Ultrastructure of the integumental melanophores of the Australian lungfish,Neoceratodus forsteri

The integumental melanophores of Australina lungfish, Neoceratodus forsteri, were examined by light and electron microscopy and found to possess essentially the same structural characteristics observed in other vertebrates. The epidermal melanophores are located in the intermediate epidermis and possess round perikarya and slender dendrites extending into nearby intercellular spaces. The dermal melanophores are found immediately below the basement membrane as well as in the deeper dermis. These cells possess flattened nuclei and dendrites running parallel to the basement membrane. Each melanophore contains numerous oval or elliptical, intensely electron-dense melanosomes, relatively large mitochondria, systems of vacuolar endoplasmic reticulum, groups of free RNP particles, and some microfilaments. Only a few, short microtubules could be demonstrated in the perinuclear cytoplasm of the dermal melanophore, while a relatively large number of late premelanosomes are found both in perikarya and dendritic processes of epidermal melanophores. These premelanosomes exhibit a particulate internal structure in cross section. Both melanosomes and premelanosomes occur singly in the cytoplasm of epidermal cells, thereby confirming the existence of the epidermal melanin unit in the lowest vertebrates thus far examined electron microscopically.     

Structure of Intestine, Pancreas, and Spleen of the Australian Lungfish, Neoceratodus forsteri (Krefft)

The structure of the intestine and its adnexa in the Australian lungfish Neoceratodus forsteri (Krefft), is described from four adult specimens and compared with that of other lungfish. In all lungfish genera the intestine is thick and straight and contains a complicated spiral valve; a pyloric fold separates the foregut from the midgut. In Neoceratodus the coiling of the intestinal lumen begins in the prepyloric or gastric region, unlike in other vertebrates with a spiral valve, where it begins behind the pylorus. The spiral valve of Neoceratodus begins as a deep groove on the right side of the foregut, just behind the glottis. Such a prepyloric groove is present but poorly developed also in the lungfishes Protopterus and Lepidosiren and contains a prepyloric spleen in all genera. A separate postpyloric spleen is situated in the free margin of the spiral valve, i.e. in the axis of the intestine. The spleen has a heavily pigmented cortex containing large amounts of iron. The pancreas, buried in the spiral valve in front of the pylorus in Neoceratodus , contains numerous islets of Langerhans, similar to those of tetrapods. This is unexpected because the islets of Protopterus , described by Gabe in 1969, are more like the large, encapsulated "Brockmann bodies" of teleosts.     

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.     

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.     

Homeobox genes in the Australian lungfish, Neoceratodus forsteri.

The aim of the present study was to determine whether the postulated gnathostome duplication from four to eight Hox clusters occurred before or after the split between the actinopterygian and sarcopterygian fish by characterizing Hox genes from the sarcopterygian lungfish, Neoceratodus forsteri. Since lungfish have extremely large genomes, we took the approach of extracting pure high molecular weight (MW) genomic DNA to act as a template for polymerase chain reaction (PCR) of the conserved homeobox domain of the highly conserved Hox genes. The 21 clones thus obtained were sequenced and translated in a BLASTX protein database search to designate Hox gene identity. Fourteen of the clones were from Hox genes, two were Hox pseudogenes, four were Gbx genes, and one most closely resembled the homeobox gene, insulin upstream factor 1. The Hox genes identified were from all four tetrapod clusters A, B, C, and D, confirming their presence in lungfish, and there is no evidence to suggest more than these four functional Hox clusters, as is the case in teleosts. A comparison of Hox group 13 amino acid sequences of lungfish, zebrafish, and mouse provides firm evidence that the expansion of Hox clusters, as seen in zebrafish, occurred after separation of the actinopterygian and sarcopterygian lineages. J. Exp. Zool. (Mol. Dev. Evol.) 285:140-145, 1999.     

