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Background The concerted activity of Meis and Hoxa11 transcription factors is essential for the subdivision of tetrapod limbs into proximo-distal (PD) domains; however, little is know about the evolution of this patterning mechanism. Here, we aim to study the expression of meis and hoxa11 orthologues in the median and paired rayed fins of zebrafish and in the lobed fins of the Australian lungfish. Results First, a late phase of expression of meis1.1 and hoxa11b in zebrafish dorsal and anal fins relates with segmentation of endochondral elements in proximal and distal radials. Second, our zebrafish in situ hybridization results reveal spatial and temporal changes between pectoral and pelvic fins. Third, in situ analysis of meis1, meis3 and hoxa11 genes in Neoceratodus pectoral fins identifies decoupled domains of expression along the PD axis. Conclusions Our data raise the possibility that the origin of stylopod and zeugopod lies much deeper in gnathostome evolution and that variation in meis and hoxa11 expression has played a substantial role in the transformation of appendage anatomy. Moreover, these observations provide evidence that the Meis/Hoxa11 profile considered a hallmark of stylopod/zeugopod patterning is present in Neoceratodus.

Summary 1. Understanding individual and population responses to climate change is emerging as an important challenge. Because many phenotypic traits are sensitive to environmental conditions, directional climate change could significantly alter trait distribution within populations and may generate an evolutionary response. 2. In species with environment-dependent sex determination, climate change may lead to skewed sex ratios at hatching or birth. However, there are virtually no empirical data on the putative link between climatic parameters and sex ratios from natural populations. 3. We monitored a natural population of viviparous lizards with temperature-dependent sex determination ( Niveoscincus ocellatus ) over seven field seasons. Sex ratios at birth fluctuated significantly among years and closely tracked thermal conditions in the field, with the proportion of male offspring increasing in colder years. 4. This is the first study to demonstrate the effect of local climatic conditions (e.g. temperature) on offspring sex ratio fluctuations in a free-living population of a viviparous ectotherm. A succession of warmer-than-usual years (as predicted under many climate-change scenarios) likely would generate female-biased sex ratios at birth, while an increase in interannual variation (as also predicted under climate change scenarios) could lead to significant fluctuations in cohort sex ratios. If cohort sex ratio bias at birth leads to adult sex ratio bias, long-term directional changes in thermal conditions may have important effects on population dynamics in this species.

Epithelial sodium channel (ENaC) is a Na + -selective, aldosterone-stimulated ion channel involved in sodium transport homeostasis. ENaC is rate-limiting for Na + absorption in the epithelia of osmoregulatory organs of tetrapods. Although the ENaC/degenerin gene family is proposed to be present in metazoans, no orthologues or paralogues for ENaC have been found in the genome databases of teleosts. We studied full-length cDNA cloning and tissue distributions of ENaCα, β and γ subunits in the Australian lungfish, Neoceratodus forsteri , which is the closest living relative of tetrapods. Neoceratodus ENaC ( n ENaC) comprised three subunits: n ENaCα, β and γ proteins. The n ENaCα, β and γ subunits are closely related to amphibian ENaCα, β and γ subunits, respectively. Three ENaC subunit mRNAs were highly expressed in the gills, kidney and rectum. Amiloride-sensitive sodium current was recorded from Xenopus oocytes injected with the n ENaCαβγ subunit complementary RNAs under a two-electrode voltage clamp. n ENaCα immunoreactivity was observed in the apical cell membrane of the gills, kidney and rectum. Thus, n ENaC may play a role in regulating sodium transport of the lungfish, which has a renin–angiotensin–aldosterone system. This is interesting because there may have been an ENaC sodium absorption system controlled by aldosterone before the conquest of land by vertebrates.

The effects of oxygen partial pressure ( $$ P_{{{\text{O}}_{2} }} $$ ) on development and respiration were investigated in the eggs of the Australian lungfish, Neoceratodus forsteri. At 20°C, embryonic survival and development was optimal at 15 and 20.9 kPa. Development was slowed at 5 and 10 kPa and embryos did not survive 2 kPa. At lower $$ P_{{{\text{O}}_{2} }} $$ , the rate of oxygen consumption also decreased. Embryos responded to hypoxia by hatching at an earlier age and stage of development, and hatching wet and dry gut-free masses were reduced. The role of oxygen conductance ( $$ G_{{{\text{O}}_{2} }} $$ ) in gas exchange was also examined under selected environmental $$ P_{{{\text{O}}_{2} }} $$ and temperatures. The breakdown of the vitelline membrane changed capsule geometry, allowed water to be absorbed into the perivitelline space and increased capsule $$ G_{{{\text{O}}_{2} }} $$ . This occurred at embryonic stage 32 under all treatments and was largely independent of both $$ P_{{{\text{O}}_{2} }} $$ and temperature (15, 20 and 25°C), demonstrating that capsule $$ G_{{{\text{O}}_{2} }} $$ cannot adaptively respond to altered environmental conditions. The membrane breakdown increased capsule diffusive $$ G_{{{\text{O}}_{2} }} $$ and stabilised perivitelline $$ P_{{{\text{O}}_{2} }} $$ , but reduced the convective $$ G_{{{\text{O}}_{2} }} $$ of the perivitelline fluid, as the large perivitelline volume and inadequate convective current resulted in a $$ P_{{{\text{O}}_{2} }} $$ gradient within the egg prior to hatch.

The rate of oxygen consumption throughout embryonic development is used to indirectly determine the ‘cost’ of development, which includes both differentiation and growth. This cost is affected by temperature and the duration of incubation in anamniote fish and amphibian embryos. The influences of temperature on embryonic development rate, respiration rate and energetics were investigated in the Australian lungfish, Neoceratodus forsteri, and compared with published data. Developmental stage and oxygen consumption rate were measured until hatching, upon which wet and dry gut-free masses were determined. A measure of the cost of development, the total oxygen required to produce 1 mg of embryonic dry tissue, increased as temperature decreased. The relationship between the oxygen cost of development (C, ml mg−1) and dry hatchling mass (M, mg) in fishes and amphibians is described by C = 0.30 M0.22 ± 0.13 (95% CI), r 2 = 0.52. The scaling exponent indicates that the cost of embryonic development increases disproportionally with increasing hatchling mass. At 15 and 20°C, N. forsteri cost of development is significantly lower than the regression mean for all species, and at 25°C is lower than the allometrically scaled data set. Unexpectedly, incubation of N. forsteri is long, despite natural development under relatively warm conditions, and may be related to a large genome size. The low cost of development may be associated with construction of a rather sluggish fish with a low capacity for aerobic metabolism. The metabolic rate is lower in N. forsteri hatchlings than in any other fishes or amphibians at the same temperature, which matches the extremely low aerobic metabolic scope of the juveniles.

