Lepidosiren Research Articles

Phylogeny of central CO2/pH chemoreception in vertebrates

The traditional view has been that respiratory chemoreceptors responsive to changes in PCO2/pH first evolved in air-breathing vertebrates at both peripheral and central sites. Levels of arterial PCO2 in water breathing fish are typically less than 2–3 torr and it has been assumed that the ventilatory responses of fish to changes in aquatic PCO2/pH were due to the effects of acidosis on haemoglobin oxygen transport. There is growing evidence, however, to suggest that fish also possess peripheral chemoreceptors responsive to changes in PCO2/pH per se that reside primarily in the gills, are innervated by the glassopharyngeal and vagus nerves, and respond primarily to changes in aquatic rather than arterial PCO2. Their distribution overlaps extensively with that of the gill O2 chemoreceptors in fish and it is not yet clear whether both responses arise from the same sensory cells. To date, there is no convincing evidence that strictly water breathing fish possess central chemoreceptors. There is, however, growing evidence to suggest that some species of air-breathing fish possess central CO2 chemoreceptors. Central chemosensitivity has been reported in in vitro brainstem-spinal cord preparations from both a primitive (holostean) and a modern (teleost) actinopterygian (ray finned) fish. Stimulation of these putative receptors had no effect on fictive gill ventilation but stimulated fictive air breathing. Unfortunately, the fictive breathing rates of these preparations were more than 25 times the resting rates reported for intact animals raising questions about the physiological significance of changes in the fictive motor output identified as air breathing under these conditions. In the South American lung-fish (a sarcopterygian fish belonging to the lineage giving rise to higher vertebrates), on the other hand, central perfusion with mock csf of differing pH stimulated air breathing at rates similar to those seen in control animals. While these data suggest that central chemoreceptors have arisen several times in evolutionary history, hand in hand with the evolution of air breathing, this issue is not yet totally resolved. An intriguing and related finding is that central CO2 chemosensitivity appears to develop slowly in amphibian tadpoles. It is not present in young tadpoles but develops over time. The receptors initially stimulate gill ventilation but transfer their influence to lung ventilation just prior to metamorphosis from the aquatic larval stage into the air breathing adult form. In association with this the primary location of the receptors in the brainstem shifts from a diffuse distribution to a rostral concentration. While central chemoreceptors have been demonstrated in reptiles and birds, not much is known of their properties or distribution. They have been well studied in mammals where there is growing evidence for multiple sites of central CO2/pH chemoreception and evidence to suggest that the mechanism of sensory transduction may vary both within and between receptor populations. While there has been much recent interest in the membrane channels, receptors and electrical coupling of several chemosensitive sites, this work has largely been on cells with downstream respiratory rhythmicity in preparations whose responses are very different (in terms of changes in frequency and amplitude of phrenic output) from that which is seen in intact animals. The phylogenetic trends that are emerging indicate that the use of CO2 chemoreception for cardio-respiratory processes may have arisen much earlier than previously believed, that CO2/pH chemoreception arose in the periphery before the evolution of central CO2/pH chemoreceptors, that the sites of CO2/pH chemoreception (both peripheral and central) have increased throughout the course of vertebrate evolution and that the mechanism of CO2/pH chemotransduction may vary. The sum of the data suggests that CO2/pH chemoreceptors have arisen multiple times, at multiple sites during vertebrate evolution.