Development of the Snout of the Australian Lungfish Neoceratodus forsteri (Krefft, 1870), with Special Reference to Cranial Nerves

Abstract The anatomy and embryology of the dipnoan snout and olfactory organ play a major role in the discussion about the phylogenetic position of Dipnoi and the question of ancestry of Tetrapoda. This is primarily due to the fact that an internal nostril is regarded as an important preadaptive organ of the ancestors of tetrapods. Two conflicting scenarios of phylogenetic change were proposed in favour of different hypotheses of relationship. One emphasizes the similarities between Dipnoi and Chondrichthyes concerning this complex of characters. The other supports the idea of a close relationship of Dipnoi and Tetrapoda and a common origin of a ‘choana’ from the posterior external nostril of fishes. Accordingly, there is need for a detailed embryological and anatomical study which could help to clarify homologies and the basis of character evaluation. This, in the first place, concentrates on Neoceratodus forsteri. A plate reconstruction of the larval head provides many new insights which are important for comparison with the lepidosirenid lungfishes Protopterus and Lepidosiren and with other vertebrate taxa. The value of the peripheral nervous system as a topographical reference is critically reviewed. The results support the hypothesis that lungfish form a ‘primitive’ teleostomate group, closely adjoining Chondrichthyes in many characteristics of the snout formation. The relations between cranial nerves in the snout and the developing nasal sac, however, do not support the conclusion that the recent selachian condition is the exceptional ancestral character state of the lungfish snout. Dipnoi lack a ‘choana’, but nevertheless a closer relationship to tetrapods is not excluded. A vestigial and transitory naso-buccal connection in larvae of Neoceratodus might indicate such common ancestry, but has a completely different functional significance among fishes. It is shared with several plagiostomes and is, therefore, probably not useful as a synapomorphy of Dipnoi and Tetrapoda.     

Spawning activity of the Australian lungfish Neoceratodus forsteri in an impoundment

This study assessed the spawning activity of the threatened Australian lungfish Neoceratodus forsteri by measuring egg densities within the artificial habitat of a large impoundment (Lake Wivenhoe, Australia). Eggs were sampled (August to November 2009) from multiple locations across the impoundment, but occurred at highest densities in water shallower than 40 cm along shorelines with a dense cover of submerged terrestrial vegetation. The numbers of eggs declined over the study period and all samples were dominated by early developmental stages and high proportions of unviable eggs. The quality of the littoral spawning habitats declined over the study as flooded terrestrial grasses decomposed and filamentous algae coverage increased. Water temperatures at the spawning site exhibited extreme variations, ranging over 20·4° C in water shallower than 5 cm. Dissolved oxygen concentrations regularly declined to <1 mg l^?1 at 40 and 80 cm water depth. Spawning habitats utilised by N. forsteri within impoundments expose embryos to increased risk of desiccation or excessive submergence through water‐level variations, and extremes in temperature and dissolved oxygen concentration that present numerous challenges for successful spawning and recruitment of N. forsteri in large impoundment environments.     

True enamel covering in teeth of the Australian lungfish Neoceratodus forsteri

Lungfish are a unique order of sarcopterygian fish cleidographically positioned between tetrapods and fish. An uninterrupted 400-million-year-old fossil record has documented lungfish skeletal elements to remain virtually unchanged since the Early Devonian. In the current study we investigated the enamel layer of lungfish teeth in order to determine whether there was evidence for higher vertebrate "true" enamel in the Australian lungfish. Juvenile lungfish from the Brisbane River were processed for light and electron microscopy and analyzed for parameters indicative of true enamel formation. Using anti-amelogenin primary antibodies for immunodetection and Western blots, enamel protein epitopes were detected in developing lungfish teeth. Using transmission electron microscopy and electron diffraction analysis, long and parallel-oriented hydroxyapatite crystals were observed in lungfish outer tooth coverings. Our findings indicate that Australian lungfish teeth are covered by a layer of true enamel. Based on the lungfish fossil record we conclude that features of true enamel formation may be as old as 400 million years. Based on taxonomic classification we confirm that true enamel is found not only in tetrapods but also in the sarcopterygian clade of the Gnathostomata.     