New observations on the axial skeleton of the extant lungfish Neoceratodus forsteri (Dipnoi; Sarcopterygii) indicate that neural and haemal arch elements develop more independently than previously believed. For example, while the cartilaginous neural arches/spines begin development anteriorly, just behind the skull, the distal supraneurals first form separately in the posterior region of the axial skeleton. Proximal supraneurals develop subsequently, but initially lack clear association with either neural arches/spines or distal supraneurals. This contradicts previous studies of Neoceratodus and other extant lungfish suggesting that the supraneurals and more distal radials develop from a single cartilage. This was suggested as a unique sarcopterygian pattern, but our new observations suggest a closer resemblance to the actinopterygian condition. With respect to the caudal fin skeleton, the heterocercal tail of Devonian lungfish is replaced in Carboniferous and younger taxa by a diphycercal tail, comprising elongate and equal dorsal and ventral lobes. Whether these lobes resulted from fusion of dorsal/anal and caudal fins or expansion of dorsal and anal fin rays at the expense of the caudal fin is uncertain. However, comparing ontogenetic development of Neoceratodus with Devonian taxa indicates that the elements of the ventral diphycercal lobe in Neoceratodus are homologous to haemal elements present in the caudal fin of Devonian forms, but loss of supporting basal plates in the dorsal fins of Devonian taxa allowed supraneural elements of the dorsal fin to expand to form the dorsal lobe of the Neoceratodus tail.

The apical ectodermal ridge (AER) in Neoceratodus develops after an initial period of mesenchymal proliferation and outgrowth of the fin bud and persists until chondrogenesis of the stylopod and zeugopod is initiated. At this time, the lateral margins of the AER convert to the fin fold leading to subsequent development of the dermal fin skeleton. Thorogood's (1991) fin fold model predicts that the AER should persist longer in Neoceratodus than it does in actinopterygians because of the comparatively extensive endochondral skeleton in lungfish. While the AER does persist into early chondrogenesis and is extended compared to actinopterygians (lost before fin radial chondrogenesis) it does not persist into further stages of chondrogenesis, providing partial support for Thorogood's model. Fgf8 appears in the lungfish fin epithelium during the initial period of fin outgrowth before a physical AER forms, when Fgf8 is restricted to the AER plus the preaxial and postaxial epithelium immediately adjacent to the AER. Fgf8 is no longer detected after the AER is replaced by a fin fold. Neoceratodus appears to provide a halfway point between ray fins and limbs during very early development as Thorogood proposed, but not precisely for the reasons his model suggests.

Our research on the evolution of head development focuses on understanding the developmental origins of morphological innovations and involves asking questions like: How flexible (or conserved) are cell fates, patterns of cell migration or the timing of developmental events (heterochrony)? How do timing changes, or changes in life history affect head development and growth? Our ‘model system’ is a comparison between lungfishes and representatives from all three extant groups of amphibians. Within anuran amphibians, major changes in life history such as the repeated evolution of larval specializations (e.g. carnivory), or indeed the loss of a free-swimming larva, allows us to test for developmental constraints. Cell migration and cell fate are conserved in cranial neural crest cells in all vertebrates studied so far. Patterning and developmental anatomy of cranial neural crest and head mesoderm cells are conserved within amphibians and even between birds, mammals and amphibians. However, the specific formation of hypobranchial muscles from ventral somitic processes shows variation within tetrapods. The evolution of carnivorous larvae in terminal taxa is correlated with changes in both pattern and timing of head skeletal and muscle development. Sequence-heterochronic changes are correlated with feeding mode in terminal taxa and with phylogenetic relatedness in basal branches of the phylogeny. Eye muscles seem to form a developmental module that can evolve relatively independently from other head muscles, at least in terms of timing of muscle differentiation.

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.

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. J. Exp. Zool. (Mol. Dev. Evol.) 310B:345–354, 2008. © 2007 Wiley-Liss, Inc.

In two species of Heterodontus, H. portusjacksoni and H. galeatus, the first scales to develop form two opposing rows along the caudal fin axis on both the left and right sides of the fin. The opposing rows originate from an initial scale located on either side of the posterior tip of the caudal fin, with subsequent scales erupting in a posterior to anterior direction along the tail axis. These scale rows may strengthen tail movements, providing aeration in the egg case, but are lost later in ontogeny. Development of subsequent body scales shows a more irregular origin and arrangement, from anterior to posterior, to cover the dorsal and ventral lobes of the caudal fin. Although the early developmental pattern of the scale associated with the Heterodontus caudal fin has not been previously described, several chondrichthyan taxa, including chimeroids, likewise possess ordered rows of flank scales early in ontogeny that are subsequently lost. These ordered scales contrast with previous suggestions that chondrichthyan scale development is entirely random. Instead, regulated and sequential development of scales may be a plesiomorphic character for both chondrichthyans and osteichthyans, with the less organized arrangement in later ontogenetic stages being a derived condition within Chondrichthyes.

The enigmatic Devonian fossil Palaeospondylus gunni was identified as a larval form, metamorphosing into the lungfish Dipterus valenciennesi. Morphological features used to identify P. gunni as a larval lungfish include enlarged cranial ribs, rudimentary limb girdles, and absence of teeth. However, this combination of features does not characterize the extant lungfish Neoceratodus forsteri, even at very young stages, nor early stages of Devonian and younger fossil lungfish. Absence of teeth is problematic because early ontogenetic stages of fossil and living lungfish possess full dentitions including marginal teeth. Also problematic are cranial ribs as a defining character of lungfish, as these also occur in certain actinopterygians. It is argued that Neoceratodus is an obligate neotene (reproductively mature larva), with the implication that metamorphosis was a feature of the ontogeny of early lungfish. Pedomorphic characters have been recognized in Neoceratodus and other post-Devonian lungfish, including large cells and correspondingly large genome size; these latter characters correlate with neoteny in salamanders. Small cells preserved in fossil bone suggest that Devonian lungfish had a smaller genome than post-Devonian lungfish, implying that they were not neotenic. As fossil lungfish cell sizes (and genomes) increased in the late Paleozoic, the diversity of lungfish morphologies decreased, so that taxa like Sagenodus and Conchopoma show morphological similarity to Neoceratodus, marking a point in phylogeny at which metamorphosis was potentially lost. Since ancestral larval characters are retained in neotenic adults, we predict that Devonian larvae should resemble these post-Devonian taxa, a prediction which Palaeospondylus does not fulfill.

