Darryl L Whitehead | The University of Queensland, Australia (original) (raw)

Papers by Darryl L Whitehead

Vertebrate sex differentiation follows a conserved suite of developmental events: the bipotential... more Vertebrate sex differentiation follows a conserved suite of developmental events: the bipotential gonads differentiate and shortly thereafter sex specific traits become dimorphic. However, this may not apply to squamates, a diverse vertebrate lineage comprising of many species with thermosensitive sexual development. Of the three species with data on the relative timing of gonad differentiation and genital dimorphism, the females of two (Niveoscincus ocellatus and Barisia imbricata) exhibit a phase of temporary pseudohermaphroditism or TPH (gonads have differentiated well before genital dimorphism). We report a third example of TPH in Pogona vitticeps, an agamid with temperature-induced male to female sex reversal. These findings suggest that for female squamates, genital and gonad development may not be closely synchronised, so that TPH may be common. We further observed a high frequency of ovotestes, a usually rare gonadal phenotype characterised by a mix of male and female structures, exclusively associated with temperature-induced sex reversal. We propose that ovotestes are evidence of a period of antagonism between male and female sex-determining pathways during sex reversal. Female sexual development in squamates is considerably more complex than has been appreciated, providing numerous avenues for future exploration of the genetic and hormonal cues that govern sexual development. Sex determination and differentiation in amniotes is widely accepted to follow a well-defined sequence 1–3. Early in development, the bipotential gonads differentiate, then secrete sex-specific steroid hormones, which are thought to prompt the development of sex-specific traits, such as the male Wolffian or female Müllerian ducts (and regression of the opposing sex ducts), and the external genitalia (e.g. hemipenes/hemiclitores) 1,2,4–6. Among reptiles the primary sex-determining cue can be either temperature or genetic 3. In temperature-dependent sex determination (TSD), incubation temperature determines the sex of the individual during the thermosensitive period, which usually occurs in the middle-third of development 7,8. The mechanism by which temperature influences sexual development in squamates (snakes and lizards) is not fully understood but is likely to involve epigenetic remodelling via altered expression and/or splicing of chromatin modifying genes 9–11. In contrast, gonadal differentiation is controlled in other squamates, by the presence, absence or dosage of as yet unidentified genes on sex chromosomes (genetic sex determination or GSD) 12–14. Regardless of whether sex is controlled by TSD or GSD, the downstream molecular processes of gonad differentiation appear to be highly conserved 15–18. Although organisms tend to be classified as either TSD or GSD in the literature, in some species sex can be determined via gene–environment interactions 19,20. This can occur when GSD is overridden by high or low incubation temperatures. In most cases of sex reversal in nature, the phenotype of the homogametic sex (ZZ or XX) becomes discordant with the sex chromosomes, though there are rare, mostly experimental examples of het-erogametic (XY or ZW) sex reversal 3,21,22. Such gene-environment interactions are possibly more common than assumed 20 , occurring in at least three squamate species-the spotted skink (Niveoscincus ocellatus), the three-lined

Molecular & Cellular Proteomics, 2013

Although it has been established that all toxicoferan squamates share a common venomous ancestor,... more Although it has been established that all toxicoferan squamates share a common venomous ancestor, it has remained unclear whether the maxillary and mandibular venom glands are evolving on separate gene expression trajectories or if they remain under shared genetic control. We show that identical transcripts are simultaneously expressed not only in the mandibular and maxillary glands, but also in the enigmatic snake rictal gland. Toxin molecular frameworks recovered in this study were threefinger toxin (3FTx), CRiSP, crotamine (beta-defensin), cobra venom factor, cystatin, epididymal secretory protein, kunitz, L-amino acid oxidase, lectin, renin aspartate protease, veficolin, and vespryn. We also discovered a novel low-molecular weight disulfide bridged peptide class in pythonid snake glands. In the iguanian lizards, the most highly expressed are potentially antimicrobial in nature (crotamine (beta-defensin) and cystatin), with crotamine (beta-defensin) also the most diverse. However, a number of proteins characterized from anguimorph lizards and caenophidian snakes with hemotoxic or neurotoxic activ-ities were recruited in the common toxicoferan ancestor and remain expressed, albeit in low levels, even in the iguanian lizards. In contrast, the henophidian snakes express 3FTx and lectin toxins as the dominant transcripts. Even in the constricting pythonid and boid snakes, where the glands are predominantly mucous-secreting, low-levels of toxin transcripts can be detected. Venom thus appears to play little role in feeding behavior of most iguanian lizards or the powerful constricting snakes, and the low levels of expression argue against a defensive role. However, clearly the incipient or secondarily atrophied venom systems of these taxa may be a source of novel compounds useful in drug design and discovery.