Evolutionary history of the enolase gene family

The enzyme enolase [EC 4.2.1.11] is found in all organisms, with vertebrates exhibiting tissue-specific isozymes encoded by three genes: alpha (α), beta (β), and gamma (γ) enolase. Limited taxonomic sampling of enolase has obscured the timing of gene duplication events. To help clarify the evolutionary history of the gene family, cDNAs were sequenced from six taxa representing major lineages of vertebrates: Chiloscyllium punctatum (shark), Amia calva (bowfin), Salmo trutta (trout), Latimeria chalumnae (coelacanth), Lepidosiren paradoxa (South American lungfish), and Neoceratodus forsteri (Australian lungfish). Phylogenetic analysis of all enolase and related gene sequences revealed an early gene duplication event prior to the last common ancestor of living organisms. Several distantly related archaebacterial sequences were designated as ‘enolase-2’, whereas all other enolase sequences were designated ‘enolase-1’. Two of the three isozymes of enolase-1, α- and β-enolase, were discovered in actinopterygian, sarcopterygian, and chondrichthian fishes. Phylogenetic analysis of vertebrate enolases revealed that the two gene duplications leading to the three isozymes of enolase-1 occurred subsequent to the divergence of living agnathans, near the Proterozoic/Phanerozoic boundary (approximately 550Mya). Two copies of enolase, designated α1 and α2, were found in the trout and are presumed to be the result of a genome duplication event.

Ascorbic acid biosynthesis in Amazonian fishes

The incapacity to synthesize ascorbic acid (AA) is due to the lack of activity of l-gulonolactone oxidase (GLO), which catalyzes the last step of AA biosynthesis. It was postulated that vertebrates unable to synthesize AA had sufficient amounts of this nutrient in their diet and consequently did not need to preserve synthetic capability. In the present study, we analyzed the GLO activity in kidney and liver of 13 fish species, including 11 teleosts, namely: freshwater stingray, Potamotrygon sp.; South American lungfish, Lepidosiren paradoxa; “sardinhão,” Pelona sp.; arowana, Osteoglossum bicirrhosum; arapaima, Arapaima gigas; “piranha caju,” Pygocentrus nattereri; “piranha mucura,” Serrasalmus elongatus; “aracu,” Schizodon fasciatus; “tambaqui,” Colossoma macropomum; “acari-pedra,” Hypostomus sp.; “sarapó,” Steatogenys elegans; electric eel, Electrophorus electricus; and the peacock bass, Cichla sp. Four representatives of the Characiformes order with distinct feeding habits were included in this study to evaluate the influence of feeding habit on GLO activity. Only two species of non-teleost fishes, the freshwater stingray (Miliobatiformes) and the South American lungfish (Lepidosireniformes), showed GLO activity in their kidneys, corroborating the hypothesis that teleosts are unable to synthesize AA. Additionally, as expected, we observed that the phylogenetic position is more important than feeding habit as a determinant of the biosynthetic ability since none of the Characiformes species analyzed synthesize AA, independent of their distinct feeding habits. Female freshwater stingrays had a significantly higher GLO activity than males ( P<0.05), but there was no difference in the biosynthetic capability regarding the portion of the kidney analyzed ( P>0.05), as previously reported in white sturgeon. This is the first report of the AA biosynthetic ability in South American lungfish and freshwater Elasmobranchs.

Isozyme distribution of ten enzymes and their loci in South American lungfish, Lepidosiren paradoxa (Osteichthyes, Dipnoi)

Scarce bibliographical data exists on the enzymes in Lepidosiren paradoxa and analysis of several enzymes was considered worthy of investigation. Distribution of ADH, ALP, FBALD, GAPDH, G3PDH, G6PDH, GPI, LDH, MDH, and PGM was identified in ten tissues (retina, heart, muscle, liver, kidney, lung, gut, gills, brain, and ovary) of the South American lungfish and compared with patterns previously described in other vertebrates. Compared with earlier results differences in the number of loci expressed were observed for ADH, G3PDH, GPI, and MDH. The number of loci expressed and/or in tissue specificity of several enzymes (ADH, FBALD, GAPDH, G3PDH, G6PDH and PGM) were found to be similar to those of other vertebrates. Differences were detected in ALP due to the absence of an intestinal-specific form typical of fish, amphibians, reptiles and birds; further differences were observed in GPI and MDH due to their tissue expression. The differences in LDH involve the LDH-A 4 isozyme which was most common in tissues. Overall, comparison with other vertebrates reveals that in L. paradoxa the tissue-restricted expressions of some enzymes are similar, while others have retained an ancestral pattern and exhibit a more widespread tissue expression of genes.