The lampreys (family Petromyzontidae) are divided into three subfamilies, the Petromyzontinae in the Northern Hemisphere and the Geotriinae and Mordaciinae in the Southern Hemisphere. We previously found two proopiomelanocortin subtypes, proopiocortin (POC) and proopiomelanotropin (POM) in sea lamprey, Petromyzon marinus (Petromyzontinae). POC encoding adrenocorticotropic hormone (ACTH) and beta-endorphin (beta-END) is expressed in the pars distalis of the pituitary, while POM encoding melanophore-stimulating hormone (MSH)-A and B together with a different beta-END is expressed in the pars intermedia of the pituitary. All these hormonal segments are encoded on the third exon in both POC and POM. Here, we demonstrate the presence of both POC and POM genes in Geotria australis (Geotriinae) and Mordacia mordax (Mordaciinae) by molecular cloning of the third exons with the polymerase chain reaction using genomic DNA or pituitary cDNA. Molecular phylogenetic analysis showed that the POC and POM are distinctly different for the Southern Hemisphere lampreys as they are for P. marinus. Moreover, the relationship of each hormonal segments in POC and POM between Geotria, Mordacia, and Petromyzon is inconsistent. Immunocytochemical studies showed that the distribution of POC and POM in the pituitaries of the Southern Hemisphere lampreys is the same as that in the Northern Hemisphere. Taken together, these findings suggest that the duplication event which generated the two genes may have occurred in a common ancestor of the three extant lamprey subfamilies.

In vertebrates, hormones released from the thyroid gland travel in the circulation to target tissues where they may be processed by deiodinating enzymes into more active or inactive iodothyronines. In mammals, there are three deiodinating enzymes described. Type1 (D1), which primarily occurs in the liver, converts reverse T3 into T2 for clearance. It also converts T4 into T3. This production of T3 is believed to contribute to the bulk of circulating T3 in mammals. The type2 (D2) enzyme may be found in many other tissues where it converts T4 to T3, which is then transferred to the receptors in the nucleus of the same cell, i.e. does not contribute to the circulating T3. The type3 (D3) enzyme converts T3 into T2. The expression of the genes for these three enzymes and/or the activity of the enzymes have been studied in several non-mammalian groups of vertebrates. From agnathans to birds, D2 and D3 appear to occur universally, with the possible exception of squamate reptiles (lack D2?). D1 has not been found in amphibians, lungfish or agnathans. All three enzymes are selenoproteins, in which a selenocysteine is found in the active centre. The nucleotide code for translation of a selenocysteine is UGA, which under normal circumstances is a stop codon. In order for UGA to code for selenocysteine, there must be a SECIS element in the 3'UTR of the mRNA. Any disruption of the SECIS will result in a truncated protein in the region of its active centre. It is suggested that such alternative splicing may be a mode of altering the expression of deiodinases in particular tissues to change the response of such tissues to thyroid hormones under differing circumstances such as stages of development.

Differentiation of the axial skeleton into distinct regions, once thought to be characteristic of the Tetrapoda, also occurs in the actinopterygian Danio rerio. In these taxa, the boundary between the cervical-thoracic regions correlates with Hoxc6 expression and morphological features such as position of the pectoral fin and associated nerves, and the absence of ribs. In the lungfish Neoceratodus, a member of the extant sister taxon to the Tetrapoda, the first vertebral element to chondrify is situated well posterior to the skull, developing from somites 6 and 7 (6/7) and associated with an enlarged cranial rib and nerves innervating the pectoral fin. Two vertebral elements develop later and more anteriorly, associated with somites 4/5 and 5/6. These three elements become incorporated into the occipital region of the skull during Neoceratodus ontogeny, until the cranial rib itself articulates to the rear of the skull. These features of early development indicate a regionalization of the Neoceratodus vertebral column: the cranial rib marks the boundary between the cervical and thoracic regions, the two more anterior vertebrae lacking ribs represent the cervical region, while somites 1-4 (cranial half), lacking any vertebral development, represent the occipital region. However, the cervical region of the vertebral column is effectively lost during ontogeny of Neoceratodus. A recognizable cervical region in the tetrapod vertebral column, as in zebrafish, suggests that cervical vertebrae are not incorporated into the skull but maintained as distinct elements of the column, representing an important shift in relative developmental timing and the influence of heterochrony in this region during the fish-tetrapod transition.

The genomes of lungfish, together with those of some urodele amphibians, are the largest of all vertebrate genomes. It has been assumed that the bulk of the DNA making up these large genomes has been derived from repeat elements, like the noncoding DNA of those genomes that have been sequenced (e.g., human). In an attempt to characterize repeat sequences in the lungfish genome, we have isolated, by restriction enzyme digestion of genomic DNA, sequences of a repeat element in Neoceratodus forsteri, the most primitive of the living lungfishes. The fragments sequenced from the EcoRI and BglII digests were used to perform genome walking PCR in order to obtain the full sequence of the repeat element. This element shares homology with the non-LTR (LINE) element, Chicken Repeat 1 (CR1), described for several vertebrates and some invertebrates; we have called it N. forsteri CR1 (NfCR1). NfCR1 shares all the domains of other CR1 elements but it also has several unique features that suggest it may no longer be active in the lungfish genome. It occurs in both full-length and 5'-truncated versions and in its present "inactive" form represents approximately 0.05% of the lungfish genome.

Among bony fishes, the ontogenetic sequence by which the actinopterygian scapulocoracoid develops has been well described, but that of the sarcopterygian scapulocoracoid is poorly known, as the majority of taxa are only known from fossils. To rectify this, the cartilaginous scapulocoracoid of the extant lungfish Neoceratodus forsteri is examined. In initial stages of its development, the scapulocoracoid of Neoceratodus has a simple rounded shape, and supports the glenoid fossa. It appears nearly contemporaneously with the proximal endochondral element (humerus) of the pectoral fin. Pectoral fin elements develop by segmentation from a continuous field of cartilaginous precursor cells extending distally from the glenoid region of the scapulocoracoid. Subsequent scapulocoracoid development produces a ventromedial process, which is not associated with this field of precursor cells. A dorsal process also develops outside this field. Thus, the scapulocoracoid of Neoceratodus may consist of at least two developmentally distinct regions; (1) the ventromedial being homologous with the coracoid of actinopterygians, tetrapods and other jawed vertebrates and (2) a smaller dorsal process, homologous to the scapular region. The two, together with the glenoid region, give an overall triangular shape. The scapulocoracoids of fossil lungfish and other sarcopterygian fishes are also triangular and are composed of scapular and coracoid regions, rather than the ‘buttresses’ associated with scapulocoracoids of the Actinopterygii and Tetrapoda.

This work presents characterisation of deiodinase type III (D3) mRNA as cDNA and the tissue distribution of D3 mRNA in the Australian lungfish, Neoceratodus forsteri . We have identified the full length of a ∼1.4 kb D3 mRNA in the liver, which has a single in-frame UGA codon and a selenocysteine insertion sequence (SECIS) form 2 in the 3 ′ -UTR. Lungfish D3 mRNA was expressed in all tested tissues (liver, lung, kidney, brain, heart, and gills) as demonstrated by Northern blot analyses. PCR conducted on genomic DNA indicated that the lungfish D3 is a single exon gene. Also, we present enzymatic characteristics of this exclusively IRD enzyme, have determined its substrate preference, DTT cofactor requirements, PTU inhibition, and kinetic properties. These results indicate that lungfish D3 has the typical enzymatic characteristics of vertebrate D3 enzymes.