The differentiated serous-secreting dental glands of caenophidian snakes are diverse in form desp... more The differentiated serous-secreting dental glands of caenophidian snakes are diverse in form despite their developmental homology. This variation makes the eluci-dation of their evolutionary history a complex task. In addition, some authors identify as many as ten discrete types/subtypes of ophidian oral gland. Over the past decade and a half, molecular systematics and toxinology have deepened our understanding of the evolution of these fascinating and occasionally enigmatic structures. This paper includes a comprehensive examination of ophidian oral gland structure and (where possible) function, as well as new data on rictal glands and their associated anatomy. Following this, appropriate use of terminology, especially that pertaining to homologous structures (including the controversial ''venom gland'' vs ''Duvernoy's gland'' debate), is considered. An interpretation of the evolutionary history of the ophidian venom system, drawing on recent results from molecular systematics, toxinology and palaeontology, concludes the paper.

Journal of Physiology Paris, 2002

The ampulla of Lorenzini of juvenile Carcharhinus leucas differ histologically from those previou... more The ampulla of Lorenzini of juvenile Carcharhinus leucas differ histologically from those previously described for other elasmobranchs. The wall of the ampullary canal consists of protruding hillock-shaped epidermal cells that appear to secrete large quantities of a mucopolysaccharide gel. The ampullary organs comprise a long canal sheathed in collagen terminating in an ampulla. Each ampulla contains six alveolar sacs, with each sac containing hundreds of receptor cells. The receptor cells are characteristic of others described for elasmobranchs being pear-shaped cells with a central nucleus and bearing a single kinocilium in the exposed apical region of the cell. The supportive cells differ from general elasmobranch ampullary histology in that some have an apical nucleus. These ampullary structures allow Carcharhinus leucas to detect and respond to artificial electrical fields. Carcharhinus leucas from freshwater habitats respond to electrical signals supplied in freshwater aquaria by abruptly turning towards low voltage stimuli (410 mA) and either swimming over or biting at the origin of the stimulus. #

Journal of morphology, Jan 18, 2015

Journal of Morphology, 2015

The morphology of ampullary organs in Plicofollis argyropleuron, collected from a southeast Queen... more The morphology of ampullary organs in Plicofollis argyropleuron, collected from a southeast Queensland estuary, was examined by light and electron microscopy to assess the morphological characteristics of teleost ampullary organs in environments with fluctuating salinities. This catfish possesses both macroampullae and microampullae. Both have the typical teleost arrangement of an ampullary pore linked by a canal to a single ampulla that is lined with receptor and supportive cells. The canal wall of macroampullae consists of a collagen sheath, a basement membrane, and two layers of squamous epithelial cells adjacent to the lumen, joined by desmosomes and tight junctions near the surface of the epithelium. Ampullary pore diameters are similar in range for both the macroampullae and the microampullae, with microampullae always arising from the larger pores within a single region of the head. Canal length of the macroampullae is dramatically longer than those of the microampullae. Macroampullae also contain approximately 10 times as many receptor cells compared with the microampullae. In both organs, these pear-shaped receptor cells alternate with supportive cells along the entire luminal surface of the ampulla. The apical region of receptor cells extends into the lumen and bears numerous microvilli. The basal region of receptor cells adjoins to either individual or multiple unmyelinated neural terminals. The coexistence of two markedly different ampullary organ morphologies within a single species support theories concerning the possible multifunctionality of these sensory organs. J. Morphol., 2015. © 2015 Wiley Periodicals, Inc.