Specialized metabolism and biochemical suppression during aestivation of the extant South American lungfish — Lepidosiren paradoxa

Specialized metabolism and biochemical suppression during aestivation of the extant South American lungfish — Lepidosiren paradoxa

Chronotropic and inotropic responses to temperature changes of ventricular myocardium of the South American lungfish.

Chronotropic and inotropic responses to temperature changes of ventricular myocardium of the South American lungfish.

Nuclear DNA content of thirty species of Neotropical fishes

The present paper reports nuclear DNA content in 30 Neotropical freshwater fish species and summarizes the data on other Neotropical species presented in the literature. Among Neotropical fishes, the nuclear DNA content ranges from 1.04 ± 0.09 pg/nucleus in Corydoras cf. simulatus (2n = 62) to 248.0 pg/nucleus in Lepidosiren paradoxa (2n = 38). A general analysis of the data obtained in the present study for each species showed that DNA measurements were practically constant at the individual level, while significant differences were observed among individuals of the same population. This observation was valid for all species analyzed and was more evident in those species that presented other karyotypic particularities such as sex chromosomes or supernumerary chromosomes. The importance of changes in nuclear DNA content in the evolutionary process of Neotropical fishes is discussed.

Lymphatic vessels in lungfishes (Dipnoi)

Lymphatic capillaries are distributed throughout the body of lepidosirenid and protopterid Dipnoi, except in the central nervous system. They form small, interconnected units which are individually evacuated into nearby blood capillaries by lymphatic micropumps. The number of lymphatic micropumps varies considerably in different parts of the body. In fin areas, 30–50 per mm3 tissue may be considered normal in Protopterus annectens, but up to 105 per mm3 have been counted in an anterior fin of Lepidosiren paradoxa. Lymphatic capillaries are formed by thin endothelial cells with fine processes into the surrounding interstitial space. Occasionally there is a faint, discontinuous basal lamina. Pericytes, however, are completely absent. Microfibrils establish contact between endothelial cells and surrounding connective tissue fibers. The lymphatic micropumps are essentially spherical, contractile organs of 35–55 μm in diameter. Their central lumen is lined by extensions of a single endothelial cell. Additional endothelial cells form inflow and outflow valves. The endothelial layer is surrounded by a single large, highly specialized muscle cell. This spherical muscle cell has many perforations, allowing the passage of thin outward processes of the endothelial cell which form part of the suspension apparatus of the lymphatic micropump. The muscle cell establishes a specialized end-to-end contact between opposing parts of its own cell membrane. This contact is very similar to an intercalated disc in vertebrate heart muscle. Each lymphatic micropump is suspended within a cell-free tissue area by microfibrils which radiate from the lymphatic micropump into the surrounding connective tissue. The microfibrils are occasionally reinforced by single collagen fibers. The cell-free area around each lymphatic micropump appears as a bright halo in both light and electron micrographs. No type of lymphatic vessel other than lymphatic capillaries could be detected in the Dipnoi studied. Lepidosireniform Dipnoi are the only Vertebrata besides the Tetrapoda in which lymphatic vessels and characteristic lymphatic pumps have been documented. In addition, these Dipnoi and all Tetrapoda share the same overall design of blood circulation, which is not divided into a primary and a secondary system of vessels, as it is in Actinopterygii, Chondrichthyes, and Agnatha. Since there are primary and secondary blood vessels in the gills of Latimeria chalumnae, while the existence of lymphatic vessels has not been confirmed, general angioarchitecture should be taken into account as an important character when phylogenetic relationships among extant Sarcopterygii are discussed.

The evolutionary significance of pulmonary surfactant in lungfish (Dipnoi).

