Establishment and characterization of two cell lines derived from primary cultures of Gekko japonicus cerebral cortex

Adult Gekko japonicus is one of those vertebrates that are able to regenerate their missing or amputated tail. The most interesting feature of this animal lies in the ability of its spinal cord to regrow a functional tail. A fundamental question is whether the neuroglial cells play a different role compared with high vertebrates. Since in vitro studies using primary neuroglial cells are hampered by the limited lifespan and miscellaneous genetic background of these cells, we generated neuroglial cell lines from primary cell cultures of cerebral cortex of G. japonicus. The SV40 (simian‐virus‐40) T antigen gene was introduced into primary cell cultures. Cell cycle analysis, cell growth and proliferation, cell colony formation and contact inhibition, as well as karyotype assays were investigated. Two cell colonies, Gsn‐1 and Gsn‐3, were immunochemically characterized as glial fibrillary acidic protein and galactocerebroside‐positive respectively. Compared with parental primary cells, the Gsn cells displayed shorter population doubling time, decreased percentage of cells in the G0/G1 phase, higher cell proliferation index, and increased cell activity. In assays of colony characteristics, Gsn cells showed increased cell activity at the lower cell densities or FBS (fetal bovine serum) supplement. The karyotype of immortalized Gsn cells exhibited transformational characteristics with hyperdiploid and polyploid chromosomes. The cell lines will provide a useful in vitro model for gecko neuroglial cells and facilitate systematic studies investigating the biological functions of specific gene products related to regeneration of the central nervous system.     

Tail regeneration in the gecko Sphaerodactylus argus shows that the formation of an axial elastic skeleton is functional for the new tail

Tail regeneration in the gecko Sphaerodactylus argus shows that the formation of an axial elastic skeleton is functional for the new tail (Acta Zoologica, Stockolm). The present autoradiographic and immunohistochemical study describes tail regeneration and formation of the axial skeleton in early regenerating tails of the Jamaican red-tailed gecko, Sphaerodactylus argus. Cell proliferation, studied by tritiated thymidine, shows intense labelling mainly in forming scales and differentiating cartilaginous, muscle and ependymal cells of the regenerating spinal cord, while the labelling is more diffuse in the apical blastema and proximal connective tissues. The slow apical proliferation maintains the tail front growing while in more proximal regions, cells initiate differentiation, losing thymidine-labelling. Cell proliferation is maximal at the beginning of scales, muscles and cartilage formation. Scales are regenerated following migration into the dermis of tritiated thymidine-labelled keratinocytes to form epithelial pegs that later split and give rise new scales. Differentiation of new corneous layers begins underneath the external corneous epidermis, starting with a shedding layer followed by a beta-layer that accumulates corneous beta proteins. Intense proliferation of apical myoblasts gives rise to long myotubes and segmented muscles. The vertebral column is substituted with a cartilaginous tube made of turgid chondrocytes accumulating chondroitin sulphate proteoglycan and elastin. Therefore, the regenerated tail remains flexible and capable of curling to maintain efficient the climbing ability in these geckos.     

Multi-Scale Compliant Foot Designs and Fabrication for Use with a Spider-Inspired Climbing Robot

Climbing robots are of potential use for surveillance, inspection and exploration in different environments. In particular, the use of climbing robots for space exploration can allow scientists to explore environments too challenging for traditional wheeled designs. To adhere to surfaces, biomimetic dry adhesives based on gecko feet have been proposed. These biomimetic dry adhesives work by using multi-scale compliant mechanisms to make intimate contact with different surfaces and adhere by using Van der Waals forces. Fabrication of these adhesives has frequently been challenging however, due to the difficulty in combining macro, micro and nanoscale compliance. We present an all polymer foot design for use with a hexapod climbing robot and a fabrication method to improve reliability and yield. A high strength, low-modulus silicone, TC-5005, is used to form the foot base and microscale fibres in one piece by using a two part mold. A macroscale foot design is produced using a 3D printer to produce a base mold, while lithographic definition of microscale fibres in a thick photoresist forms the ＇hairs＇ of the polymer foot. The adhesion of the silicone fibres by themselves or attached to the macro foot is examined to determine best strategies for placement and removal of feet to maximize adhesion. Results demonstrate the successful integration of micro and macro compliant feet for use in climbing on a variety of surfaces.     

Comparative studies on the structure and adhesion of setae in G. gecko and G. swinhonis

Scanning electron microscopy (SEM) and histological techniques were used to observe and study the setae structures of two gecko species (G. gecko and G. swinhonis) and the relationships between these structures and the adhesive forces. The SEM results showed that the setae of these two species were densely distributed in an orderly fashion, and branched with curved tips. The setae of G. gecko had cluster structures, each cluster containing 4-6 setae whose terminal branches curved towards the center of the toes at ~ 10o, the tips of the branches like spatulae and densely arrayed at an interval of less than 0.2―0.3 μm. On the contrary, the branch tips in the setae of G. swinhonis were curled, and the terminal parts of setae curved towards the center of the toes at various angles. Usually the setae of these gecko species branch twice at the top at intervals greater than that of G. gecko. The histological observation found that inside the setae of these two species there were plenty of unevenly distributed contents, such as epithelia, fat cells, pigmental cells and muscle tissue, but no gland cells existed. The results of functional experiments suggested that modifying the structure of gecko's setae could reduce its adhesive ability dramatically, demonstrating the positive correlation between the structure of the gecko's setae and its adhesive ability. The above results provide important information in designing bio-mimic setae and bio-gecko robots.     