Venom research and technology has advanced greatly, rapidly transforming our knowledge of reptile... more Venom research and technology has advanced greatly, rapidly transforming our knowledge of reptile venoms. Research advances, like the development of molecular systematics, provide the framework necessary to reconstruct the evolutionary history of glands and fangs. Such research developments have expanded our understanding of venom's evolution and its usefulness in therapeutic development. The results of this punctuated toxin molecular evolutionary expansion include protein neofunctionalization. While these changes may impact antivenom efficacy, this molecular diversity also facilitates their usefulness in the development of novel drug therapies. Venomous Reptiles And Their Toxins brings together the world's leading toxinologists in this comprehensive study of the entire scope of reptile venoms, from clinical effects to evolution to drug design and development. The book contains detailed applied chapters on clinical care of the envenomed patient, ineffective traditional or mo...

Journal of Physiology-Paris, 2002

Journal of Fish Biology, 2008

A study observing the foraging behaviours and prey discrimination of a common demersal stingray, ... more A study observing the foraging behaviours and prey discrimination of a common demersal stingray, the bluespotted maskray Dasyatis kuhlii was performed under controlled laboratory conditions. A selection of prey species and masses were offered at depths of 10 and 50 mm in sand. Foraging efficiency and prey selection at both burial depths were compared. Dasyatis kuhlii selected the ghost shrimps, Trypaea australiensis and T. australiensis >2Á5 g, range AE 0Á2 g though foraging errors represented by prey being excavated and not consumed suggested a limited discriminatory ability at the point of detection. Burial depth did not influence prey species, mass selection or discriminatory ability.

Biomaterials, 2014

Previous reports in the literature investigating chondrogenesis in mesenchymal progenitor cell (M... more Previous reports in the literature investigating chondrogenesis in mesenchymal progenitor cell (MPC) cultures have confirmed the chondro-inductive potential of pentosan polysulphate (PPS), a highly sulphated semi-synthetic polysaccharide, when added as a soluble component to culture media under standard aggregate-assay conditions or to poly(ethylene glycol)/hyaluronic acid (PEG/HA)-based hydrogels, even in the absence of inductive factors (e.g. TGFb). In this present study, we aimed to assess whether a 'bound' PPS would have greater activity and availability over a soluble PPS, as a media additive or when incorporated into PEG/HA-based hydrogels. We achieved this by covalently pre-binding the PPS to the HA component of the gel (forming a new molecule, HA-PPS). We firstly investigated the activity of HA-PPS compared to free PPS, when added as a soluble factor to culture media. Cell proliferation, as determined by CCK8 and EdU assay, was decreased in the presence of either bound or free PPS whilst chondrogenic differentiation, as determined by DMMB assay and histology, was enhanced. In all cases, the effect of the bound PPS (HA-PPS) was more potent than that of the unbound form. These results alone suggest wider applications for this new molecule, either as a culture supplement or as a coating for scaffolds targeted at chondrogenic differentiation or maturation. We then investigated the incorporation of HA-PPS into a PEG/HA-based hydrogel system, by simply substituting some of the HA for HA-PPS. Rheological testing confirmed that incorporation of either HA-PPS or PPS did not significantly affect gelation kinetics, final hydrogel modulus or degradation rate but had a small, but significant, effect on swelling. When encapsulated in the hydrogels, MPCs retained good viability and rapidly adopted a rounded morphology. Histological analysis of both GAG and collagen deposition after 21 days showed that the incorporation of the bound-PPS into the hydrogel resulted in increased matrix formation when compared to the addition of soluble PPS to the hydrogel, or the hydrogel alone. We believe that this new generation injectable, degradable hydrogel, incorporating now a covalently bound-PPS, when combined with MPCs, has the potential to assist cartilage regeneration in a multitude of therapeutic targets, including for intervertebral disc (IVD) degeneration.

