Reptilian transferrins: evolution of disulphide bridges and conservation of iron-binding center

Transferrins, found in invertebrates and vertebrates, form a physiologically important family of proteins playing a major role in iron acquisition and transport, defense against microbial pathogens, growth and differentiation. These proteins are bilobal in structure and each lobe is composed of two domains divided by a cleft harboring an iron atom. Vertebrate transferrins comprise of serotransferrins, lactoferrins and ovotransferrins. In mammals serotransferrins transport iron in physiological fluids and deliver it to cells, while lactoferrins scavenge iron, limiting its availability to invading microbes. In oviparous vertebrates there is only one transferrin gene, expressed either in the liver to be delivered to physiological fluids as serotransferrin, or in the oviduct with a final localization in egg white as ovotransferrin. Being products of one gene sero- and ovotransferrin are identical at the amino-acid sequence level but with different, cell specific glycosylation patterns. Our knowledge of the mechanisms of transferrin iron binding and release is based on sequence and structural data obtained for human serotransferrin and hen and duck ovotransferrins. No sequence information about other ovotransferrins was available until our recent publication of turkey, ostrich, and red-eared turtle (TtrF) ovotransferrin mRNA sequences [Ciuraszkiewicz, J., Olczak, M., Watorek, W., 2006. Isolation, cloning and sequencing of transferrins from red-eared turtle, African ostrich and turkey. Comp. Biochem. Physiol. 143 B, 301-310]. In the present paper, ten new reptilian mRNA transferrin sequences obtained from the Nile crocodile (NtrF), bearded dragon (BtrF), Cuban brown anole (AtrF), veiled and Mediterranean chameleons (VtrF and KtrF), sand lizard (StrF), leopard gecko (LtrF), Burmese python (PtrF), African house snake (HtrF), and grass snake (GtrF) are presented and analyzed. Nile crocodile and red-eared turtle transferrins have a disulphide bridge pattern identical to known bird homologues. A partially different disulphide bridge pattern was found in the Squamata (snakes and lizards). The possibility of a unique interdomain disulphide bridge was predicted for LtrF. Differences were found in iron-binding centers from those of previously known transferrins. Substitutions were found in the iron-chelating residues of StrF and TtrF and in the synergistic anion-binding residues of NtrF. In snakes, the transferrin (PtrF, HtrF and GtrF) N-lobe "dilysine trigger" occurring in all other known transferrins was not found, which indicates a different mechanism of iron release.     

Vertebrate evolution reflected in the evolution of nuclear ribosomal internal transcribed spacer 2

In eukaryotes, mature rRNA sequences are produced from single large (45S) precursor (pre-rRNA) as the result of successive removal of spacers through a series of rapid and intricate actions of endo- and exonucleases. The excision of internal transcribed spacer (ITS2), a eukaryotic-specific insertion, remains the most elusive processing step. ITS2 is the element mandatory for all eukaryotic pre-rRNAs that contain at least three processing cleavage sites for precise 5.8S and 28S formation. Conserved core sequences (cis-elements) binding to trans-factors provide for precise rRNA processing, whereas rapidly diverging regions between the core sequences preserve internal complementarity, which guarantees the spatial integrity of ITS2. Characteristic differences in the formation of such insertions during evolution should reflect the relationships between taxa. The phylogeny of the reptiles and the relationships between taxa proposed by scientists are controversial. To delineate the structural and functional features preserved among reptilian ITS2s, we cloned and sequenced 58 ITS2s belonging to four reptile orders: Squamata, Crocodilians, Aves, and Testudines. We studied the subsequent alignment and folding of variable regions. The sizes and packing of the loop–stems between conserved consensus segments in reptiles vary considerably between taxa. Our phylogenetic trees constructed on the basis of the reptile ITS2s primary structural alignments revealed a split between Iguania clade and all other taxa. True lizards (suborder Scleroglossa) and snakes (suborder Serpentes) show sister relationships, as well as the two other reptilian orders, Crocodilia + Aves and Testudines. In summary, our phylogenetic trees exhibit a mix of specific features deduced or, to the contrary, rejected earlier by other authors.     

