Morphometric comparison of the respiratory organs in the South American lungfish Lepidosiren paradoxa (Dipnoi)

In light of the relationship of lungfish to the origin of tetrapods, information on the respiratory biology of lungfish can give insight into the functional morphological and physiological prerequisites for the conquest of land by the first tetrapods. Stereological methods were employed in order to determine the respiratory surface area and thickness of the water-blood barrier or air-blood of the gills, lungs, and skin, respectively, of the South American lungfish Lepidosiren paradoxa. The morphometric diffusing capacity was then determined by multiplying by the appropriate Krogh diffusion constants (K). Our results indicate a total diffusing capacity of all respiratory organs of 0.11 mL min(-1) mmHg(-1) kg(-1), which is more than twice the value of the physiological diffusion capacity (approximately 0.04 mL min(-1) mmHg(-1) kg(-1)). Of this, 99.15% lies in the lungs, 0.85% in the skin, and only 0.0013% in the gills. Since K for CO(2) is 20-25 times greater than for O(2), diffusing capacity of CO(2) through the skin is potentially important. That of the gills, however, is negligible, raising the question as to their function. Our results indicate that the morphological prerequisites for terrestrial survival with regard to supporting aerobic metabolism already existed in the lungfish.     

Blood and inflammatory cells of the lungfish Lepidosiren paradoxa

A special interest exists concerning lungfish because they may possess characteristics of the common ancestor of land vertebrates. However, little is known about their blood and inflammatory cells; thus the fine structure, cytochemistry and differential cell counts of coelomic exudate and blood leucocytes were studied in Lepidosiren paradoxa. Blood smear analyses revealed erythrocytes, lymphocytes, monocytes, polymorphonuclear agranulocytes, thrombocytes and three different granulocytes. Blood monocytes and lymphocytes had typical vertebrate morphology. Thrombocytes had large vacuoles filled with a myelin rich structure. The polymorphonuclear agranulocyte had a nucleus morphologically similar to the human neutrophil with no apparent granules. Types I and II granulocytes had eosinophilic granules. Type I granulocytes had round or elongated granules heterogeneous in size, while type II had granules with an electron dense core. Type III granulocyte had many basophilic granules. The order of frequency was: type I granulocyte, followed by lymphocyte, type II granulocyte, monocyte, polymorphonuclear agranulocyte and type III granulocyte. Peroxidase localized mainly at the periphery of the granules from type II granulocytes, while no peroxidase expression was detected in type I granulocytes. Alkaline phosphatase was localized in the granules of type II granulocyte and acid phosphatase cytochemistry also labelled a few vacuoles of polymorphonuclear agranulocyte. About 85% of the coelomic inflammatory exudate cell population was type II granulocyte, 10% polymorphonuclear agranulocyte and 5% macrophages as judged by the nucleus and granule morphology. These results indicate that this lungfish utilises type II granulocytes as its main inflammatory granulocytes and that the polymorphonuclear agranulocyte may also be involved in the inflammatory response. The other two granulocytes appear similar to the mammalian eosinophil and basophil. In summary, this lungfish appears to possess the typical inflammatory granulocytes of teleosts, however, further functional studies are necessary to better understand the polymorphonuclear agranulocyte.     

Eimeria lepidosirenis n.sp. (Apicomplexa:Eimeriidae) of the South American lungfish Lepidosiren paradoxa (Osteichthyes:Dipnoi) from Amazonian Brazil

The mature oocysts of Eimeria lepidosirenis n.sp. are described in faeces removed from the lower region of the intestine of a single specimen of the South American lungfish Lepidosiren paradoxa, from Belém, state of Pará, Amazonian Brazil. Oocysts with endogenous sporulation: spherical to slightly subspherical, 30.8 x 30.3 microm (28.1 x 25.9 -33.3 x 31.8), shape-index (ratio length/width) 1.0, n = 25. Oocyst wall a very thin, single layer approximately 0.74 microm thick, smooth, colourless, with no micropyle and rapidly breaking down to release the sporocysts. Oocyst residuum a bulky ovoid to spherical mass of approximately 20.0 x 15 microm, composed of fine granules and larger globules and enclosed by a very fine membrane: no polar bodies seen. Sporocysts 15.5 x 9.0 microm (14.5 x 8.0 16.0 x 9.0), shape index 1.7 (1.6-1.8), n = 30, ovoid, with one extremity rather pointed and with a very delicate Stieda body but no sub-Stieda body: sporocyst wall a single extremely thin layer with no valves. Sporocyst residuum a spherical to ovoid mass of approximately 5.0 x 4.0 microm, composed of fine granules and small globules and enclosed by a very fine membrane. Sporozoites strongly recurved at their ends and apparently with only a single refractile body. Site of development in the host uncertain: no evidence of endogenous stages was found in fresh scrapings and stained smears of the intestinal epithelium.     