Biomaterials, 2013

Intervertebral disc (IVD) degeneration is one of the leading causes of lower back pain and a majo... more Intervertebral disc (IVD) degeneration is one of the leading causes of lower back pain and a major health problem worldwide. Current surgical treatments include excision or immobilisation, with neither approach resulting in the repair of the degenerative disc. As such, a tissue engineering-based approach in which stem cells, coupled with an advanced delivery system, could overcome this deficiency and lead to a therapy that encourages functional fibrocartilage generation in the IVD. In this study, we have developed an injectable hydrogel system based on enzymatically-crosslinked polyethylene glycol and hyaluronic acid. We examined the effects of adding pentosan polysulphate (PPS), a synthetic glycosaminoglycan-like factor that has previously been shown (in vitro and in vivo) to this gel system in order to induce chondrogenesis in mesenchymal precursor cells (MPCs) when added as a soluble factor, even in the absence of additional growth factors such as TGF-β. We show that both the gelation rate and mechanical strength of the resulting hydrogels can be tuned in order to optimise the conditions required to produce gels with the desired combination of properties for an IVD scaffold. Human immunoselected STRO-1+ MPCs were then incorporated into the hydrogels. They were shown to retain good viability after both the initial formation of the gel and for longer-term culture periods in vitro. Furthermore, MPC/hydrogel composites formed cartilage-like tissue which was significantly enhanced by the incorporation of PPS into the hydrogels, particularly with respect to the deposition of type-II-collagen. Finally, using a wild-type rat subcutaneous implantation model, we examined the extent of any immune reaction and confirmed that this matrix is well tolerated by the host. Together these data provide evidence that such a system has significant potential as both a delivery vehicle for MPCs and as a matrix for fibrocartilage tissue engineering applications.

Abstract: Cnidarian venom research has lagged behind other toxinological fields due to technical ... more Abstract: Cnidarian venom research has lagged behind other toxinological fields due to
technical difficulties in recovery of the complex venom from the microscopic nematocysts.
Here we report a newly developed rapid, repeatable and cost effective technique of venom
preparation, using ethanol to induce nematocyst discharge and to recover venom contents
in one step. Our model species was the Australian box jellyfish (Chironex fleckeri), which
has a notable impact on public health. By utilizing scanning electron microscopy and
light microscopy, we examined nematocyst external morphology before and after ethanol
treatment and verified nematocyst discharge. Further, to investigate nematocyst content or
“venom” recovery, we utilized both top-down and bottom-up transcriptomics–proteomics
approaches and compared the proteome profile of this new ethanol recovery based method
to a previously reported high activity and recovery protocol, based upon density purified
intact cnidae and pressure induced disruption. In addition to recovering previously
characterized box jellyfish toxins, including CfTX-A/B and CfTX-1, we recovered putative
metalloproteases and novel expression of a small serine protease inhibitor. This study not
only reveals a much more complex toxin profile of Australian box jellyfish venom but also
suggests that ethanol extraction method could augment future cnidarian venom proteomics
research efforts.

Journal of morphology, Jan 22, 2015

We hypothesized that due to the relative conductivity of the environment, and to maintain sensory... more We hypothesized that due to the relative conductivity of the environment, and to maintain sensory function, ampullary organs of marine Neoarius graeffei would differ morphologically from those described previously for estuarine and freshwater conspecifics. Unlike the ampullary systems of N. graeffei from freshwater and estuarine habitats, the ampullary pores of marine specimens occur in two distinct patterns; numerous pores seemingly randomly scattered on the head and ventro-lateral regions of the body, and pores arranged in distinctive vertical lines above the lateral line on the dorso-lateral body of the fish. Light and electron microscopy revealed that the ampullary organs also differed morphologically from estuarine and freshwater specimens in the presence of longer ampullary canals, a hitherto unreported canal wall composition, and in the collagen sheath surrounding both the canal and the ampulla proper within dermal connective tissues. Ampullary pores were wider in marine indi...

