papillae consist of thick, unmineralised connective tissue, enclosing fine blood vessels packed with lymphocytes. Tissues in the epidermis and the dermis of N. forsteri are not associated with deposits of calcium, which is below detectable limits in the skin of the snout at all stages of the life cycle. Canals of the sensory line system, with mechanoreceptors, are separate from the tubules, the plexus and the dermal papillae, as are the electroreceptors in the epidermis. The system of tubules, plexus, dermal papillae and lymphatic capillaries may function to protect the tissues of the snout from infection.     

Nitric oxide synthase immunoreactivity in the nematode Trichinella britovi. Evidence for nitric oxide production by the parasite

Nitric oxide has been extensively studied as an effector molecule of the host immune response against both protozoa and helminths, but parasites can also produce this molecule, through the action of nitric oxide (NO) synthases or NO synthases-like enzymes. The aim of this study was to verify the possible production of NO by Trichinella britovi L(1) larvae and the enzymes involved in this process. The NO synthase immunoreactivity and putative nitric oxide synthase-activity was analysed using antibodies to mammalian NO synthase III and to nitrotyrosine with immunohistochemistry, gold immunocytochemistry and immunoblot analysis and NADPH-diaphorase histochemistry. Our results show that T. britovi L(1) larvae possess an enzymatic activity capable of producing NO. The localisation of this activity, according to the NADPH-diaphorase histochemistry, is both at the cuticular and the internal level. This localisation is confirmed by nitrotyrosine immunohistochemistry both under optical and electron microscopy. Using the NO synthase III antibody, a similar pattern of labelling was found: in particular, electron microscopy showed a localisation of this immunoreactivity in the cuticle and in the stichocytes, where only the alpha2 granules contained gold particles, mainly concentrated at their periphery. Four polypeptides reacting to the NO synthase III antibody are revealed by Western blotting. Their molecular weight ranged from 38 to 50 kDa. A significant reaction of the anti-nitrotyrosine antibody to polypeptides 95, 60, 48 and 39 kDa from the same sample suggested the presence of different nitrosylated proteins.     

Cephalic muscle development in the Australian lungfish,Neoceratodus forsteri

Lungfishes are the extant sister group of tetrapods. As such, they are important for the study of evolutionary processes involved in the water to land transition of vertebrates. The evolution of a true neck, that is, the complete separation of the pectoral girdle from the cranium, is one of the most intriguing morphological transitions known among vertebrates. Other salient changes involve new adaptations for terrestrial feeding, which involves both the cranium and its associated musculature. Historically, the cranium has been extensively investigated, but the development of the cranial muscles much less so. Here, we present a detailed study of cephalic muscle development in the Australian lungfish, Neoceratodus forsteri, which is considered to be the sister taxon to all other extant lungfishes. Neoceratodus shows several developmental patterns previously described in other taxa; the tendency of muscles to develop from anterior to posterior, from their region of origin toward insertion, and from lateral to ventral/medial (outside‐in), at least in the branchial arches. The m.protractor pectoralis appears to develop as an extension of the most posterior m.levatores arcuum branchialium, supporting the hypothesis that the m.cucullaris and its derivatives (protractor pectoralis, levatores arcuum branchialium) are branchial muscles. We present a new hypothesis regarding the homology of the ventral branchial arch muscles (subarcualis recti and obliqui, transversi ventrales) in lungfishes and amphibians. Moreover, the morphology and development of the cephalic muscles confirms that extant lungfishes are neotenic and have been strongly influenced via paedomorphosis during their evolutionary history.     

