South American Lungfish Lepidosiren Research Articles

Physiological adaptations of red blood cells during aestivation in the south American lungfish Lepidosiren paradoxa

The South American lungfish Lepidosiren paradoxa inhabits areas with variable pluvial regimes. During aestivation (dormancy state observed in some species during dry seasons), the prolonged period of dryness imposes osmotic stress. We aimed to investigate the physiological and morphological adaptations of RBCs in Lepidosiren paradoxa during aestivation. Here, the lungfish were subjected to aestivation for 20 and 40 days and compared to a control group in an active period. The osmotic fragility, blood osmolality, and pH were measured. Blood smears were performed to assess morphological changes in the RBCs. Lepidosiren paradoxa presented lower hemolysis when compared with a teleost fish and tegu lizard. Hemolysis increased when the lungfish was subjected to 40 days of aestivation (from 6.04 % to 16.51 %; control vs 40-day aestivation). Hematocrit rose in both aestivation groups compared to the control (26.36 %, 41.36 % and 41 %, control, 20 and 40 days, respectively; p < 0.05) indicating changes in RBC volume and hydration status. Moreover, the results revealed increased osmolality in the 40-day aestivation group (244.4 vs 372.1 mmol/Kg; control vs 40-day aestivation). 40 days of aestivation led to a decrease in blood pH when compared to the control and 20-day aestivation groups. Both aestivation durations resulted in a reduction in the perimeter and cell diameters in at least one direction of the RBCs (24 % mean reduction in size from control to 40 days aestivation). These findings suggest that South American lungfish possess remarkable physiological and morphological adaptations in their RBCs during aestivation.

The biogeography of extant lungfishes traces the breakup of Gondwana

AbstractAimLungfishes are one of the two surviving clades from the once diverse grade of lobe‐finned fishes leading to tetrapods. This classic living fossil lineage, which is the living sister to four‐limbed terrestrial vertebrates, appeared approximately 425 million years ago and rapidly diversified. However, the evolution of lungfishes after their initial radiation is poorly understood, and whether their present distribution tracks ancient geographical change is a classic problem in biogeography.LocationGlobal.TaxonLungfishes (Dipnoi).MethodsHere, we combine mitogenomic, nuclear gene and fossil data to reconstruct the timing of lungfish diversification a Bayesian tip‐dating approach to quantitatively test hypotheses of lungfish historical biogeography and divergence times. We sample all major living and extinct lungfish lineages, including three of the four species of African lungfishes (Protopterusspp.), the Australian lungfishNeoceratodus forsteri, the South American lungfishLepidosiren paradoxaand 13 fossils representing extinct lineages from across the globe.ResultsOur results demonstrate that the divergences of the three major living lungfish clades closely recapitulate the stepwise fragmentation of the Gondwana during the Mesozoic. All of our model‐based biogeographical reconstructions support a Gondwanan vicariance model for the origins of the present distribution of lungfish lineages.ConclusionsIn turn, lungfishes provide an excellent example of how the integration of fossil data may drastically change support for historical biogeographical hypotheses previously discounted by molecular data and are one of the few living animal lineages that record incredibly ancient geographical changes in their phylogeny.

The South American lungfish Lepidosiren paradoxa as a new host for Trichodina quelenii.

Recently, the South American lungfish Lepidosiren paradoxa is being found inside aquaculture ponds, and even though there are a few studies on their parasite fauna, there is still much to be reported. Thus, the objective of this study is to report parasitism by trichodinids in L. paradoxa, as these ciliate protozoa are related to injuries and mortality in fish farming. The lungfish were collected from experimental tanks, had their tegument scraped and the resultant mucus was analyzed under an optical microscope for morphological and morphometrical analyses in Giemsa and silver nitrate stained slides. The species found was identified as Trichodina quelleni. This is the first report of this parasite in L. paradoxa, and the second report in cultivated fish in Brazil.