In this study, we characterized surfactant lipids from the lungs of the lungfish, Protopterus annectens, Lepidosiren paradoxa, and Neoceratodus fosteri (Sarcopterygiia: Dipnoi). We quantified the types of phospholipids present, the amounts of total phospholipid, disaturated phospholipid (DSP)--purported to be the primary surface tension-controlling agent--and cholesterol (CHOL), an important fluidizer. The surfactant phospholipid profiles of all three lungfish were very similar to each other and those of many other animals reported previously. Phosphatidylcholine was the dominant phospholipid (60% to 80%); phosphatidylglycerol was virtually absent; and there was a significant proportion of the combination of phosphatidylserine and phosphatidylinositol (10%). The Australian lungfish N. forsteri possessed a surfactant 5 times richer in CHOL and 2 and 3 times poorer in DSP than the surfactant of the African lungfish P. annectens and the South American lungfish L. paradoxa, respectively. Hence, the CHOL/DSP mass ratio of N. forsteri was 12 and 20 times greater than that of P. annectens and L. paradoxa, respectively. Therefore, the surfactant composition of the two derived species of lungfish (P. annectens and L. paradoxa) very closely resembles that of amphibians, whereas surfactant from the primitive lungfish (N. forsteri) is almost identical to that of the primitive air-breathing actinopterygiian fish. Thus, it is likely that pulmonary surfactant had only a single origin, coinciding with that of the vertebrates. As with most nonmammalian vertebrates, it is possible that lungfish surfactant functions as an antiglue at low lung volumes or when the lungs are completely collapsed. Furthermore, it appears that within a species, an increase in lung development correlates with an increase in the relative amount of surfactant cholesterol and a decrease in the phospholipid saturation level.

Lipids and Myelin Proteins in the Brains of Coelacanth (Latimeria chalumnae), lungfish (Lepidosiren paradoxaandProtopterus aetiopicus), bichir (Polypterus senegalus), and sturgeon (Acipenser ruthenus) (Osteichthyes): Phylogenetic Implications

The chemical components of nerve membranes of primitive bony fishes (coelacanth (Latimeria chalumnae), lungfish (Lepidosiren paradoxa and Protopterus aetiopicus), bichir (Polypterus senegalus), and sturgeon (Acipenser ruthenus)) were analyzed to investigate their phylogenetic relations. Representative myelin components such as cerebroside and myelin proteins and enzyme exhibited characteristic profiles in the different species. Coelacanth was characterized by an abundance of hydroxy components of cerebroside fatty acids and by the occurrence of a 28 000 molecular weight band (possibly P0-like protein) and proteolipid protein (PLP). Lungfish possessed glycosylated PLP, but lacked PLP; bichir and sturgeon showed the same protein compositions, PLP and P0-like protein being major components. The activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP), a myelin marker enzyme, was detected in coelacanth myelin, but was almost negligible in the others. The ganglioside compositions were simple in coelacanth and lungfish compared with those in bichir and sturgeon, which were abundant in polysialo components. These findings suggest that coelacanth nerve membranes are the most developed in these species; thus, coelacanth may be more advanced phylogenetically than the others. Bichir and sturgeon were suggested to occupy unique phylogenetic positions, showing similar chemical compositions of nerve membranes which were distinct from those of coelacanth and lungfish.

Relationship among coelacanths, lungfishes, and tetrapods: A phylogenetic analysis based on mitochondrial cytochrome oxidase I gene sequences

To clarify the relationship among coelacanths, lungfishes, and tetrapods, the amino acid sequences deduced from the nucleotide sequences of mitochondrial cytochrome oxidase subunit I (COI) genes were compared. The phylogenetic tree of these animals, including the coelacanth Latimeria chalumnae and the lungfish Lepidosiren paradoxa, was inferred by several methods. These analyses consistently indicate a coelacanth/lungfish clade, to which little attention has been paid by previous authors with the exception of some morphologists. Overall evidence of other mitochondrial genes reported previously and the results of this study equally support the coelacanth/lungfish and lungfish/tetrapod clades, ruling out the coelacanth/tetrapod clade.

Ultrastructure of the granulocytes of the South American lungfish, Lepidosiren paradoxa: Morphogenesis and comparison to other leucocytes.