Extremely Low Microsatellite Diversity but Distinct Population Structure in a Long-Lived Threatened Species,the Australian Lungfish Neoceratodus forsteri (Dipnoi)

The Australian lungfish is a unique living representative of an ancient dipnoan lineage, listed as ‘vulnerable’ to extinction under Australia’s Environment Protection and Biodiversity Conservation Act 1999. Historical accounts indicate this species occurred naturally in two adjacent river systems in Australia, the Burnett and Mary. Current day populations in other rivers are thought to have arisen by translocation from these source populations. Early genetic work detected very little variation and so had limited power to answer questions relevant for management including how genetic variation is partitioned within and among sub-populations. In this study, we use newly developed microsatellite markers to examine samples from the Burnett and Mary Rivers, as well as from two populations thought to be of translocated origin, Brisbane and North Pine. We test whether there is significant genetic structure among and within river drainages; assign putatively translocated populations to potential source populations; and estimate effective population sizes. Eleven polymorphic microsatellite loci genotyped in 218 individuals gave an average within-population heterozygosity of 0.39 which is low relative to other threatened taxa and for freshwater fishes in general. Based on F _ST values (average over loci = 0.11) and STRUCTURE analyses, we identify three distinct populations in the natural range, one in the Burnett and two distinct populations in the Mary. These analyses also support the hypothesis that the Mary River is the likely source of translocated populations in the Brisbane and North Pine rivers, which agrees with historical published records of a translocation event giving rise to these populations. We were unable to obtain bounded estimates of effective population size, as we have too few genotype combinations, although point estimates were low, ranging from 29 - 129. We recommend that, in order to preserve any local adaptation in the three distinct populations that they be managed separately.     

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.     

Cranial neural crest cell migration in the Australian lungfish, Neoceratodus forsteri

SUMMARY A crucial role for the cranial neural crest in head development has been established for both actinopterygian fishes and tetrapods. It has been claimed, however, that the neural crest is unimportant for head development in the Australian lungfish ( Neoceratodus forsteri   ), a member of the group (Dipnoi) which is commonly considered to be the living sister group of the tetrapods. In the present study, we used scanning electron microscopy to study cranial neural crest development in the Australian lungfish. Our results, contrary to those of Kemp, show that cranial neural crest cells do emerge and migrate in the Australian lungfish in the same way as in other vertebrates, forming mandibular, hyoid, and branchial streams. The major difference is in the timing of the onset of cranial neural crest migration. It is delayed in the Australian lungfish in comparison with their living sister group the Lissamphibia. Furthermore, the delay in timing between the emergence of the hyoid and branchial crest streams is very long, indicating a steeper anterior-posterior gradient than in amphibians. We are now extending our work on lungfish head development to include experimental studies (ablation of selected streams of neural crest cells) and fate mapping (using fluoresent tracer dyes such as DiI) to document the normal fate as well as the role in head patterning of the cranial neural crest in the Australian lungfish.     

Skin and Blood Vessels of the Snout of the Australian Lungfish,Neoceratodus forsteri,and their Significance for Interpreting the Cosmine of Devonian Lungfishes

Improved structural and functional interpretations regarding the dermal skeleton of Paleozoic lungfishes (Dipnoi) can be derived from a direct comparison of Recent and fossil tissues. In particular, skin from the snout of adult Australian lungfish (Neoceratodus forsteri) contains horizontal plexuses and vertical capillary loops which resemble in structure, size and density components of the cosmine layer in such Paleozoic lungfishes as Dipterus valenciennesi and Chirodipterus australis. In addition to these dermal papillae, the skin of the snout also contains ampullary electroreceptors, goblet cells, compound mucus glands, and terminal branches and openings of the mechanoreceptive lateral line system. Pore canal systems of fossil lungfishes previously have been interpreted as housing electroreceptors or other cutaneous sense organs of the lateral line system. In contrast, we regard pore canal systems as evidence of a complex cutaneous vasculature involved in the deposition of mineralized tissues. Prevailing ideas on the structure and biological role of cosmine are reinterpreted, including the theory that electroreceptors played an important part in the origin of the dermal skeleton.     