The β subunits of the two pituitary gonadotropins LH and FSH and of thyroid-stimulating hormone (TSH) were cloned from Australian lungfish (Neoceratodus forsteri) pituitary glands. These three glycoprotein hormone β subunits possess the main characteristics common to their counterparts in other vertebrates. Taking advantage of the phylogenetic position of the lungfish, close to the root of tetrapods, a maximum parsimony tree was inferred from these new sequences and sequences from representatives of the diversity of vertebrates. The topology of the tree was imposed so that it reflected as closely as possible the real evolutionary history of the subunits. This tree was used to estimate the relative evolution rate of the three subunits in vertebrates. Cumulated amino acid substitutions from the basal subunit node (ancestral subunit sequence) to the species node were calculated and compared. It showed that a burst in evolutionary rate occurred for the LHβ subunit in the tetrapod lineage sometime after the emergence of amphibians. The rate of evolution of the LHβ subunit was particularly high throughout the radiation of mammals while FSH and TSHβ subunits kept quite stable in this lineage. A burst in evolutionary rate was also observed for the FSHβ subunit in the lineage leading to teleosts sometime after the emergence of chondrosteans and the dynamic of evolution was high throughout the radiation of teleosts. These results were consistent with data obtained from pairwise comparisons.

In mammals the opioids Met-enkephalin and Leu-enkephalin are derived from a common precursor, proenkephalin, and as a result these neuropeptides are co-localized in enkephalinergic neurons. The mammalian scheme for enkephalinergic networks is not universal for all classes of sarcopterygian vertebrates. In an earlier study, distinct Met- and Leu-enkephalin-positive neurons were detected in the central nervous system (CNS) of the African lungfish, Protopterus annectens. More recently, characterization of proenkephalin cDNAs separately cloned from the CNS of P. annectens and the Australian lungfish, Neoceratodus forsteri, revealed that the proenkephalin gene in these species encodes only Met-enkephalin-related opioids. In the current study a full-length prodynorphin cDNA (accession No. AY 445637) was cloned and sequenced from the CNS of N. forsteri. In addition to encoding α-neoendorphin, dynorphin A and dynorphin B sequences unique to the lungfish, two Leu-enkephalin sequences, flanked by paired basic amino acid proteolytic cleavage sites, were detected in this precursor. The partial sequence of a P. annectens prodynorphin cDNA (accession No. AY445638) also encoded a Leu-enkephalin sequence and a novel YGGFF sequence. The presence of the Leu-enkephalin sequence in the lungfish prodynorphin precursors would explain the origin of the distinct Leu-enkephalin-positive neurons found in the African lungfish CNS. The realization that Met-enkephalin and Leu-enkephalin can be derived from distinct opioid-coding precursor genes calls into question the interpretation of comparative immunohistochemical studies that have mapped ‘enkephalinergic’ networks in non-mammalian vertebrates.

Male jacky dragons, Amphibolurus muricatus, indicate territoriality to rivals during the mating season through the use of stereotyped motor displays. The relationship between corticosterone (B) and testosterone (T) and its effects on territorial display expression were investigated in captive lizards. Results demonstrated that territorial display production was most effectively predicted by elevated baseline T levels. This parallels the heightened T concentrations exhibited by males in the field during the spring mating period. The effect of social interaction on B and T levels was also examined by presenting territory-holding males with a size-matched male intruder. Resident males exhibited clear differences in the level of territorial response evoked by a male intruder, which were correlated with differences in physiological activity. Males that gave no territorial response had moderate B levels that did not change with social interaction, and significantly lower T levels than males that responded to the intruder with territorial displays. Among displaying males, those exhibiting low levels of territorial responses showed no change in B or T. In contrast, high-level territorial responders exhibited acute B increases when faced with an intruder, which might assist in supporting sustained metabolic activity and could possibly reflect differences in the perception of social stress. These same males also exhibited a reduction in T levels at such times, probably due to the acute rise in B. Combined, these results suggest that high circulating T is acting in a preparatory manner to increase the likelihood of producing a territorial response upon engaging in a social encounter. However, once this response has been initiated T may not need to remain elevated to allow continued expression of territorial behavior.

Many lizards produce visual displays to mediate social behavior. However, most studies have focused upon displays used by iguanid lizards. We investigated and quantified the displays used by jacky dragons, an agamid lizard from southeastern Australia. By establishing male jacky dragons within individual territories and conducting experimental presentations of male and female intruders, we were able to demonstrate that territorial male Amphibolurus muricatus utilize a fixed action pattern display comprising discrete motor components to indicate territoriality and aggression. Displays directed to intruders contain an extra introductory component and modifying postural elements, whereas non-directed displays given in the absence of intruders lack these features and appear to be used to advertise territory ownership. Although the sequence of display components remained the same across non-directed, male-directed and female-directed displays, resident males differed in pushup duration and the number of pushups per display in relation to individual body size. This display variance might function to provide an honest signal revealing the body size or the physical condition of each individual, permitting opponents to assess each other effectively through their displays. Visual signals produced by intruding lizards were qualitatively distinct from the displays given by residents, and appear to indicate submission. However, male and female intruders differed in the frequency with which each type of submissive gesture was performed, implying that this might convey intruder sex.

Antechinus stuartii is a small marsupial with a brief, highly synchronised mating period believed to be controlled by the rate of change of photoperiod. Two experiments were performed to explore aspects of photoperiodic control of the seasonal cycle. In the first experiment the pineal hormone, melatonin, administered in the drinking water from the winter solstice, changed the normal response of A. stuartii to increasing rate of change of photoperiod. Melatonin administration shifted the induction of estrus in the females from the first week of August (controls) to an earlier time of mid-July and the consequent pouch changes associated with pregnancy and pseudo-pregnancy were also shifted by the same length of time. Post-mating decline and consequent death of males were also accelerated. In the second experiment melatonin was administered from the autumnal equinox, and this experimental protocol resulted in a desynchronisation of reproductive events. Melatonin administration desynchronised the female reproductive cycle, such that the mating period was extended to eight weeks, instead of the two weeks displayed by control females. Pouch changes and birth of young reflected this desynchronisation. Melatonin administration in males resulted in desynchronisation of reproductive parameters. While the normal yearly reproductive cycle was approximated in these males, the high syncronisation of reproductive maturation and male mortality events observed in control males, was not evident in melatonin-treated males. These results indicate that the pineal gland by way of the hormone melatonin is important in the synchronisation of the unusual life history of this marsupial mammal.