Globin E is a myoglobin-related, respiratory protein highly expressed in lungfish oocytes

Globins are a classical model system for the studies of protein evolution and function. Recent studies have shown that – besides the well-known haemoglobin and myoglobin – additional globin-types occur in vertebrates that serve different functions. Globin E (GbE) was originally identified as an eye-specific protein of birds that is distantly related to myoglobin. GbE is also present in turtles and the coelacanth but appeared to have been lost in other vertebrates. Here, we show that GbE additionally occurs in lungfish, the closest living relatives of the tetrapods. Each lungfish species harbours multiple (≥5) GbE gene copies. Surprisingly, GbE is exclusively and highly expressed in oocytes, with mRNA levels that exceed that of myoglobin in the heart. Thus, GbE is the first known oocyte-specific globin in vertebrates. No GbE transcripts were found in the ovary or egg transcriptomes of other vertebrates, suggesting a lungfish-specific function. Spectroscopic analysis and kinetic studies of recombinant GbE1 of the South American lungfish Lepidosiren paradoxa revealed a typical pentacoordinate globin with myoglobin-like O2-binding kinetics, indicating similar functions. Our findings suggest that the multiple copies of GbE evolved to enhance O2-supply in the developing embryo of lungfish, analogous to the embryonic and fetal haemoglobins of other vertebrates. In evolution, GbE must have changed its expression site from oocytes to eyes, or vice versa.

Epithelial crypts: A complex and enigmatic olfactory organ in African and South American lungfish (Lepidosireniformes, Dipnoi).

African lungfish (Protopterus) seem unique among osteognathostomes in possessing a potential vomeronasal organ homolog in form of accessory epithelial crypts within their nasal cavity. Many details regarding structural and functional properties of these crypts are still unexplored. In this study, we reinvestigate the issue and also present the first data on epithelial crypts in the South American lungfish Lepidosiren paradoxa. The nasal cavities of L. paradoxa and Protopterus annectens were studied using histology, scanning electron microscopy, and alcian blue and PAS staining. In both species, the epithelial crypts consist of a pseudostratified sensory epithelium and a monolayer of elongated glandular cells, in accordance with previously published data on Protopterus. In addition, we found a new second and anatomically distinct type of mucous cell within the duct leading into the crypt. These glandular duct cells are PAS positive, whereas the elongated glandular cells are stainable with alcian blue, suggesting distinct functions of their respective secretions. Furthermore, the two lungfish species show differently structured crypt sensory epithelia and external crypt morphology, with conspicuous bilaterally symmetrical stripes of ciliated cells in L. paradoxa. Taken together, our data suggest that stimulus transport into the crypts involves both ciliary movement and odorant binding mucus.

Effects of aerial hypoxia and temperature on pulmonary breathing pattern and gas exchange in the South American lungfish, Lepidosiren paradoxa

The South American lungfish Lepidosiren paradoxa is an obligatory air-breathing fish possessing well-developed bilateral lungs, and undergoing seasonal changes in its habitat, including temperature changes. In the present study we aimed to evaluate gas exchange and pulmonary breathing pattern in L. paradoxa at different temperatures (25 and 30°C) and different inspired O2 levels (21, 12, 10, and 7%). Normoxic breathing pattern consisted of isolated ventilatory cycles composed of an expiration followed by 2.4±0.2 buccal inspirations. Both expiratory and inspiratory tidal volumes reached a maximum of about 35mlkg−1, indicating that L. paradoxa is able to exchange nearly all of its lung air in a single ventilatory cycle. At both temperatures, hypoxia caused a significant increase in pulmonary ventilation (V̇E), mainly due to an increase in respiratory frequency. Durations of the ventilatory cycle and expiratory and inspiratory tidal volumes were not significantly affected by hypoxia. Expiratory time (but not inspiratory) was significantly shorter at 30°C and at all O2 levels. While a small change in oxygen consumption (V̇O2) could be noticed, the carbon dioxide release (V̇CO2, P=0.0003) and air convection requirement (V̇E/V̇O2, P=0.0001) were significantly affected by hypoxia (7% O2) at both temperatures, when compared to normoxia, and pulmonary diffusion capacity increased about four-fold due to hypoxic exposure. These data highlight important features of the respiratory system of L. paradoxa, capable of matching O2 demand and supply under different environmental change, as well as help to understand the evolution of air breathing in lungfish.

Combined ventilatory responses to aerial hypoxia and temperature in the South American lungfish Lepidosiren paradoxa