Nest-site selection in two eublepharid gecko species with temperature-dependent sex determination and one with genotypic sex determination

At present, most turtles, all crocodilians, and several lizards are known to have temperature-dependent sex determination (TSD). Due to the dependence of sex determination on incubation temperature, the long-term survival of TSD species may be jeopardized by global climate changes. The current study was designed to assess the degree to which this concern is justified by examining nest-site selection in two species of Pattern II TSD geckos (Eublepharis macularius and Hemitheconyx caudicinctus) and comparing these preferences with those of a species with genotypic sex determination (GSD) (Coleonyx mitratus). Temperature preferences for nest sites were found to be both species-specific and female-specific. While H. caudicinctus females selected a mean nest-site temperature (32.4°) very close to the upper pivotal temperature (32°C) for the species, E. macularius females selected a mean nest-site temperature (28.7°C) well below this species' lower pivotal temperature (30.5°C). Thus, the resultant sex ratios are expected to differ between these two TSD species. Additionally, nest-site temperatures for the GSD species were significantly more variable (SE=+0.37) than were temperatures for either of the TSD species (E. macularius SE=±0.10; H. caudicinctus SE =+ 0.17), diereby further demonstrating temperature preferences within the TSD species.     

Male sexual behavior does not require elevated testosterone in a lizard (Coleonyx elegans, Eublepharidae)

Male sexual behavior depends on gonadal androgens in species of all major vertebrate lineages, including reptiles. However, male sexual behavior includes distinct appetitive and consummatory phases, typically denoted as courtship and mounting, with potentially different hormonal control. Different proximate controls of courtship versus mounting could enable disconnected evolutionary losses and gains of various aspects of male sexual behavior. Male courtship display, which is activated by testosterone (T) in many species, is an ancestral trait in the lizard family Eublepharidae. However, Coleonyx elegans (Yucatan Banded Gecko) lost the courtship display, while retaining a highly simplified male sexual behavior that involves only mounting for copulation. We performed surgical manipulations (castration with and without T replacement in adult males; implantation of adult females with exogenous T) to investigate hormonal mechanisms involved in this evolutionary novelty. Our results indicate that the expression of simplified sexual behavior in C. elegans does not require elevated circulating levels of T, a finding that is previously unreported in lizards. In females, however, exogenous T induced male-like mounting. Thus, the mounting phase of sexual behavior is not activated by T in the traditional sense of this term but probably requires post-natal, maturational organization (if not periodic reorganization) by androgens. We conclude that the simplification of male sexual behavior and its independence from elevated levels of circulating androgens in C. elegans evolved via 1) evolutionary loss of the androgen-activated courtship display and 2) retention of the mounting phase, which has a longer “functional memory” for the effects of androgenic steroids.     

Body-size effect on egg size in eublepharid geckos (Squamata: Eublepharidae), lizards with invariant clutch size: negative allometry for egg size in ectotherms is not universal

Within a single clutch, smaller species of ectotherms generally lay a smaller number of relatively larger eggs than do larger species. Many hypotheses explaining both the interspecific negative allometry in egg size and egg size–number trade-off postulate the existence of an upper limit to the egg size of larger species. Specifically, in lizards, large eggs of large species could have too long a duration of incubation, or they could be too large to pass through the pelvic opening, which is presumably constrained mechanically in larger species. Alternatively, negative allometry could be a result of limits affecting eggs of smaller species. Under the latter concept, hatchling size in smaller species may be close to the lower limit imposed by ecological interactions or physiological processes, and therefore smaller species have to invest in relatively larger offspring. Contrary to these lower limit hypotheses, explanations based on the existence of an upper limit always predict negative egg-size allometry even in animals with invariant clutch size, in which naturally there is no egg size–number trade-off. We studied egg-size allometry in lizards of the family Eublepharidae, a monophyletic group of primitive geckos with large variance in body size and an invariant number of two eggs per clutch. We found an isometric relationship between egg and female size that does not support the upper limit hypotheses.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 88 , 527–532.     