The lungfish dentition is different from other osteichthyan fish because it has a characteristic and unique pattern of teeth arranged as toothplates. Growth, addition of teeth, and retention as part of a statodont dentition are determined by the initiation pattern. In adult lungfish new teeth are only added laterally to each radial row in the dentition. This is in marked contrast to marginal rows of teeth with regular, alternating replacement in most osteichthyans. We analyze development from fossil hatchling forms of the Late Devonian dipnoan Andreyevichthys and compare with those of Neoceratodus, the Australian lungfish. The specific pattern of development, unique within lungfish, is also present in the transitory, marginal, anterior dentition in both, reflecting a strongly conserved developmental pattern. These marginal teeth form but are then lost in both, so that also this program of development is conserved within lungfish for 360 million years, from the earliest known form.

A peptide with mammalian substance P (SP)-like immunoreactivity was isolated from an extract of the spiral intestine of the Australian lungfish, Neoceratodus forsteri. The primary structure of this peptide was established as Lys-Pro-Arg-Pro-Asp-Glu-Phe-Tyr-Gly-Leu-Met . NH2, showing 64% identity with mammalian SP. In isolated preparations of lungfish foregut circular muscle, lungfish SP produced a slow, long-lasting tonic contraction, with a pD2 value of 8.19. Lungfish midgut circular muscle preparations responded to lungfish SP rapidly and in a more complex manner. There was an increase in the frequency of spontaneous activity (pD2 = 8.76), associated with diminished amplitude of the spontaneous contractions (pD2 = 9.24), also coupled in some preparations with a tonic contraction (pD2 = 8.43). The response patterns of foregut and midgut circular muscle to acetylcholine (ACh) were very similar to those seen to lungfish SP. Lungfish SP and ACh, however, had very weak effects on both foregut and midgut longitudinal muscle. These data demonstrate that lungfish SP may be a physiologically important regulator of gastrointestinal motility in Neoceratodus. This study further confirmed that the structures of SP-related peptides have been strongly conserved under the pressure of vertebrate evolution, particularly in preserving the functionally important sequence, Phe-Xaa-Gly-Leu-Met . amide, at the C-terminus. The sequence of lungfish SP is identical to that of bufokinin, a SP-related peptide previously isolated from the intestine of the cane toad, Bufo marinus, supporting the hypothesis that lungfishes and amphibians share a common ancestor.

The present study has characterized gonadotropic releasing hormone (GnRH)-like molecules in the brains of representatives of the two southern hemisphere families of lampreys, Geotriidae and Mordaciidae. Chromatographic and immunocytochemical evidence showed that the brains of Geotria australis and Mordacia mordax contain two forms of GnRH-like molecules. These two forms correspond to lamprey GnRH-I and -III, which were first sequenced from the brain of the anadromous sea lamprey Petromyzon marinus, a representative of the family Petromyzontidae that is found only in the northern hemisphere. In chromatographic studies (HPLC) using lamprey GnRH-I and -III antiserum, two early eluting GnRH forms coeluted with synthetic lamprey GnRH-I and -III standards. Our studies thus indicate that, despite their apparently long period of separation, the three families of extant lampreys have each retained both of the lamprey GnRH (-I and -III forms) molecules. Moreover, immunocytochemical localization of lamprey GnRH indicated that the pattern of its distribution in the adult brain of at least one of these southern hemisphere lampreys (G. australis) is similar to that previously described for P. marinus. Distribution of GnRH in the brain of larval G. australis was not as extensive as that in larval P. marinus, which may account for the later gonadal development in the former species. The fact that lamprey GnRH-I and -III are the dominant GnRH forms in all three families of lampreys implies that these neurohormones have an ancient origin.

The previous detection of Met-enkephalin and Leu-enkephalin in the CNS of the Australian lungfish, Neoceratodus forsteri, in a molar ratio comparable to mammals suggested that the lungfish proenkephalin precursor should contain the sequences of both Met-enkephalin and Leu-enkephalin as seen for mammalian proenkephalin. However, the cloning of a full-length proenkephalin cDNA from the CNS of the Australian lungfish indicates that the organization of this precursor is more similar to amphibian proenkephalin than mammalian proenkephalin. The Australian lungfish cDNA is 1284 nucleotides in length and the open reading frame (267 amino acids) contains seven opioid sequences (GenBank #AF232671). There are five copies of the Met-enkephalin sequence flanked by sets of paired basic amino acid proteolytic cleavage sites and two C-terminally extended forms of Met-enkephalin: YGGFMRSL and YGGFMGY. As seen for amphibians, no Leu-enkephalin sequence was detected in the Australian lungfish proenkephalin cDNA. The fact that Leu-enkephalin has been identified by radioimmunoassay and HPLC analysis in the CNS of the Australian lungfish indicates that a Leu-enkephalin-coding gene, distinct from proenkephalin, must be expressed in lungfish. Potential candidates may include a prodynorphin- or other opioid-like gene. Furthermore, the absence of a Leu-enkephalin sequence in lungfish and amphibian proenkephalin would suggest that the mutations that yielded this opioid sequence in tetrapod proenkephalin occurred at some point in the radiation of the amniote vertebrates.

Pulmonary surfactant, a mixture consisting of phospholipids (PL) and proteins, is secreted by type II cells in the lungs of all air-breathing vertebrates. Virtually nothing is known about the factors that control the secretion of pulmonary surfactant in nonmammalian vertebrates. With the use of type II cell cultures from Australian lungfish, North American bullfrogs, and fat-tailed dunnarts, we describe the autonomic regulation of surfactant secretion among the vertebrates. ACh, but not epinephrine (Epi), stimulated total PL and disaturated PL (DSP) secretion from type II cells isolated from Australian lungfish. Both Epi and ACh stimulated PL and DSP secretion from type II cells of bullfrogs and fat-tailed dunnarts. Neither Epi nor ACh affected the secretion of cholesterol from type II cell cultures of bullfrogs or dunnarts. Pulmonary surfactant secretion may be predominantly controlled by the autonomic nervous system in nonmammalian vertebrates. The parasympathetic nervous system may predominate at lower body temperatures, stimulating surfactant secretion without elevating metabolic rate. Adrenergic influences on the surfactant system may have developed subsequent to the radiation of the tetrapods. Furthermore, ventilatory influences on the surfactant system may have arisen at the time of the evolution of the mammalian bronchoalveolar lung. Further studies using other carefully chosen species from each of the vertebrate groups are required to confirm this hypothesis.