The control of pulmonary ventilation in South American lungfish Lepidosiren paradoxa is poorly understood. Interactions between temperature and hypoxia are particularly relevant due to large seasonal variations of its habitat. Therefore, we tested the hypothesis that the ventilatory responses to aerial hypoxia of Lepidosiren are highly dependent on ambient temperature. We used a pneumotachograph to measure pulmonary ventilation ( V E), tidal volume ( V T) and respiratory frequency ( f R) during normoxic (21% O 2) and hypoxic (12%, 10% and 7% O 2) conditions at two temperatures (25 and 35 °C). Blood gases, arterial PO 2 (PaO 2), arterial PCO 2 (PaCO 2) and arterial pH (pH a) were also evaluated. At 25 °C, V E increased significantly at 10% and 7% hypoxic levels when compared to the control value (21% O 2). At 35 °C, all hypoxic levels elicited a significant increase of V E relative to control values. V E is augmented mostly by increases of respiratory frequency ( f R), and there were significant interactions ( p<0.001) between aerial hypoxia and temperature. PaCO 2 increased from ∼22 mmHg (normoxic value at 25 °C) to ∼32 mmHg (normoxic value at 35 °C). Concomitantly, the pH a decreased from 7.51 (25 °C) to 7.38 (35 °C). Hypoxia-induced hyperventilation caused a reduction in PaCO 2 and an increase in pH a, which were more pronounced at 35 °C than at 25 °C, reflecting an increased hyperventilation under the high temperature. In conclusion, the magnitude of ventilatory response is highly temperature-dependent in L. paradoxa, which is important for an animal experiencing large seasonal variations.

Breathing pattern, pulmonary and intrabuccal gases of the South American lungfish Lepidosiren paradoxa

Genetic and physiological studies favor the lungfish as a probable sister group to the land vertebrates (Tetrapoda), and some fossil evidence is also available. As a physiological aspect, land vertebrates and lungfish possess both central and peripheral respiratory chemoreceptors. However, ventilatory pattern as well as buccal and pulmonary gases of the South American lungfish Lepidosiren paradoxa is not well studied. Therefore, our objective was to characterize respiratory pattern along with pulmonary and intrabuccal gases in the South American lungfish. Experiments were performed with five animals (n=5). Animals were submitted to surgical procedures to implant a cannula into pulmonary and buccal compartments for gases sampling (PO2 and PCO2). Pulmonary Ventilation (Ve) was measured using pneumotachograph method. The average Ve was 90.38 ± 24.59 ml ‐1 BTPS Kg h ‐1. The respiratory cycle of L. paradoxa begins with expiration followed by inspiration. The expiratory phase is marked by a series of small events. The PCO2 in the lung and buccal compartments had average values of 2.78 ± 0.3% and 3.14 ± 0.35% respectively. PO2 values were 13.78 ± 0.72% for pulmonary and 12.57 ± 1% for the buccal compartment. These results suggest a close similarity of breathing pattern between L. paradoxa and Amphibian group. Financial support: FAPESP and CNPq

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.

Components to the acid–base related ventilatory drives in the South American lungfish Lepidosiren paradoxa

Lungfish are closely related to terrestrial vertebrates (tetrapoda). Like tetrapods, the South American lungfish (Lepidosiren paradoxa) has central chemoreceptors involved in regulation of acid-base status. However, no data were available on peripheral CO(2)/[H(+)] receptors. Therefore, we tested the hypothesis that such receptors exist by measuring the ventilatory responses during a 5h exposure to combined aquatic/gas phase hypercarbia 7% (approximately 49 mmHg). Normocarbic control ventilation was 22 ml BTPS kg(-1)h(-1), and hypercarbia increased ventilation to 175 ml BTPS kg(-1)h(-1) at 5h. This procedure was repeated with the modification that normocarbic mock CSF (pH 7.45; P(CO2) = 20.7 mmHg) was applied to superfuse the cerebral ventricular system during the last 2h of the experiment. This served to eliminate the hypercarbic stimulus to the central chemoreceptors, while possible responses from peripheral chemoreceptors would remain intact. Peripheral receptors were detected, since ventilation became reduced to 62 ml BTPS kg(-1)h(-1) (P<0.05), which exceeds the initial normocarbic control ventilation (P<0.05). Based on this, the peripheral contribution accounted for 20% of the total response to hypercarbia, similar to the contribution of these receptors in man.

Morphometric Comparison of the Respiratory Organs in the South American LungfishLepidosiren 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.

Pulmonary Oxygen Diffusing Capacity of the South American LungfishLepidosiren 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.

Role of the sarcoplasmic reticulum in calcium dynamics of the ventricular myocardium of Lepidosiren paradoxa (Dipnoi) at different temperatures

This study analyzed the impact of changes in stimulation frequency on the inotropic responses of the South American lungfish Lepidosiren paradoxa ventricle strips. The effects of prolonged diastolic pauses and stepwise increases in stimulation frequency upon force development were determined at different temperatures (15°C, 25°C, and 35°C) and in response to 10 μM of ryanodine. A post-rest potentiation of force was observed only at 15°C, but ryanodine was able to depress twitch force at low stimulation frequencies, irrespective of temperature. Moreover, when in vivo stimulation frequencies were reached, the sarcoplasmic reticulum was relevant to the Ca 2+ management only at 15°C and 25°C. In contrast, the lack of effect of ryanodine at the in vivo frequencies at 35°C indicates that the excitation–contraction coupling depends exclusively on the transarcolemmal Ca 2+ influx. Therefore, this organelle seems to have a slower Ca 2+-cycling capacity, in spite of being anatomically well developed and potentially functional in all the temperatures tested.

