Epithelial action potentials in embryos of the Australian lungfish

The epithelial cells of the skin of embryonic Australian lungfish (Neoceratodus forsteri) between stages 30 and 39, are mechanosensitive and excitable, showing overshooting action potentials 400-800 ms long with a rapid rise followed by a slower repolarization (usually with a shoulder on the repolarizing phase), which propagate at around 10 mm s-1. This skin impulse system is very similar to that found in embryonic and larval Amphibia; the significance of this similarity is discussed.     

Scale structure in the Australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

Scales of the Australian lungfish, Neoceratodus forsteri, are secreted within the dermis by a capsule of scleroblasts, and enclosed in a pouch made of collagen fibers, in contact with the epidermis over the posterior third of the scale. Each scale grows from a focus, which represents the first formed part of the scale. On the internal surface of the scale is elasmodin, made of collagen fiber bundles arranged in layers. Elasmodin, unmineralized in N. forsteri, contains cells in the living animal, and the number of layers increases as the scales grow. Squamulin, on the thin external part of the scale, is also laid down in layers, and based on a matrix of fine collagen fibrils, mineralized with a poorly crystalline biogenic calcium hydroxylapatite. Squamulin is divided into separate sections called squamulae, and contains long tubules with cells applied to the wall of the tubule. The anterior and lateral surfaces of the squamulin are ornamented with pediculae, and the posterior surface has longitudinal ridges, from which collagen fibers extend to anchor the scale within the pouch. Elasmodin and squamulin are linked by unmineralized collagen fibrils. The layers, formed at irregular intervals, are connected around the margin of the scale, effectively converting the whole scale into a flat structure resembling a pearl, with the first formed tissues deeply embedded inside the scale, and the youngest on the outer surface. Incremental lines in the hard tissue, and the number of layers in the elasmodin, do not reflect the chronological age of the fish. J. Morphol. 276:1137–1145, 2015. © 2015 Wiley Periodicals, Inc.     

Formation and structure of scales in the Australian lungfish,neoceratodus forsteri (Osteichthyes: Dipnoi)

The large elasmoid scales of the Australian lungfish, Neoceratodusforsteri, are formed within the dermis by unpigmented scleroblasts, growing within a collagenous dermal pocket below a thick glandular epidermis. The first row of scales, on the trunk of the juvenile lungfish, appears below the lateral line of the trunk, single in this species, at around stage 53. The scales, initially circular in outline, develop anteriorly and posteriorly from the point of initiation in the mid‐trunk region, and rows are added alternately below the line, and above the line, until they reach the dorsal or ventral midline, or the margins of the fins. Scales develop later on the ventral surface of the head, from a separate centre of initiation. Scales consist of three layers, all produced by scleroblasts of dermal origin. The outermost layer of interlocking plates, or squamulae, consists of a mineralised matrix of fine collagen fibrils, covered by unmineralised collagen and a single layer of cells. Squamulae of the anterior and lateral surfaces are ornamented with short spines, and the mineralised tissue of the posterior surface is linked to the pouch by collagen fibrils. The innermost layer, known as elasmodin, consists of bundles of thick collagen fibrils and cells arranged in layers. An intermediate layer, made up of collagen fibrils, links the outer and inner layers. The elasmoid scales of N. forsteri can be compared with scale types among other osteichthyan groups, although the cellsand canaliculi in the mineralised squamulae bear littleresemblance to typical bone. J.Morphol., 2012. © 2011 Wiley Periodicals, Inc.     

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.     

Skin structure in the snout of the Australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

Many fossil lungfish have a system of mineralised tubules in the dermis of the snout, branching extensively and radiating towards the epidermis. The tubules anastomose in the superficial layer of the dermis, forming a plexus consisting of two layers of vessels, with branches that expand into pore canals and flask organs, flanked by cosmine nodules where these are present. Traces of this system are found in the Australian lungfish, Neoceratodus forsteri, consisting of branching tubules in the dermis, a double plexus below the epidermis and dermal papillae entering the epidermis without reaching the surface. In N. forsteri, the tubules, the plexus and the dermal papillae consist of thick, unmineralised connective tissue, enclosing fine blood vessels packed with lymphocytes. Tissues in the epidermis and the dermis of N. forsteri are not associated with deposits of calcium, which is below detectable limits in the skin of the snout at all stages of the life cycle. Canals of the sensory line system, with mechanoreceptors, are separate from the tubules, the plexus and the dermal papillae, as are the electroreceptors in the epidermis. The system of tubules, plexus, dermal papillae and lymphatic capillaries may function to protect the tissues of the snout from infection.     