Ultrastructure of the integumental melanophores of the South American lungfish (Lepidosiren paradoxa) and the African lungfish (Protopterus sp.)

The integumental melanophores of two genera of lungfish, Lepidosiren paradoxa and Protopterus sp. were examined by light and electron microscopy. Both species possess both epidermal and dermal melanophores with fine structural characteristics basically similar to those of other vertebrates. The epidermal melanophores of both species are located in the intermediate epidermis, and possess thin perikarya containing round nuclei, and slender dendrites extending into the nearby intercellular spaces. The dermal melanophores occur immediately beneath the basement membrane, and possess flat perikarya and dendrites running horizontally between the collagen fibers of the dermis. The integument of both species does not possess an epidermal melanin unit or a dermal chromatophore unit. As in other vertebrates, each melanophore contains numerous oval, electron-opaque melanosomes, relatively large mitochondria, vacuolar endoplasmic reticula, and groups of RNP particles. Although micro filaments running randomly between other organelles occur regularly, microtubules were not demonstrated. Premelanosomes at various stages of differentiation were best illustrated in the dermal melanophores of Protopterus, and it is concluded from the observation of their fine structure that the morphological development of lungfish melanosomes closely parallels that of higher vertebrates. On the basis of melanophore morphology, Lepidosiren and Protopterus appear to be more closely related to each other than to Neoceratodus.     

Histological and enzymatic studies on the nephron of the lungfish,Lepidosiren paradoxa

Nephron of South American lungfish was examined historically and enzyme-histochemically. Cells of the first and second proximal segments exhibited poor interdigitation forming narrow intercellular spaces, whereas the distal segment consisted of deeply interdigitated cells with wide intercellular spaces. Activities of aerobic enzymes (malate, isocitrate, NADH, and β-hydroxybutyrate dehydrogenases), Na-K-ATPase, and carbonic anhydrase were mostly detected in the distal segment. In contrast, hexokinase activity was mostly seen in the 1st and 2nd proximal segments. In the collecting tubule, two types of cells were distinguished by their histological and enzyme-histochemical features. One type showed deep interdigitation and intense carbonic anhydrase activity. The other did not have heavy interdigitation and carbonic anhydrase activity. However, both cell type exhibited intense activities of aerobic enzymes. These structures and enzyme distributions in the lungfish nephron indicate that the lungfish is more specialized in nephron than teleosteans and elasmobranchs, though, slightly similar to the latter.     

Primary structure, biochemical and physiological aspects of hemoglobin from South American lungfish (Lepidosiren paradoxus, Dipnoi)

The South American Lungfish has only one hemoglobin component. The complete amino-acid sequence of this hemoglobin is presented. A large quantity of carbonate dehydratase from the lungfish erythrocytes was also isolated. The carboxymethylated chains, obtained by separation of globin on DEAE-Sephacel, were submitted to tryptic digestion and chemical cleavage. The isolation of tryptic peptides was achieved either by Dowex-50 chromatography or by high performance liquid chromatography. The alignment of peptides was performed by homology with the previously established sequences of the carp and goldfish hemoglobins. The overlapping peptides confirmed this sequence. The alpha chains have 143 residues, the beta chains 147. The relation between the primary structure and the physiological properties of lungfish hemoglobin are discussed.     

