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.     

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.     

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.     

Effect of acute temperature transitions on chronotropic and inotropic responses of the South American lungfish Lepidosiren paradoxa

The in vivo and in vitro cardiac responses of Lepidosiren paradoxa were analyzed during temperature variations from 25°C (acclimation temperature) to 15°C and 35°C, and subsequent return to 25°C. Chronotropic (heart frequency) and inotropic (twitch force) responses varied directly with temperature, decreasing from 25°C to 15°C and increasing from 25°C to 35°C. However, time to peak tension (TPT) and time to half relaxation (THR) showed an inverse tendency. The results indicate that the myocardium of L. paradoxa responds more appropriately to acute elevations in temperature, which results in an increased cardiac performance due to both positive chronotropism and inotropism, in spite of the temperature-induced curtailment of TPT and THR.     

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.     

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.     

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.     

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.     

Pulmonary oxygen diffusing capacity of the South American lungfish Lepidosiren paradoxa: physiological values by the Bohr method

Lungfish (Dipnoi) may represent the sister group to all land vertebrates and are therefore important for reconstructing the conquest of land by tetrapods. We determined venous and arterial blood gases, pulmonary O(2) uptake, and the form of the hemoglobin-O(2) dissociation curves in the South American lungfish Lepidosiren paradoxa. Measurements were performed at 25 degrees and 35 degrees C. Based on this information, we calculated its pulmonary O(2) diffusing capacity (D(L)O(2)), using the Bohr integration procedure. D(L)O(2) increased with temperature to reach about 0.04 mL stpd kg(-1) min(-1) mmHg(-1) at 35 degrees C. This value represents about 40% of the morphometric diffusing capacity and is similar to physiological values in some amphibians and reptiles.     

Equilibria and kinetic of oxygen and carbon monoxide binding to the haemoglobin of the South American lungfish,Lepidosiren paradoxa

• 1. The haemoglobin of the South American lungfishLepidosiren paradoxa has a single component.
• 2. The equilibria of this respiratory protein with oxygen have been investigated both in the blood and with the purified haemoglobin. There is a substantial, normal, alkaline Bohr effect and marked sensitivity to organic phosphates in the haemoglobin solutions.
• 3. Studies on the pH dependence of the kinetics of oxygen dissociation can be interpreted in terms of a normal Bohr effect.
• 4. The kinetics of combination of carbon monoxide have an unusual pH dependence.
• 5. These findings are discussed in terms of the two-state model of Monodet al. (1965)

     

Acid-base regulation in the South American lungfish Lepidosiren paradoxa: effects of prolonged hypercarbia on blood gases and pulmonary ventilation

The South American lungfish (Lepidosiren paradoxa) has well-developed lungs and highly reduced gills. To evaluate acid-base regulation, we applied hypercarbia while blood gases and pulmonary ventilation were measured for up to 48 h. Dorsal aortic blood was analyzed, and pulmonary ventilation was measured by pneumotachography. Two protocols were used: (1) normocarbia (control) followed by aquatic hypercarbia (7% CO2 approximately 49 mmHg), gas phase normocarbic; and (2) normocarbia (control) followed by combined aquatic/gas phase hypercarbia (7% CO2). Normocarbic values were pHa~7.5, Paco2 approximately 17 mmHg, and [HCO-3]pl approximately 22 mM. For protocol 1, the first hour of exposure increased Paco2 from 17.0 to 37.4 mmHg, and pHa fell to 7.21 and remained there for the rest of the experiment. At 3 h, pulmonary ventilation reached sixfold the normocarbic value but then decreased. For protocol 2, combined gas phase/water hypercarbia had a large effect on acid-base status. Thus, Paco2 increased gradually to 74 mmHg (pHa=7.15) at 48 h. At 3 h, ventilation reached a sixfold increase relative to normocarbic control but then rose further to a 60-fold peak at 6 h, followed by a gradual decline. As in some salamanders and air-breathing teleosts, there was no evidence of active extracellular modulation bicarbonate.     

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 and function of the feeding apparatus of the lungfish, Lepidosiren paradoxa (Dipnoi)

The feeding mechanism of the South American lungfish, Lepidosiren paradoxa retains many primitive teleostome characteristics. In particular, the process of initial prey capture shares four salient functional features with other primitive vertebrates: 1) prey capture by suction feeding, 2) cranial elevation at the cranio-vertebral joint during the mouth opening phase of the strike, 3) the hyoid apparatus plays a major role in mediating expansion of the oral cavity and is one biomechanical pathway involved in depressing the mandible, and 4) peak hyoid excursion occurs after maximum gape is achieved. Lepidosiren also possesses four key morphological and functional specializations of the feeding mechanism: 1) tooth plates, 2) an enlarged cranial rib serving as a site for the origin of muscles depressing the hyoid apparatus, 3) a depressor mandibulae muscle, apparently not homologous to that of amphibians, and 4) a complex sequence of manipulation and chewing of prey in the oral cavity prior to swallowing. The depressor mandibulae is always active during mouth opening, in contrast to some previous suggestions. Chewing cycles include alternating adduction and transport phases. Between each adduction, food may be transported in or out of the buccal cavity to position it between the tooth plates. The depressor mandibulae muscle is active in a double-burst pattern during chewing, with the larger second burst serving to open the mouth during prey transport. Swallowing is characterized by prolonged activity in the hyoid constrictor musculature and the geniothoracicus. Lepidosiren uses hydraulic transport achieved by movements of the hyoid apparatus to position prey within the oral cavity. This function is analogous to that of the tongue in many tetrapods.     

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.