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.     

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.     

Early development of the serotonergic and dopaminergic nervous system in Spisula solidissima (surf clam) larvae.

We have defined the development of the serotonergic and dopaminergic components of the central nervous system in the early Spisula solidissima (surf clam) embryo using HPLC and immunocytochemistry. HPLC analysis reveals norepinephrine, dopamine, and serotonin are present at 24 h post-fertilization. Immunocytochemistry shows that the serotonergic nervous system emerges during the late trochophore stage with the development of a single serotonergic cell, C/A1, in the cerebral/apical ganglion. After 48 h, a second serotonergic cell forms, C/A2, which is connected to C/A1 by two serotonergic processes, and a single serotonergic cell emerges in the visceral ganglion, V1. At 72 h, a new serotonergic cell body develops in the cerebral/apical ganglion, C/A3. After 96 h, the cerebral/apical ganglion and visceral ganglion are connected by a serotonergic process. Expression of the dopamine receptor, D2, begins by 24 h with a generalized expression in the region of the developing gut. D2 expression in the gut ceases by 48 h. At 48 h, a network of fibers forms dorsolateral to the mouth. By 72 h, D2 expressing projections emerge from this network.     

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.     

Monitoring age‐related trends in genomic diversity of Australian lungfish

An important challenge for conservation science is to detect declines in intraspecific diversity so that management action can be guided towards populations or species at risk. The lifespan of Australian lungfish (Neoceratodus forsteri) exceeds 80 years, and human impacts on breeding habitat over the last half century may have impeded recruitment, leaving populations dominated by old postreproductive individuals, potentially resulting in a small and declining breeding population. Here, we conduct a “single‐sample” evaluation of genetic erosion within contemporary populations of the Australian lungfish. Genetic erosion is a temporal decline in intraspecific diversity due to factors such as reduced population size and inbreeding. We examined whether young individuals showed signs of reduced genetic diversity and/or inbreeding using a novel bomb radiocarbon dating method to age lungfish nonlethally, based on ¹⁴C ratios of scales. A total of 15,201 single nucleotide polymorphic (SNP) loci were genotyped in 92 individuals ranging in age from 2 to 77 years old. Standardized individual heterozygosity and individual inbreeding coefficients varied widely within and between riverine populations, but neither was associated with age, so perceived problems with recruitment have not translated into genetic erosion that could be considered a proximate threat to lungfish populations. Conservation concern has surrounded Australian lungfish for over a century. However, our results suggest that long‐lived threatened species can maintain stable levels of intraspecific variability when sufficient reproductive opportunities exist over the course of a long lifespan.     

Early embryonic development of the central nervous system in the Australian crayfish and the Marbled crayfish (Marmorkrebs)

This study sets out to provide a systematic analysis of the development of the primordial central nervous system (CNS) in embryos of two decapod crustaceans, the Australian crayfish Cherax destructor (Malacostraca, Decapoda, Astacida) and the parthenogenetic Marbled crayfish (Marmorkrebs, Malacostraca, Decapoda, Astacida) by histochemical labelling with phalloidin, a general marker for actin. One goal of our study was to examine the neurogenesis in these two organisms with a higher temporal resolution than previous studies did. The second goal was to explore if there are any developmental differences between the parthenogenetic Marmorkrebs and the sexually reproducing Australian crayfish. We found that in the embryos of both species the sequence of neurogenetic events and the architecture of the embryonic CNS are identical. The naupliar neuromeres proto-, deuto-, tritocerebrum, and the mandibular neuromeres emerge simultaneously. After this “naupliar brain” has formed, there is a certain time lag before the maxilla one primordium develops and before the more caudal neuromeres follow sequentially in the characteristic anterior–posterior gradient. Because the malacostracan egg-nauplius represents a re-capitulation of a conserved ancestral information, which is expressed during development, we speculate that the naupliar brain also conserves an ancestral piece of information on how the brain architecture of an early crustacean or even arthropod ancestor may have looked like. Furthermore, we compare the architecture of the embryonic crayfish CNS to that of the brain and thoracic neuromeres in insects and discuss the similarities and differences that we found against an evolutionary background.     

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.     