The polypeptide hormone precursor, proopiomelanocortin (POMC), was cloned and sequenced from the pituitary of the Australian lungfish, Neoceratodus forsteri, the only surviving species of the oldest extant lineage of lungfish. The Australian lungfish POMC cDNA had an open reading frame that coded for a 255-amino acid precursor. A comparison of POMC sequences from the Australian lungfish and the African lungfish indicated that the deduced amino acid sequences for ACTH, beta-MSH, and beta-endorphin were over 90% identical. Furthermore, within the open reading frames of the two lungfish POMCs, there was 84% identity at the nucleotide level. Although a gamma-MSH-like region was detected in the Australian lungfish POMC cDNA, this sequence contained mutations that have been detected in the gamma-MSH sequences of some ray-finned fish and are not found in the gamma-MSH sequence of the African lungfish or those of tetrapods. In addition, the sequence of beta-endorphin in the two species of lungfish has amino acid motifs that are found in the beta-endorphin sequences of cartilaginous fish and ray-finned fish but not in tetrapods. However, maximum parsimony analysis of the entire POMC open reading indicated that the lungfish POMC sequences form a clade with two amphibian POMC sequences rather than with POMC sequences from ray-finned fish. This result is consistent with the accepted view that the sarcopterygians (lungfishes and tetrapods) are a monophyletic assemblage. Analysis of rates of divergence for various POMC sequences indicate that point mutations are accumulating in the lungfish POMC sequences at a slower rate than in either amphibian or mammalian POMC sequences. The phylogenetic implications of these observations are discussed.

A diverse range of animals, including elasmobranchs and nonteleost fish, use passive electroreception to locate hidden prey. The Australian lungfish, Neoceratodus forsteri (Krefft 1870), has ampullary organs analogous in form to the electroreceptors of other nonteleost fish. Afferents from these ampullae project to regions in the brain that are known to process electrosensory information in other species, suggesting that N. forsteri possesses an electric sense that may be used during prey location. To explore this hypothesis directly, we first characterized food-locating behaviour in N. forsteri and then conducted an experiment designed to quantify the effects of manipulating electrical and olfactory stimuli from live prey. A small crayfish, Cherax destructor, was housed in a specially constructed chamber hidden beneath the substrate, which prevented emission of chemical, mechanical and visual cues, but allowed transmission of bioelectric fields. Control treatments included presentation of electrically shielded prey, a dead crayfish and an empty chamber. In some treatments, a competing olfactory signal was presented simultaneously at the other end of the test tank to assess the relative salience of this sensory modality. The lungfish responded to the crayfish in the unshielded chamber with accurate and sustained feeding movements, even with a competing olfactory signal. By contrast, the abolition of electrical cues in the three control treatments reduced the accuracy and frequency of feeding movements in the vicinity of the target chamber. These results show that N. forsteri is capable of perceiving the weak electric fields surrounding living animals, and suggest that it uses this information when foraging to locate prey hidden from view. Copyright 1999 The Association for the Study of Animal Behaviour.

Tissues from a range of fish were examined for the presence of parathyroid hormone-related protein (PTHrP) to investigate PTHrP protein distribution and PTHrP gene expression in jawless fish, cartilaginous fish, and bony fish. Immunoreactive PTHrP was localized using antisera to N-terminal and mid-molecule regions of human PTHrP and PTHrP gene expression examined using a digoxigenin labeled riboprobe to a conserved region of the mammalian PTHrP gene. In all of the fish studied, PTHrP protein and messenger RNA (mRNA) were localized to the skin, kidney, and skeletal muscle, following the pattern seen in higher vertebrates. Additional sites of localization for both protein and mRNA included gill, nerve cord, and pituitary, as well as developing dermal denticles and rectal gland in the elasmobranch species. The sites of PTHrP distribution indicate that PTHrP may have roles in ionoregulation as well as growth and differentiation in fish, as has been suggested in higher vertebrates. The results imply that the distribution of PTHrP is widespread in fish and that there is homology between the PTHrP molecules found in humans and fish. The conservation of localization and possible similarity of the PTHrP molecules between tetrapods and fish suggests that PTHrP has a number of fundamental roles in vertebrates. J. Exp. Zool. 284:541-548, 1999.

The Australian lungfish Neoceratodus forsteri, a facultative air breather, is considered to be the most primitive of the extant Dipnoi and so occupies a uniquely important evolutionary position in the transition from fish to tetrapods. Insulin was isolated from an extract of the pancreas of N. forsteri and its primary structure established as: A-Chain, Gly-Ile-Val-Glu-Gln-Cys-Cys-His-Thr-Pro 10 -Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys 20 -Asn-Glu-Thr-Glu; B-Chain, Ala-Ala-Val-Asn-Gln-His-Leu-Cys-Gly-Ser 10 -His-Leu-Val-Glu-Ala-Leu- Tyr-Phe-Val-Cys 20 -Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Leu-Pro- Lys 30 -Gly. This amino acid sequence is more similar to that of human insulin than to insulins from present-day amphibians. All the residues in human insulin that are considered to be important in receptor binding, dimerization, and hexamerization are conserved in lungfish insulin except for the substitution (Leu → Phe) at the position corresponding to B17 in human insulin. Consistent with the assertion that the Dipnoi is a monophyletic group, insulins from N. forsteri and from the African lungfish Protopterus annectens contain extensions to the C-terminus of the A-chain and to the N-terminus of the B-chain that have not been found in other sarcopterygian species. However, the unusual amino acid substitutions found in insulin from P. annectens (e.g., GlyB21 → Ala, GluB22 → Asp, and ArgB23 → Asn) are not present in N. forsteri insulin, suggesting that they occurred in the Protopterus lineage after divergence of the genera.

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.

Angiotensin I (ANG I) was produced from the incubation of lungfish plasma with homologous kidney extracts. The purified peptide was found to have the sequence of H-Asn-Arg-Val-Tyr-Val-His-Pro-Phe-Thr-Leu-OH, which is homologous for the first eight residues with all teleost angiotensins so far sequenced, although lungfish generally possess tetrapod-type hormones. The lungfish decapeptide (ANG I) induced dose-dependent increases in arterial pressure in the rat. The lungfish octapeptide (ANG II) released aldosterone from kidney–adrenal tissue in vitro in a dose-dependent manner and induced dose-dependent increases in arterial pressure of the lungfish. Substitution of asparagine with aspartic acid in the first position (tetrapod-type ANG II) did not alter the blood pressure response significantly, but a second substitution of the valine in the (5)-position with isoleucine (ANG II form found in human and rat) abolished the rise in arterial pressure in lungfish over the same dose range.