Autocrine and paracrine Shh signaling are necessary for tooth morphogenesis, but not tooth replacement in snakes and lizards (Squamata)

Here we study the role of Shh signaling in tooth morphogenesis and successional tooth initiation in snakes and lizards (Squamata). By characterizing the expression of Shh pathway receptor Ptc1 in the developing dentitions of three species (Eublepharis macularius, Python regius, and Pogona vitticeps) and by performing gain- and loss-of-function experiments, we demonstrate that Shh signaling is active in the squamate tooth bud and is required for its normal morphogenesis. Shh apparently mediates tooth morphogenesis by separate paracrine- and autocrine-mediated functions. According to this model, paracrine Shh signaling induces cell proliferation in the cervical loop, outer enamel epithelium, and dental papilla. Autocrine signaling within the stellate reticulum instead appears to regulate cell survival. By treating squamate dental explants with Hh antagonist cyclopamine, we induced tooth phenotypes that closely resemble the morphological and differentiation defects of vestigial, first-generation teeth in the bearded dragon P. vitticeps. Our finding that these vestigial teeth are deficient in epithelial Shh signaling further corroborates that Shh is needed for the normal development of teeth in snakes and lizards. Finally, in this study, we definitively refute a role for Shh signaling in successional dental lamina formation and conclude that other pathways regulate tooth replacement in squamates.     

Ultrastructure and distribution of chromatophores in the skin of the leopard gecko (Eublepharis macularius)

The aim of this study was to describe the ultrastructure and arrangement of pigment cells in the leopard gecko (Eublepharis macularius) skin to explain how wild‐type coloration is formed. The study also attempted to explain, on a morphological level, how skin colour changes occur. Samples of leopard gecko skin were collected from wild‐type coloration adult specimens. The morphology of pigmented cells was determined using light microscopy on haematoxylin and eosin (H&E) stained sections and in transmission electron microscopy. These studies indicate that skin of E. macularis contains xanthophores and melanophores but lacks iridophores and that this is probably related to nocturnal activity. The number and distribution of xanthophores and melanophores determines the skin colour and pigmentation pattern. The colour changes depend on the arrangement of characteristic protrusions of melanophores and the degree of filling them with melanosomes.     

Posture, speed, and habitat structure: three-dimensional hindlimb kinematics of two species of padless geckos

Differences in habitat use are often correlated with differences in morphology and behavior, while animals in similar habitats often exhibit similarities in form and function. However, this has not been tested extensively among lizards, especially geckos. Most studies of gecko locomotion have focused on the ability to adhere to surfaces. However, there are several species of geckos that have either secondarily lost adhesive capabilities or simply lack the capability. We quantified the three-dimensional locomotor kinematics for two desert-dwelling padless geckos, Teratoscincus scincus and Eublepharis macularius, on a level trackway over a range of speeds. Our results indicate that T. scincus landed with a high relative hip height of 48.7 ± 2.4% of total limb length at footfall, while E. macularius exhibited hip heights averaging only 36.0 ± 1.8% of total limb length for footfall. The three-dimensional knee angle of T. scincus averaged 120.6 ± 3.9° at footfall, while E. macularius averaged only 101.6 ± 1.8° at footfall. In addition, the femur of E. macularius was elevated to a much greater extent (i.e., was closer to being perpendicular to the long axis of the body) than that of T. scincus and every other lizard that has been studied, suggesting they move with a “hyper-sprawled” posture. Both of these gecko species live in deserts, but T. scincus is psammophilic while E. macularius inhabits a rocky, more densely vegetated environment. Benefits of the more upright posture of T. scincus on open sandy habitat may include a greater field of view and more efficient locomotion. The more sprawled posture of E. macularius may lower its center of gravity and aid in balance while climbing on rocks or shrubs.     

Body size, male combat and the evolution of sexual dimorphism in eublepharid geckos (Squamata: Eublepharidae)

Lizards of the family Eublepharidae exhibit interspecific diversity in body size, sexual size dimorphism (SSD), head size dimorphism (HSD), occurrence of male combat, and presence of male precloacal pores. Hence, they offer an opportunity for testing hypotheses for the evolution and maintenance of sexual dimorphism. Historical analysis of male agonistic behaviour indicates that territoriality is ancestral in eublepharid geckos. Within Eublepharidae, male combat disappeared twice. In keeping with predictions from sexual selection theory, both events were associated with parallel loss of male-biased HSD and ventral scent glands. Eublepharids therefore provide new evidence that male-biased dimorphic heads are weapons used in aggressive encounters and that the ventral glands probably function in territory marking rather than in intersexual communication. Male-biased SSD is a plesiomorphic characteristic and was affected by at least three inversions. Shifts in SSD and male combat were not historically correlated. Therefore, other factors than male rivalry appear responsible for SSD inversions. Eublepharids demonstrate the full scope of Rensch's rule (small species tend to be female-larger, larger species male-larger). Most plausibly, SSD pattern hence seems to reflect body size variation. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 76 , 303–314.