Lungfish albumin is more similar to tetrapod than to teleost albumins: purification and characterisation of albumin from the Australian lungfish, Neoceratodus forsteri

Lobe-finned fish, particularly lungfish, are thought of as the closest extant relatives to tetrapods. Albumin, the major vertebrate plasma protein, has been well studied in tetrapods, but there exists no comparative study of the presence and characteristics of albumin in lobe-finned fish versus other vertebrates. There is a controversy over the presence of albumin in fish, although it is present in salmonids and lamprey. The presence of albumin in lungfish has also recently been documented. We identified albumin in plasma of the Australian lungfish, Neoceratodus forsteri, using a combination of agarose gel electrophoresis, [(14)C]palmitic acid binding and SDS-PAGE. Lungfish albumin was purified using DEAE-ion exchange chromatography, and has a mass of 67 kDa, is present at approximately 8 g/L in plasma and like other fish albumins, does not bind nickel. However, like tetrapod albumins, it is not glycosylated. N-terminal and internal peptide sequencing generated 101 amino acids of sequence, which showed a high degree of identity with tetrapod albumins. Despite the similarity in sequence but congruent with the evolutionary distances separating them, lungfish albumin did not cross-react with anti-chicken or anti-tuatara A albumin antisera. Lungfish albumin has characteristics more akin with tetrapod albumin and less like those of other fish.     

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.     

Keratinization of the epidermis of the Australian lungfish Neoceratodus forsteri (dipnoi)

The differentiation of the epidermis in sarcopterigian fish may reveal some trend of keratinization followed by amphibian ancestors to adapt their epidermis to land. Therefore, the process of keratinization of the epidermis of the Australian lungfish Neoceratodus forsteri was studied by histochemistry, electron microscopy, and keratin immunocytochemistry. The epidermis is tri-stratified in a 2-3-month-old tadpole but becomes 6-8 stratified in young adults. Keratin filaments increase from basal to external cells where loose tonofilament bundles are present. This is shown also by the comparison of positivity to sulfhydryl groups and increasing immunoreactivity to alpha-keratins in more external layers of the epidermis. Two broad-spectrum anti alpha-keratin monoclonal antibodies (AE1 and AE3) stain all epidermal layers as they do in actinopterigian fish. In the adult epidermis, but not in that of the larva, the AE2 antibody (a marker of keratinization in mammalian epidermis) often immunolabels more heavily the external keratinized layers where sulfhydryl groups are more abundant. Mucous granules are numerous and concentrate on the external surface of the epidermis to be discharged and contribute to cuticle formation. Keratin is therefore embedded in a mucus matrix, but neither compact keratin masses nor cell corneous envelope were seen in external cells. It is not known whether specific matrix proteins are associated with mucus. There was no immunolocalization of the keratin-associated proteins, filaggrin and loricrin, which suggests that the epidermis of this species lacks the matrix and cell corneus envelope proteins characteristic of that of amniotes. In conclusion, while specific keratins (AE2 positive) are probably produced in the uppermost layers as in amphibian epidermis, no interkeratin, matrix proteins seem to be present in external keratinocytes of the lungfish other than mucus.     