We have isolated cDNA clones encoding a putative glycoprotein hormone alpha subunit precursor molecule from a pituitary cDNA library of the Australian lungfish ( Neoceratodus forsteri ) by a two-step plaque hybridization technique initially using cDNA encoding the toad glycoprotein hormone alpha subunit precursor molecule as the hybridization probe. The inserts (799 bp) of two of the isolated cDNA clones contained sequences of 5′ and 3′ untranslated regions, including a poly(A) stretch, and the entire coding sequence of the alpha subunit precursor molecule. The deduced amino acid sequence showed that its signal peptide consists of 24 residues and its mature protein 97 residues. In the mature protein, there is an insert of one residue (Ala) just after the 9th residue. This insert is unique to the Australian lungfish among vertebrate species studied. The amino acid sequence of the mature protein shares the common, or the same-group, amino acid residues at 9 positions with tetrapod and not actinopterygian vertebrates, while only one residue is common to some teleosts and the lungfish to the exclusion of the tetrapods. The overall sequence of the mature protein of the Australian lungfish also shares more similarity with those of tetrapods (69 to 84%) than it does with teleosts (57 to 74%). These results on the relation of the alpha subunit precursor molecule between the lungfish and other vertebrates are consistent with the recent molecular phylogenetic studies suggesting a closer relationship between lungfish and tetrapods than between lungfish and teleosts. We also found that the primary structure of the lungfish alpha subunit is slightly but significantly more similar to that of homeothermic vertebrates than to that of amphibians. This may be due to specialization of the amphibian alpha subunit molecules.

The development of the pineal, pituitary and thyroid glands of the extant lungfish, Neoceratodus forsteri , are being studied both morphologically and functionally. This paper presents data from hatching to 40–52 weeks for a standardised series of lungfish, bred at Macquarie University. At hatching, the pineal comprises a single organ attached to the roof of the diencephalon, with well-developed photoreceptor, supporting and ganglion cells. The photoreceptors gradually degenerate, giving way to secretory cells which contain electron dense granules. These latter are immunoreactive to melatonin antibodies and digestable with protease. The pituitary at hatching comprises a hollow ball of cells lying beneath the infundibular region of the hypothalamus. Ultrastructurally, four cell types can be distinguished by cytoplasmic granule size after the first four weeks of development posthatching. By 20 weeks, a further three cell types are recognisable. Inununogold labelling has identified corticotropes and melanotropes at four weeks and, at 20 weeks, prolactin cells, thyrotropes and somatotropes also can be identified. The thyroid is only just apparent at hatching, containing 2–3 follicles. Thenumbers of follicles increases gradually, and variably between animals, with age. Iodine uptake in methimazole-treated animals did not exceed that of controls at any of the three stages tested, indicating a lack of feedback control between thyroid hormones and pituitary thyrotropes at, or before, 40 weeks of age. Thyroid hormone receptors in the liver at 40 weeks are predominantly immunoreactive to human TRac antibodies. These findings taken together suggest that, up to 40 weeks post hatching, lungfish development is equivalent to amphibian premetamorphic development. This would be consistent with lungfish neoteny, but cannot be taken as evidence for neoteny until confirmed at later stages of development

In view of the well-established role of neurohypophysial hormones in osmoregulation of terrestrial vertebrates, lungfishes are a key group for study of the molecular and functional evolution of the hypothalamo-neurohypophysial system. Here we report on the primary structure of the precursors encoding vasotocin (VT) and [Phe 2 ]mesotocin ([Phe 2 ]MT) of the Australian lungfish, Neoceratodus forsteri . Genomic sequence analysis and Northern blot analysis confirmed that [Phe 2 ]MT is a native oxytocin family peptide in the Australian lungfish, although it has been reported that the lungfish neurohypophysis contains MT. The VT precursor consists of a signal peptide, VT, that is connected to a neurophysin by a Gly-Lys-Arg sequence, and a copeptin moiety that includes a Leu-rich core segment and a glycosylation site. In contrast, the [Phe 2 ]MT precursor does not contain a copeptin moiety. These structural features of the lungfish precursors are consistent with those in tetrapods, but different from those in teleosts where both VT and isotocin precursors contain a copeptin-like moiety without a glycosylation site at the carboxyl terminals of their neurophysins. Comparison of the exon/intron organization also supports homology of the lungfish [Phe 2 ]MT gene with tetrapod oxytocin/MT genes, rather than with teleost isotocin genes. Moreover, molecular phylogenetic analysis shows that neurohypophysial hormone genes of the lungfish are closely related to those of the toad. The present results along with previous morphological findings indicate that the hypothalamo-neurohypophysial system of the lungfish has evolved along the tetrapod lineage, whereas the teleosts form a separate lineage, both within the class Osteichthyes .

Gambusia holbrooki is a small, aggressive fish introduced into Australia in 1925 to control mosquitoes. It has been suggested that this species may also prey on tadpoles and be a contributing factor in the decline of some Australian frog populations. We examined the influence of tadpole body size (10 mm. 15 mm and 20 mm). predator prey ratio (1 : 1, 1 :2 and 1 :4) and nutritional status of the predator on the level of predation by G. holbrooki on tadpoles of Limnodynasfas peronii. Unfed fish attacked all three size classes of tadpoles without any significant preference but did so more vigorously when the predator/prey ratio was densest. Predator/prey ratio had no significant overall effect on attack of tadpoles by fed fish but there was a preference for the smaller tadpoles during the first six hours of the experiment. Unfed fish also consumed more of the tadpoles they attacked than did the fed fish.Field surveys were carried out on 10 permanent water bodies in north-west Sydney to examine any correlation...

Gonadal sex differentiation is temperature-dependent in Alligator mississippiensis; testis differentiation occurs in embryos incubated at 33°C and ovary differentiation occurs in embryos incubated at 30°C. Laminin and cytokeratin were examined immunohistochemically in the gonads of alligator embryos incubated at these temperatures. The aim of this study was to determine whether these structural proteins show the same sex-specific expression patterns reported for mammalian embryos, and to assess their usefulness as early markers of gonadal differentiation in species with temperature-dependent sex determination. Laminin delineated enlarged seminiferous cords in differentiating testes from developmental stage 23 to hatching. Laminin distribution was more diffuse and revealed smaller cords of cells in differentiating ovaries. Cytokeratin was also detected in developing gonads of both sexes. Cytokeratin became concentrated in the basal cytoplasm of differentiating Sertoli cells in developing testes. In developing ovaries, prefollicular cells of the ovarian cortex and cell cords in the medulla stained strongly for cytokeratin. Cytokeratin did not show the same basal distribution in female medullary cord cells as seen in the Sertoli cells of testes, however. These sex-specific patterns of laminin and cytokeratin distribution in embryonic alligator gonads may serve as early markers of sexual differentiation.