Structural and biochemical characterization of the lungfish (Lepidosiren paradoxa) liver basic fatty acid binding protein

Only one fatty acid-binding protein (FABP) from the liver of the lungfish (Lepidosiren paradoxa) was isolated and characterized. The sequence comparison of lungfish FABP with that of the known members of the liver FABP (L-FABP) and liver basic FABP (Lb-FABP) subfamilies indicates that it is more closely related to chicken, iguana, frog, axolotl, catfish, and shark Lb-FABPs than to mammalian and axolotl L-FABPs. Lungfish liver expression of this single Lb-FABP contrasts with the other fish studied so far which coexpress an Lb-FABP with heart-adipocyte and/or intestinal FABP types. The lungfish liver FABP expression pattern resembles that of tetrapods, which only expresses liver type FABPs. Lungfish Lb-FABP is one of the two FABPs reported to have a disulfide bridge. The molecular modeling of lungfish Lb-FABP predicts that nine of the conserved residues of Lb-FABPs are oriented toward the binding cavity, thus suggesting they are related to the protein binding characteristics.     

Morphology and growth of lepidosirenid lungfish tooth plates (Pisces: Dipnoi)

The structure of the tooth plates of Protopterus and Lepidosiren was investigated to determine the causes and consequences of postlarval shape change. During growth, the basal area of the tooth plates increases, some cusps become more prominent, and shearing surfaces are sharpened. The jaw articulation restricts the range of movements of the lower jaw, and causes the tooth plates to occlude precisely; the resulting wear patterns are regular. The tooth plates are composed of enamel, trabecular dentine, and petrodentine. A petrodentine column forms the core of a tooth plate; it is flanked by trabecular dentine. Microhardness measurements show that trabecular dentine is comparable in hardness to mammalian dentine, whereas the petrodentine is comparable to enamel. The location and differential wear of these tissues produce the prominent cusps and self-sharpened blades of the adult tooth plates.     

Immunocytochemical demonstration of mammalian lutropin-like material in the pituitary of the lungfish,Lepidosiren paradoxa

Summary The presence of lutropin (LH)-like material in the pituitary gland of the South-American lungfish, Lepidosiren paradoxa, has been demonstrated by means of the unlabeled antibody-enzyme method, by use of rabbit antiovine LH as first antibody. The LH-like substance was revealed in a single PAS-positive cell type primarily located in the anterior part of the distal lobe. Dot blot tests as well as conventional liquid-phase absorption experiments indicate that the anti-ovine LH antibodies possess specificity against the oLH/gb subunit. These observations indicate that dipnoans (Lepidosiren) share a number of antigenic determinants with those of mammalian LH/gb and support the concept that mammalian LH/gb, or part of it, was established early in evolution. The exact nature and physiological function of the substance detected remains to be defined.     

The mechanism of breathing in the South Asmerican lungfish, Lepidosiren paradoxa; radiological study

In filling the lungs by means of the buccal force-pump, air drawn into the buccal cavity by the lowering of the floor of the mouth is forced into the lungs when the mouth is firmly closed and the buccal floor raised. This mechanism depends on the ability of the fish to prevent air being forced out of the mouth through the lips and it is shown that the tongue is pressed against the roof of the mouth to form a seal. Radiologically it has been shown that the hyoid apparatus (ceratohyal of some authors) plays a large part in the movements necessary to draw air into the mouth, seal it in and force it into the lungs. Another skeletal element involved is the pectoral girdle which makes considerable movements in association with the filling of the lungs. Expiration of the air from the lungs, which as shown by radiographs may be remarkably complete, is brought about by the elasticity of the lung tissue.     

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.     

Relationship between cerebro-spinal fluid pH and pulmonary ventilation of the South American lungfish, Lepidosiren paradoxa (Fitz.)

The respiratory control in land vertebrates (Tetrapoda) is mainly linked to regulation of acid-base status, which involves peripheral and central chemoreceptors. The lungfish (Dipnoi) might constitute the sister group of all land vertebrates (Tetrapoda) and possess a combination of real lungs and reduced gills. In this context, we evaluated the possible presence of central respiratory chemoreceptors in the South American Lungfish, Lepidosiren paradoxa. Pulmonary ventilation and respiratory frequency increased significantly with reductions of CSF pH by means of mock CSF solutions. This suggests that Lepidosiren possess central acid-base receptors.     

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.     

Parathyroid hormone preparations, salmon calcitonin, and urine flow in the South American lungfish, Lepidosiren paradoxa.

In the South American lungfish, Lepidosiren paradoxa, injections of salmon calcitonin or bovine parathyroid extract have no effect on serum calcium levels. However, salmon calcitonin elicits a distinct diuresis. Crude or purified parathyroid extract or synthetic parathyroid hormone (1-34 fraction) produce a distinct antidiuresis.