Inhibition of nitric oxide synthase by cobalamins and cobinamides

Cobalamins are important cofactors for methionine synthase and methylmalonyl-CoA mutase. Certain corrins also bind nitric oxide (NO), quenching its bioactivity. To determine if corrins would inhibit NO synthase (NOS), we measured their effects on -l-[¹⁴C]arginine-to-l-[¹⁴C]citrulline conversion by NOS1, NOS2, and NOS3. Hydroxocobalamin (OH-Cbl), cobinamide, and dicyanocobinamide (CN₂-Cbi) potently inhibited all isoforms, whereas cyanocobalamin, methylcobalamin, and adenosylcobalamin had much less effect. OH-Cbl and CN₂-Cbi prevented binding of the oxygen analog carbon monoxide (CO) to the reduced NOS1 and NOS2 heme active site. CN₂-Cbi did not react directly with NO or CO. Spectral perturbation analysis showed that CN₂-Cbi interacted directly with the purified NOS1 oxygenase domain. NOS inhibition by corrins was rapid and not reversed by dialysis with l-arginine or tetrahydrobiopterin. Molecular modeling indicated that corrins could access the unusually large heme- and substrate-binding pocket of NOS. Best fits were obtained in the “base-off” conformation of the lower axial dimethylbenzimidazole ligand. CN₂-Cbi inhibited interferon-γ-activated Raw264.7 mouse macrophage NO production. We show for the first time that certain corrins directly inhibit NOS, suggesting that these agents (or their derivatives) may have pharmacological utility. Endogenous cobalamins and cobinamides might play important roles in regulating NOS activity under normal and pathological conditions.     

The liver fluke Opisthorchis viverrini expresses nitric oxide synthase but not gelatinases

Host-parasite interaction during infection with the liver fluke Opisthorchis viverrini plays an important role in opisthorchiasis-associated cholangiocarcinoma via nitric oxide (NO) production. Host cells induce nitric oxide synthase (NOS)-dependent DNA damage and secrete Ras-related C3 botulinum toxin substrate (Rac)1, heme oxygenase (HO)-1, and gelatinases (matrix metalloproteinase (MMP)2 and MMP9). We evaluated whether these enzymes are expressed in O. viverrini. Colocalization of NOS and Rac1 was most prominently detected on day 30 post-infection (p.i.) in the gut, reproductive organ, eggs, acetabular and tegument. Expression of HO-1, an antioxidative enzyme, increased in a similar pattern to NOS, but was not present in the tegument. The levels of nitrate/nitrite, end products of NO, and ferric reducing antioxidant capacity, an indicator of antioxidant enzyme capacity, in parasite homogenates were highest on day 30 p.i. and then decreased on day 90 p.i. In contrast, zymography revealed that MMP2 and MMP9 were not present in parasite homogenates at all time points. In conclusion, O. viverrini induces NOS expression and NO production, but does not express gelatinases. The study may provide basic information and an insight into drug design for prevention and/or intervention approaches against O. viverrini infection.     

The epithelial sodium channel in the Australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

Epithelial sodium channel (ENaC) is a Na⁺-selective, aldosterone-stimulated ion channel involved in sodium transport homeostasis. ENaC is rate-limiting for Na⁺ absorption in the epithelia of osmoregulatory organs of tetrapods. Although the ENaC/degenerin gene family is proposed to be present in metazoans, no orthologues or paralogues for ENaC have been found in the genome databases of teleosts. We studied full-length cDNA cloning and tissue distributions of ENaCα, β and γ subunits in the Australian lungfish, Neoceratodus forsteri, which is the closest living relative of tetrapods. Neoceratodus ENaC (nENaC) comprised three subunits: nENaCα, β and γ proteins. The nENaCα, β and γ subunits are closely related to amphibian ENaCα, β and γ subunits, respectively. Three ENaC subunit mRNAs were highly expressed in the gills, kidney and rectum. Amiloride-sensitive sodium current was recorded from Xenopus oocytes injected with the nENaCαβγ subunit complementary RNAs under a two-electrode voltage clamp. nENaCα immunoreactivity was observed in the apical cell membrane of the gills, kidney and rectum. Thus, nENaC may play a role in regulating sodium transport of the lungfish, which has a renin–angiotensin–aldosterone system. This is interesting because there may have been an ENaC sodium absorption system controlled by aldosterone before the conquest of land by vertebrates.