Ultrastructure and peroxidase cytochemistry of the granulocytes of the lungfish Lepidosiren paradoza were investigated. In peripheral blood and hemopoietic tissue (kidney and spleen) only three granulocytic types were found. After comparison with light microscopic images of Giemsa-stained semithin sections the granulocytes were tentatively designated as eosinophilic I, eosinophilie II, and a basophilic type. The eosinophilc I cell was the most numerous granulocyte and was characterized by granules ranging in diameter from 0.2-1.8 μm. A developmental series was observed, leading from small, peroxidase (PO) positive granules to large ones with PO-negative cores and only peripheral PO-positive reactivity. The central electron-dense cores of the large granules were often eroded. The eosinophlic II cell was less numerous than the eosinophilic I type and had smaller (diameter 0.4-0.9 μm) granules than the eosinophilic I type, some of them elongated and rod like. The basophilic cell was rare and contained large, homogenously dense granules measuring up to 1.2 μm in diameter. Immature stages of the granulocytic types showed vesicular rough endoplasmic reticulum. Nuclear identation and segmentation occurred early in development, in contrast to the relatively late onset of these processes in mamals. Monocytes and macrophages had an irregular or slightly indented nucleus and lacked the specific granules of the granulocytes. Phylogenetic relationships were discussed with special reference to the question of the evolution of only one or possibly of two separate heterophilic/neutrophilic cell lines in fishes. © 1993 Wiley-Liss, Inc.

The origin of the tetrapods

The origin of tetrapods is one of the longest standing (and still not fully resolved) fields in vertebrate evolution (Thomson, 1993). The discovery of living lungfishes in the nineteenth century sparked interest because of their apparent intermediate position between other kinds of fishes and amphibians. (As is well known, the South American lungfish Lepidosiren was actually first described as a degenerate amphibian.) The discovery of the Australian lungfish provided a direct link between a living form (a “living fossil” to use Darwin's term) and well-known Triassic fossils. The lungfishes therefore accorded well with Darwinian theory, just as did Archaeopteryx. The discovery of the living coelacanth Latimeria in 1938 continues to spark public interest (Thomson, 1991a).

Aspectos do desenvolvimento ovocitário no peixe-pulmonado Sul-americano, Lepidosiren paradoxa Fitzinger (Dipnoi)

Histological changes in the oocyte development of South American lungfish are described. During maturation, pre-vitellogenic and vitellogenic phases could be distinguished, and they are similar to those of teleost fishes.

Tachykinins and intestinal motility in different fish groups

The presence and function of tachykinins were studied in the intestine of hagfish ( Myxine glutinosa), lamprey ( Lampetra fluviatilis), starry ray ( Raja radiata), lungfish ( Lepidosiren paradoxa), bichir ( Polypterus senegalensis), and rainbow trout ( Oncorhynchus mykiss), which represent different systematic groups of fish. Immunohistochemistry revealed cells containing substance P (SP)-like material in the intestine of lamprey and lungfish, and in the stomach of the ray. The intestinal motility was studied using isolated muscle strip preparations. SP had no effect on hagfish or lamprey intestine. In the other four species SP produced intestinal contractions. In ray, bichir, and lungfish the tachykinins may be released from endocrine cells and act, at least in the bichir and lungfish, directly onto the smooth muscle cells. In the rainbow trout intestine, where SP-like material may be released from both nerve fibres and endocrine cells, it is indicated that the contractile effect is in part direct upon the smooth muscle and in part via stimulation of cholinergic and serotonergic neurons.

Ultrastructure of the agranular cells in the adenohypophysis of the South American lungfish, Lepidosiren paradoxa.

Light and electron microscopic examination demonstrated two types of non-endocrine agranular cells, cavity boundary cells and stellate cells, in the adenohypophysis of the South American lungfish, Lepidosiren paradoxa. The cavity boundary cells line the hypophyseal cleft and diverticulum and display few microvilli, occasional cilia, prominent junctional complexes, and many cytoplasmic microfilaments. The stellate cells are scattered in the glandular parenchyma and are devoid of microvilli and cilia. When adjacent, they are connected to one another by desmosomes. Pinocytotic vesicles or caveolae are frequently seen along the plasma membrane of the agranular cells adjoining the endocrine cells or abutting on the basement membrane. Possible roles of the agranular cells, physically and metabolically supportive functions, are discussed on the basis of their ultrastractural features.