Early locomotor behaviour in genetic stocks of chickens with different growth rates

Reduction in exercise increases the occurrence of lameness in meat-type chickens. Locomotor activity is dramatically reduced during the finishing period in chickens from fast-growing genetic types compared to slow-growing genetic types, but it is not known whether this difference is already present during the starting period and may be influenced by genetic factors. In order to define the effect of genetic origin on early locomotor behaviour, exercise was compared from 1 to 22 days of age in two meat-type chicken stocks differing in growth rate: male broilers (B) which grow fast and are often lame, and male "label rouge" chickens (L) which grow slowly and are rarely lame.Time budget (lying, standing, drinking, eating, walking) was measured by scanning in six repetitions of five birds (density=2.5 birds/m(2)) at 1, 8, 15 and 17 days of age. Standing bouts were analysed by focal sampling at 2-3, 6-7, 13-14 and 20-21 days of age.B chicks spent less time standing than L chicks at 15 days of age (B=13+/-2%, L=24+/-1%, P<0.01) and 17 days of age, and spent more time lying at 17 days of age (B=73+/-3%, L=60+/-4%, P<0.05).The major part (74%) of the total active time observed by focal sampling was linked to feeding activity. At 2 and 3 days, the activity of B chicks was half that of L chicks during standing bouts (duration of walking per bout: 19+/-4 s for B; 45+/-4 s for L, P<0.05). The activity observed by focal sampling during non-feeding bouts at 20-21 days was significantly correlated with the corresponding data recorded at 2-3 days in the same chicks in the B stock but not in the L stock.We concluded that (1) both B and L genetic stocks have the same overall activity during the first 3 days of age (scanning) but they exhibit different organisation and composition of standing bouts (focal sampling). (2) Genetic factors are probably involved in the expression of locomotor behaviour in very young chicks. (3) The correlations between the levels of activity at early and later ages suggest that selection of young mobile broiler chicks might increase activity at a later age and might therefore reduce the occurrence of leg abnormalities.     

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.     

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     

Vascular distribution of nitric oxide synthase and vasodilation in the Australian lungfish, Neoceratodus forsteri

The presence of nitric oxide synthase (NOS) and role of nitric oxide (NO) in vascular regulation was investigated in the Australian lungfish, Neoceratodus forsteri. No evidence was found for NOS in the endothelium of large and small blood vessels following processing for NADPH-diaphorase histochemistry. However, both NADPH-diaphorase histochemistry and neural NOS immunohistochemistry demonstrated a sparse network of nitrergic nerves in the dorsal aorta, hepatic artery, and branchial arteries, but there were no nitrergic nerves in small blood vessels in tissues. In contrast, nitrergic nerves were found in non-vascular tissues of the lung, gut and kidney. Dual-wire myography was used to determine if NO signalling occurred in the branchial artery of N. forsteri. Both SNP and SIN-1 had no effect on the pre-constricted branchial artery, but the particulate guanylyl cyclase (GC) activator, C-type natriuretic peptide, always caused vasodilation. Nicotine mediated a dilation that was not inhibited by the soluble GC inhibitor, ODQ, or the NOS inhibitor, L-NNA, but was blocked by the cyclooxygenase inhibitor, indomethacin. These data suggest that NO control of the branchial artery is lacking, but that prostaglandins could be endothelial relaxing factors in the vasculature of lungfish.     

Development of the vasculature of the pectoral fin in the Australian lungfish, Neoceratodus forsteri

The development of the vasculature of the pectoral fin in the Australian lungfish, Neoceratodus forsteri, was studied by the dye-injection method. Only a single primitive subclavian artery appears from the dorsal aorta for the fin anlage, and it passes laterally through the postaxial region of the structure. The venous channel draining into the posterior cardinal vein is located in the preaxial region medially. As development proceeds, the arteriovenous arrangement in the pectoral fin anlage changes as follows: 1) one artery and one venous plexus, 2) two arteries and one vein, 3) three arteries and one vein, 4) four arteries and one vein, 5) three arteries and two veins, and 6) two arteries (radial and ulnar) and three veins (radial, ulnar, and ulnar marginal). The fin anlage through embryonic first rotation has gradually changed its postaxial margin to face dorsally and its preaxial margin to face ventrally. The second rotation causes the original preaxial margin to become dorsal and the original postaxial margin to become ventral. As a result, the radial and ulnar arteries are observed in the dorsal and ventral regions, respectively, in the medial side of the fin instead of in the lateral side as seen in the previous stage.