The microhardness and fracture surface of the petrodentine of Lepidosiren (Dipnoi), and of other mineralised tissues

The South American lungfish Lepidosiren has toothplates bearing an extremely hard version of dentine: petrodentine. The hardness of this tissue, and its associated ordinary dentine, was compared with that of the enamel, dentine and cement of mammalian teeth, and also with that of other mammalian bony tissues. The hardnesses of petrodentine and dentine of Lepidosiren were found to be similar to those of enamel and dentine in other, mammalian, teeth. Furthermore, the anatomical arrangement of the Lepidosiren tissues was similar to that found in the incisors of rodents, and they presumably function in the same way to keep a sharp chisel edge at the tip of the tooth. Comparison of fracture surfaces of Lepidosiren petrodentine and that of rat incisor showed, however, that petrodentine does not have the refined, crack-stopping structure found in rat incisor enamel.

Adrenergic receptors, Na+/H+ exchange and volume regulation in lungfish erythrocytes.

Aestivation in African and South American lungfish (Protopterus and Lepidosiren, respectively) is associated with elevations of extracellular osmolarity. Osmotic shrinkage of Protopterus red blood cells (RBCs) caused a small but significant stimulation of the Na influx that was amiloride-sensitive. suggesting involvement of the Na+/H+ exchanger (NHE). The associated in vitro regulatory volume increase was insignificant within a time frame of 120 min, but the shrinkage-activated Na+ influx may be sufficient for slow regulatory volume increase during aestivation in vivo. Osmotic swelling of the RBCs induced an incomplete regulatory volume decrease that was statistically significant after 180 min. The RBCs of Protopterus were very large (mean cellular volume of 6939 +/- 294 microm3) and possessed 23,066 +/- 7,326 beta-adrenoceptors cell(-1) with a Kd value of 6.1 +/- 3.2 nM. The number of receptors per unit surface area of lungfish RBCs was calculated to be twice that of trout RBCs and 70% that of cod RBCs. There was, however, no adrenergic stimulation of the NHE in either Protopterus or Lepidosiren. Acidification of the extracellular medium also failed to activate the NHE.

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.

Effects of environmental hypercapnia on pulmonary ventilation of the South American lungfish

Aquatic hypercapnia at PCO2 of 55 mmHg significantly increased pulmonary ventilation in the South American lungfish Lepidosiren paradoxa, whereas no significant increases occurred when hypercapnia was applied to the gas phase with or without concomitant aquatic hypercapnia. On return from gas phase hypercapnia to inspiration of air there was a marked transient increase of ventilation. This post‐hypercapnic response is discussed in relation to the possible presence of upper airway or pulmonary CO2 receptors that inhibit pulmonary ventilation during hypercapnia. Post‐hypercapnic hyperpnea has been reported for various groups of reptiles and for anuran amphibians. The occurrence of post‐hypercapnic hyperpnea in Lepidosiren adds new information related to the evolution of respiratory regulation in vertebrates.

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).

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.

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Liver tumors were observed in 2 South American lungfish (Lepidosiren paradoxa) and in 1 African lungfish (Protopterus amphibius) kept in 3 different aquariums in Japan for 2-5 years. In the first case a single, large nodule (70 X 60 X 55 mm) in the liver had been noted externally as a swelling 1 year prior to death. In the second case 2 rounded nodules (25 X 15 X 15 mm and 15 X 10 X 10 mm) were incidentally found at autopsy. In the third case, which also demonstrated abdominal swelling for 1 year preceding death, 2 green nodules (50 X 50 X 40 mm and 17 X 17 X 17 mm) were found in the liver together with multiple metastatic lesions. Histologically, variation was evident from tumor to tumor, even within the same animal. The most common histologic type was typical trabecular hepatomas. Admixtures of glandular or papillary structures were noted in some tumors. A region of poorly differentiated hepatocellular carcinoma with spindle-shaped cells in a sheetlike arrangement was noted in one case. The cause of these tumors is unknown. Since liver tumors were discovered at relatively high incidence (3/14 autopsied cases, including 6 species), lungfish seem to be predisposed to the development of liver tumors.