The fine structure and formation of hypermineralized petrodentine in the tooth plate of extant lungfish (Lepidosiren paradoxa and Protopterus sp.).

Histological features of the tooth plate of lungfish were investigated by light and electron microscopy and contact microradiography with primary reference to the fine structure and formation of petrodentine, one of the principal constituents of the tooth plates. Petrodentine is a highly mineralized tissue noticeably analogous to that of enameloid, and is deposited intermittently in a proximal direction by the sole participation of mesenchymal petroblasts. The petroblasts appear very likely to have prominent biphasic functions of secreting the petrodentine matrix and of eliminating the matrix by resorption. The petrodentine thus may be capable of attaining hypermineralization with the participation mesenchymal cells alone. Mineral crystals constituting the petrodentine are large, hexagonal or similar but irregularly shaped columns. Initially these crystals appeared extremely thin, with curled ones frequently noted. The petrodentine of lungfish tooth plates is considered to be structurally distinct from the pleromin (Kosmin) of holocephalian tooth plates because of the difference between their constituent crystals.

Morphology of central compartment and vasculature of the gills of Lepidosiren paradoxa (Fitzinger)

The four paired gill arches of the South American lungfish Lepidosiren paradoxa contain single branchial arteries directly connecting dorsal and ventral arteries. In gill arches 3 and 4 the branchial arteries also supply looped arlerioles and capillaries to much‐reduced gill filaments. Regulation of blood between these routes is thought to be by alteration of vascular resistance. Within the filaments, extensive subepithelial capillary networks and numerous small pumps connect lymphatic vessels in the central connective tissue compartment with venules which, in turn, drain to paired branchial veins.The features of the endothelium of many of the filament blood vessels suggest extensive transporting, haematolytic and granulopoeitic functions. Large numbers of macrophages pack the connective tissue. Many contain extensive quantities of haemosiderin.

On the cardiac control in the South American lungfish, Lepidosiren paradoxa

1. 1. The mechanisms behind cardiac control were investigated in the South American lungfish, Lepidosiren paradoxa, using fish with chronically implanted cannulae and electromagnetic flow probes. In addition, a preliminary study was made of the cardiovascular events associated with air breathing. 2. 2. The study suggests that the heart of Lepidosiren is controlled by cholinergic vagal fibres which, in some animals, exert a tonic influence in the resting fish. Cyclic changes in heart rate in association with air breaths is due to modulation of this cholinergic tonus. 3. 3. In addition to the variable cholinergic tonus, there appears to be a relatively stable adrenergic tonus on the heart, which causes an elevated heart rate. The adrenergic tonus is likely to be due to local release of catecholamines from endogenous chromaffin cells within the atrium. 4. 4. Preliminary results suggest that pulmonary arterial flow increases by about 50% immediately following an air breath. The mechanism behind this increase probably involves both an elevation of the heart rate and a redistribution of blood flow into the pulmonary circuit.

A glycosylated proteolipid protein is common to CNS myelin of recent lungfish ( Ceratodiae, Lepidosirenidae)

1. 1. Myelin proteins from the CNS of recent lungfish ( Lepidosiren paradoxa, Protopterus dolloi, Neoceratodus forsteri) were separated and analysed by staining and immunoblotting. 2. 2. All species showed a glycosylated component (g-PLP) that cross-reacted with antibodies against tetrapod proteolipid protein (PLP), indicating phylogenetic relationships with amphibia. 3. 3. Actinopterygian IP or teleostean 36 k components were not detectable in lungfish CNS myelin. 4. 4. The identical size of g-PLPs from Lepidosiren and Protopterus ( M r = 29,000) underlines the close relationship of the Lepidosirenidae. The smaller size of g-PLP from the ceratodidan Neoceratodus forsteri ( M r = 27,500) pointed to an earlier diversion.