Toxins, 2015

Cnidarian venom research has lagged behind other toxinological fields due to technical difficulti... more Cnidarian venom research has lagged behind other toxinological fields due to technical difficulties in recovery of the complex venom from the microscopic nematocysts. Here we report a newly developed rapid, repeatable and cost effective technique of venom preparation, using ethanol to induce nematocyst discharge and to recover venom contents in one step. Our model species was the Australian box jellyfish (Chironex fleckeri), which has a notable impact on public health. By utilizing scanning electron microscopy and light microscopy, we examined nematocyst external morphology before and after ethanol treatment and verified nematocyst discharge. Further, to investigate nematocyst content or "venom" recovery, we utilized both top-down and bottom-up transcriptomics-proteomics approaches and compared the proteome profile of this new ethanol recovery based method to a previously reported high activity and recovery protocol, based upon density purified intact cnidae and pressure induced disruption. In addition to recovering previously characterized box jellyfish toxins, including CfTX-A/B and CfTX-1, we recovered putative metalloproteases and novel expression of a small serine protease inhibitor. This study not only reveals a much more complex toxin profile of Australian box jellyfish venom but also suggests that ethanol extraction method could augment future cnidarian venom proteomics research efforts.

Animal Biology, 2004

Passive electroreception is a complex and specialised sense found in a large range of aquatic ver... more Passive electroreception is a complex and specialised sense found in a large range of aquatic vertebrates primarily designed for the detection of weak bioelectric elds. Particular attention has traditionally focused on cartilaginous shes, but a range of teleost and non-teleost shes from a diversity of habitats have also been examined. As more species are investigated, it has become apparent that the role of electroreception in shes is not restricted to locating prey, but is utilised in other complex behaviours. This paper presents the various functional roles of passive electroreception in non-electric shes, by reviewing much of the recent research on the detection of prey in the context of differences in species' habitat (shallow water, deep-sea, freshwater and saltwater). A special case study on the distribution and neural groupings of ampullary organs in the omnihaline bull shark, Carcharhinus leucas, is also presented and reveals that prey-capture, rather than navigation, may be an important determinant of pore distribution. The discrimination between potential predators and conspeci cs and the role of bioelectric stimuli in social behaviour is discussed, as is the ability to migrate over short or long distances in order to locate environmentally favourable conditions. The various theories proposed regarding the importance and mediation of geomagnetic orientation by either an electroreceptive and/or a magnetite-based sensory system receives particular attention. The importance of electroreception to many species is emphasised by highlighting what still remains to be investigated, especially with respect to the physical, biochemical and neural properties of the ampullary organs and the signals that give rise to the large range of observed behaviours.

Journal of Morphology, 2014

Ampullae of Lorenzini were examined from juvenile Carcharhinus leucas (831-1,045 mm total length)... more Ampullae of Lorenzini were examined from juvenile Carcharhinus leucas (831-1,045 mm total length) captured from freshwater regions of the Brisbane River. The ampullary organ structure differs from all other previously described ampullae in the canal wall structure, the general shape of the ampullary canal, and the apically nucleated supportive cells. Ampullary pores of 140-205 mm in diameter are distributed over the surface of the head region with 2,681 and 2,913 pores present in two sharks that were studied in detail. The primary variation of the ampullary organs appears in the canal epithelial cells which occur as either flattened squamous epithelial cells or a second form of pseudostratified contour-ridged epithelial cells; both cell types appear to release material into the ampullary lumen. Secondarily, this ampullary canal varies due to involuted walls that form a clover-like canal wall structure. At the proximal end of the canal, contour-ridged cells abut a narrow region of cuboidal epithelial cells that verge on the constant, six alveolar sacs of the ampulla. The alveolar sacs contain numerous receptor and supportive cells bound by tight junctions and desmosomes. Pear-shaped receptor cells that possess a single apical kinocilium are connected basally by unmyelinated neural boutons. Opposed to previously described ampullae of Lorenzini, the supportive cells have an apical nucleus, possess a low number of microvilli, and form a unique, jagged alveolar wall. A centrally positioned centrum cap of cuboidal epithelial cells overlies a primary afferent lateral line nerve. J. Morphol. 000:000-000,