Characterization of the hemoglobins of the Australian lungfish Neoceratodus forsteri (Krefft)

We examined for the first time the hemoglobin components of the blood of the Australian lungfish, Neoceratodus forsteri and their functional responses to pH and the allosteric modulators adenosine triphosphate (ATP), guanosine triphosphate (GTP), 2,3-bisphosphoglyceric acid (BPG) and inositol hexaphosphate (IHP) at 25 degrees C. Lysates prepared from stripped, unfractionated hemolysate produced sigmoidal oxygen equilibrium curves with high oxygen affinity (oxygen partial pressure required for 50% hemoglobin saturation, p(50)=5.3 mmHg) and a Hill coefficient of 1.9 at pH 7.5. p(50) was 8.3 and 4.5 mmHg at pH 6 and 8, respectively, which corresponded to a modest Bohr coefficient (Delta log p(50)/Delta pH) of -0.13. GTP increased the pH sensitivity of oxygen binding more than ATP, such that the Bohr coefficient was -0.77 in the presence of 2 mmol L(-1) GTP. GTP was the most potent regulator of hemoglobin affinity, with concentrations of 5 mmol L(-1) causing an increase in p(50) from 5 to 19 mm Hg at pH 7.5, while the order of potency of the other phosphates was IHP>ATP>BPG. Three hemoglobin isoforms were present and each contained both alpha and beta chains with distinct molecular weights. Oxygen affinity and pH-dependence of isoforms I and II were essentially identical, while isoform III had a lower affinity and increased pH-dependence. The functional properties of the hemoglobin system of Neoceratodus appeared consistent with an active aquatic breather adapted for periodic hypoxic episodes.     

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     

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

Tooth plate formation in the Queensland lungfish, Neoceratodus forsteri, Krefft begins with simple groups of isolated cusps, three in each tooth plate. The cusps fuse in ridges radiating from a point situated posterolingually. During growth, cusps are added to the labial ends of the ridges, and more ridges are added posteriorly, giving a total of seven in each tooth plate. Each tooth grows in thickness by the addition of layers of material, in line with the new cusps, beneath the tooth plate. The tooth plate grows outwards and is resorbed from the inner angle at the same time. The crushing surface is formed by the growth of cusps between the ridges. Angles between the ridges become progressively smaller, and angles between more posterior ridges are consistently less than between more anterior ridges. Similar but less pronounced changes in angles between ridges occur in a fossil genus, Sagenodus inaequalis, examined for comparison.
Vomerine teeth grow in the same way, by fusion of isolated cusps and the addition of new cusps to one end (labial) of the tooth plate. Layers of material are also added beneath the tooth plate. The vomerine tooth plates are initially low-based with long cusps but develop into high-based low cusped incisiform tooth plates in fully grown adults.
The labial dentition of the lower jaw starts to develop like the vomerine teeth, but degenerates by stage (vi) of tooth development. The single medial tooth is resorbed even earlier.
The pattern of tooth plate formation described in this paper is consistent with illustrations published by Semon (1901) and Greil (1908, 1913) but the inferred developmental processes are different.
Implications of the results for the Zahnreihe hypothesis of Edmund and for the phylogeny of Dipnoi are discussed.     

The fate of cranial neural crest cells in the Australian lungfish, Neoceratodus forsteri

The cranial neural crest has been shown to give rise to a diversity of cells and tissues, including cartilage, bone and connective tissue, in a variety of tetrapods and in the zebrafish. It has been claimed, however, that in the Australian lungfish these tissues are not derived from the cranial neural crest, and even that no migrating cranial neural crest cells exist in this species. We have earlier documented that cranial neural crest cells do migrate, although they emerge late, in the Australian lungfish. Here, we have used the lipophilic fluorescent dye, DiI, to label premigratory cranial neural crest cells and follow their fate until stage 43, when several cranial skeletal elements have started to differentiate. The timing and extent of their migration was investigated, and formation of mandibular, hyoid and branchial streams documented. Cranial neural crest was shown to contribute cells to several parts of the head skeleton, including the trabecula cranii and derivatives of the mandibular arch (e.g., Meckel's cartilage, quadrate), the hyoid arch (e.g., the ceratohyal) and the branchial arches (ceratobranchials I-IV), as well as to the connective tissue surrounding the myofibers in cranial muscles. We conclude that cranial neural crest migration and fate in the Australian lungfish follow the stereotyped pattern documented in other vertebrates.