Sexual differentiation of the gonads in Alligator mississippiensis and many other oviparous reptiles is controlled by egg incubation temperature. Estrogens are thought to play a role in this process, and it has been hypothesized that estrogen production is thermosensitive in species with temperature-dependent sex determination (TSD). Using the tritiated water assay, we measured the activity of the critical estrogen-synthesising enzyme, P450 aromatase, in the gonad-adrenal-mesonephric kidney complex (GAM) of alligator embryos incubated at male- and female-producing temperatures. Aromatase activity increased in the GAM of developing embryos incubated at 30° C (100% female-producing) and 34.5° C (predominantly female-producing), while it remained very low throughout development in embryos incubated at the intermediate temperature of 33° C (100% male-producing). However, it is unclear whether enhanced aromatase activity represents the initial signal for ovary differentiation or whether it lies downstream in the female developmental pathway. For embryos incubated at 30° C (female-producing), there was no detectable increase in aromatase activity until developmental stage 24, which is after the temperature-sensitive period for sex determination. This suggests that aromatase may be a downstream component of the ovary-determining cascade. In female alligator hatchlings, most of the aromatase activity was localised in the ovary, activity being low in the adrenal-mesonephros. Aromatase assays carried out at 30° C and at 33° C indicated that, at viable incubation temperatures, aromatase activity is not thermosensitive. This suggests that temperature directly or indirectly influences enzyme synthesis. Radioimmunoassay of estradiol synthesised by the GAM during the aromatase assay confirmed increased enzyme activity during female development but not during male development. Increased aromatase activity and estrogen synthesis during female development were correlated with the timing of ovary differentiation, particularly proliferation of the gonadal cortex. These findings implicate aromatase in temperature-dependent gonadal sex differentiation in alligator embryos, higher enzyme activity being associated with ovary development.

Our current understanding of the lungfish immune system is limited. This study is characterizing the immune cells separated from primary and secondary immune organs of the Australian lungfish, Neoceratodus forsteri . Our functional studies utilized flow cytometry to study the immune cells extracted from the thymus, spiral valve intestine, spleen, and kidney. The different characteristics of lymphocytes and granulocytes were analyzed by utilization of viability, phagocytosis, oxidative burst, and apoptosis assays. Most of the nonviable intestinal cells were lymphocytes. Depending on the organ, 6–25% of the total population, predominantly granulocytes, underwent phagocytosis where the splenic cells were the most and intestinal cells the least phagocytic cells. Cells responded positively but differently to stimulation with phorbol myristate acetate (PMA) to produce radical oxygen species, an indication of their oxidative burst activity, which was mainly associated with granulocytes. Although cells were induced by dexamethasone to undergo apoptosis, such an induction did not follow a consistent pattern of dose of dexamethasone or incubation time between the different organs. In the absence of monoclonal antibodies against lungfish immune cells, these functional flow cytometric analyses aid our understanding on the functionality of immune cells.

Haploid genomes greater than 25,000 Mb are rare, within the animals only the lungfish and some of the salamanders and crustaceans are known to have genomes this large. There is very little data on the structure of genomes this size. It is known, however, that for animal genomes up to 3,000 Mb, there is in general a good correlation between genome size and the percent of the genome composed of repetitive sequence and that this repetitive component is highly dynamic. In this study, we sampled the Australian lungfish genome using three mini-genomic libraries and found that with very little sequence, the results converged on an estimate of 40% of the genome being composed of recognizable transposable elements (TEs), chiefly from the CR1 and L2 long interspersed nuclear element clades. We further characterized the CR1 and L2 elements in the lungfish genome and show that although most CR1 elements probably represent recent amplifications, the L2 elements are more diverse and are more likely the result of a series of amplifications. We suggest that our sampling method has probably underestimated the recognizable TE content. However, on the basis of the most likely sources of error, we suggest that this very large genome is not largely composed of recently amplified, undetected TEs but may instead include a large component of older degenerate TEs. Based on these estimates, and on Thomson's (Thomson K. 1972. An attempt to reconstruct evolutionary changes in the cellular DNA content of lungfish. J Exp Zool. 180:363-372) inference that in the lineage leading to the extant Australian lungfish, there was massive increase in genome size between 350 and 200 mya, after which the size of the genome changed little, we speculate that the very large Australian lungfish genome may be the result of a massive amplification of TEs followed by a long period with a very low rate of sequence removal and some ongoing TE activity.

Egg incubation temperature controls sexual differentiation of the gonads in the saltwater crocodile, Crocodylus porosus. Embryos incubated at 30°C develop ovaries and hatch as females while most embryos incubated at 32–32.5°C develop testes and hatch as males. Several lines of evidence suggest that estrogen is involved in gonadal sex differentiation in crocodilians and other reptiles with temperature-dependent sex determination (TSD). In vivo autoradiography was used here to identify sites of 3H-estradiol uptake in the urogenital tissues of C. porosus embryos incubated at 30°C (female-producing) and at 32.5°C (predominantly male-producing). During the period of gonadal differentiation (developmental stage 23), 3H-estradiol was specifically concentrated in the gonads of presumptive females at 30°C and presumptive males at 32.5°C. Binding was localised primarily in the medulla of the gonad in both sexes, with little or no uptake in the cortex. No uptake was detected in the adjoining adrenal glands, at either temperature, although some uptake was observed in the mesonephros at the female temperature (30°C). No specific binding of estradiol by the gonads was found prior to (stage 19) or after (stage 28) the period of gonadal differentiation. These results are consistent with the hypothesis that estrogen binding sites are present in the gonads of both sexes during a specific period of embryogenesis, which coincides with the time of gonadal sex differentiation. Furthermore, these observations suggest that the role of estrogen in TSD is not mediated through differential uptake of the steroid by presumptive ovaries and testes. It is therefore hypothesised that estrogen synthesis is temperature-sensitive, rather than estrogen binding by the gonad. © 1994 Wiley-Liss, Inc.

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.

Temperature-dependent sex determination (TSD) was first described for a crocodilian in Alligator mississippiensis, in which egg incubation at 33°C produces 100% male hatchlings and incubation at 30°C produces 100% females. Ultrastructural signs of testis differentiation in embryos incubated at 33°C are first detected during days 28–34 (developmental stages 21–22), when small numbers of enlarged “presumptive” Sertoli cells (pre-Sertoli cells) appear in the gonadal medulla of most embryos. These pre-Sertoli cells proliferate and differentiate into Sertoli cells during stage 23 at 33°C (days 35–41), marking seminiferous cord formation. At 30°C, some pre-Sertoli-like cells are also present in the medulla, but they are less numerous than at 33°C and they do not differentiate into Sertoli cells. Ovary differentiation at 30°C is characterized by proliferation of cortical germs cells from day 39 (stage 22), closely followed by proliferation of somatic (pre-follicular) cells in the cortex and regression of the medullary cords from day 43 (stage 23). We hypothesize that TSD in the alligator might be a function of a temperature-derived mismatch between Sertoli cell differentiation and germ cell/cortex proliferation. © 1994 Wiley-Liss, Inc.

The following is a brief description of how lungfish research at Macquarie University began, of the period in which it flourished, and, most recently, of the winding down of the University’s involvement with this research. During this latter period, the Australian lungfish in the wild were threatened by the construction of a megadam in their very limited habitat. Fortunately, this was averted in December 2009, after 3 years of lobbying the Federal Government. They now await another “Aussie” to make them accessible for further research by Australian and international researchers.