Zoomorphology, 2009

Small epidermal pores of the electrosensory ampullae of Lorenzini located both ventrally and dors... more Small epidermal pores of the electrosensory ampullae of Lorenzini located both ventrally and dorsally on the disk of Aptychotrema rostrata (Shaw and Nodder, 1794) open to jelly-Wlled canals, the distal end of which widens forming an ampulla that contains 6 § 0.7 alveolar bulbs (n = 13). The sensory epithelium is restricted to the alveolar bulbs and consists of receptor cells and supportive cells. The receptor cells are ellipsoid and their apical surfaces are exposed to the alveolar lumen with each bearing a single central kinocilium. Presynaptic bodies occur in the basal region of the receptor cell immediately proximal to the synaptic terminals. The supportive cells that surround receptor cells vary in shape. Microvilli originate from their apical surface and extend into the alveolar lumen. Tight junctions and desmosomes connect the supportive cells with adjacent supportive and receptor cells in the apical region. The canal wall consists of two cell layers, of which the luminal cells are squamous and interconnect via desmosomes and tight junctions, whereas the cells of the deeper layer are heavily interdigitated, presumably mechanically strengthening the canal wall. Columnar epithelial cells form folds that separate adjacent alveoli. The same cells separate the ampulla and canal wall. An aVerent sensory nerve composed of up to nine myelinated nerve axons is surrounded by several layers of collagen Wbers and extends from the ampulla. Each single aVerent neuron can make contacts with multiple receptor cells. The ultrastructural characteristics of the ampullae of Lorenzini in Aptychotrema rostrata are very similar to those of other elasmobranch species that use electroreception for foraging.

Journal of Morphology, 1999

Whole body staining of Arius graeffei revealed that ampullary pores cover the body with their hig... more Whole body staining of Arius graeffei revealed that ampullary pores cover the body with their highest densities occurring on the head and lowest densities on the mid-ventral surface. Each ampullary organ consists of a long canal (0.2-1.75 mm) passing perpendicular to the basement membrane, through the epidermis into underlying dermal connective tissues, curving thereafter to run roughly parallel to the epidermis. Histochemical staining techniques (Alcian blue and LillieЈs allochrome) indicate that the canals contain a neutral to acidic glycoprotein-based mucopolysaccharide gel that varies in composition along the length of the canal. Collagen fibers, arranged in a sheath, surround a layer of squamous epithelium that lines each ampullary canal. At the proximal end of the canal, squamous cells are replaced by cuboidal epithelial cells that protrude into the lumen, thus constricting the lumen to form a small pore into the ampulla. The ampulla is lined with receptor and supportive cells. The numerous (60-120) pear-shaped receptor cells bear microvilli on their luminal surface. Two forms of receptor cells exist in each ampullary organ: basal and equatorial receptor cells. Each receptor cell is connected to an unmyelinated nerve. Each receptor cell is surrounded by supportive cells on all but the apex. Tight junctions and underlying desmosomes occur between adjacent receptor and supportive cells. This form of ampullary organ has not previously been described for teleosts.

Journal of Morphology, 2003

Whole body studies of Plotosus tandanus revealed that ampullary pores occur over the entire body ... more Whole body studies of Plotosus tandanus revealed that ampullary pores occur over the entire body of the fish, but are in higher concentrations in the head region. These pores give rise to a short canal (50 -60 m) produced by columnar epithelial cells bound together by tight junctions and desmosomes. At the junction of the canal and the ampulla, cuboidal epithelial cells make up the wall. The ampulla consists of layers of collagen fibers that surround flattened epithelial cells in the lateral regions and give rise to supportive cells that encase a small number of receptor cells (10 -15). The ampullary wall comprises several types of cells that are adjoined via tight junctions and desmosomes between cell types. The ovoid receptor cells possess microvilli along the luminar apical area. Beneath this area, the cells are rich in mitochondria and rough endoplasmic reticulum. An unmyelinated neuron adjoins with each receptor cell opposite multiple presynaptic bodies. This form of microampulla has not been previously described within the Family Plotosidae. J. Morphol. 255:253-260, 2003.