Quantification of three steroid hormone receptors of the leopard gecko (Eublepharis macularius), a lizard with temperature-dependent sex determination: their tissue distributions and the effect of environmental change on their expressions

Sex steroid hormones play a central role in the reproduction of all vertebrates. These hormones function through their specific receptors, so the expression levels of the receptors may reflect the responsibility of target organs. However, there was no effective method to quantify the expression levels of these receptors in reptilian species. In this study, we established the competitive-PCR assay systems for the quantification of the mRNA expression levels of three sex steroid hormone receptors in the leopard gecko. These assay systems were successfully able to detect the mRNA expression level of each receptor in various organs of male adult leopard geckoes. The expression levels of mRNA of these receptors were highly various depending on the organs assayed. This is the first report regarding the tissue distributions of sex steroid hormone receptor expressions in reptile. The effects of environmental conditions on these hormone receptor expressions were also examined. After the low temperature and short photoperiod treatment for 6 weeks, only the androgen receptor expression was significantly increased in the testes. The competitive-PCR assay systems established in this report should be applicable for various studies of the molecular mechanism underlying the reproductive activity of the leopard gecko.     

Using testis size to predict the mating systems of New Zealand geckos

Abstract

The mating system is unknown for the majority of New Zealand geckos (Family Gekkonidae). I investigated interspecific variation in relative testis volume across 19 New Zealand gecko taxa and 63 additional species of squamate reptiles from other countries to make predictions about the social mating system of the New Zealand geckos. Relative testis size varied greatly between species of New Zealand gecko, with testes ranging from 119% larger than expected to 75% smaller than expected. This variation was even greater across other squamates, with testes ranging from 55 to 152% of expected size. This variation could not be explained by seasonality of breeding, clutch size or the sex ratio of populations. As species subject to more intense sperm competition sport relatively larger testes in other vertebrate groups, it is likely that similar variation in gecko testis size is due to differences in mating systems between species. Based on testis volume, it is predicted that at least half of the New Zealand gecko taxa studied are likely to have polygynandrous mating systems.     

Flip,flop and fly: modulated motor control and highly variable movement patterns of autotomized gecko tails

Many animals lose and regenerate appendages, and tail autotomy in lizards is an extremely well-studied example of this. Whereas the energetic, ecological and functional ramifications of tail loss for many lizards have been extensively documented, little is known about the behaviour and neuromuscular control of the autotomized tail. We used electromyography and high-speed video to quantify the motor control and movement patterns of autotomized tails of leopard geckos (Eublepharis macularius). In addition to rhythmic swinging, we show that they exhibit extremely complex movement patterns for up to 30 min following autotomy, including acrobatic flips up to 3 cm in height. Unlike the output of most central pattern generators (CPGs), muscular control of the tail is variable and can be arrhythmic. We suggest that the gecko tail is well suited for studies involving CPGs, given that this spinal preparation is naturally occurring, requires no surgery and exhibits complex modulation.     

Phylogenetic relationships among gekkotan lizards inferred from C-mos nuclear DNA sequences and a new classification of the Gekkota

A phylogeny of gekkotan lizards was derived from C- mos nuclear DNA sequence data. Forty-one currently recognized genera, representing all major gekkotan lineages, were included in the study. A total of 378 bp of partial C- mos gene sequences was obtained and aligned. Maximum parsimony (MP) and maximum likelihood (ML) trees were generated based on unweighted analysis using P AUP *; similar tree topologies were recovered by both methods. The Eublepharidae were monophyletic and its relationship to other major clades was poorly resolved. The Pygopodidae of Kluge (1987) was monophyletic, but relationships within this group differed from those retrieved by previous analyses. The Diplodactylini + padded carphodactylines were the sister group of pygopods + padless carphodactylines. The Gekkonidae were monophyletic, but we found no evidence in support of the Teratoscincinae, as Teratoscincus was embedded well within the gekkonids. Both MP and ML analyses supported the basal position of Sphaerodactylus within the gekkonids, in contrast to morphologically based hypotheses. We propose a new higher order classification of the Gekkota that reflect these results. Five gekkotan families: Eublepharidae, Gekkonidae, Pygopodidae, Diplodactylidae, and Carphodactylidae are recognized. The higher order status of the sphaerodactyls will require more intensive sampling of this group. Our results support the hypothesis that the early cladogenesis of the Gekkota was associated with the split of Eastern Gondwanaland from Western Gondwanaland. Divergences among living genera in the Eublepharidae and the Eastern Gondwanan lineages (Diplodactylidae, Pygopodidae and Carphodactylidae) may be older than those in the Gekkonidae.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 83 , 353–368.     

Multiscale modelling and simulation of the deformation and adhesion of a single gecko seta

A 3D multiscale model is presented which describes the adhesion and deformation of a gecko seta. The multiscale approach combines three models at different length scales: at the top level, on the order of several micrometers, a nonlinear finite element beam model is chosen to capture the branched microstructure of the gecko seta. At the intermediate level, on the order of several nanometers, a second finite element model is used to capture the detailed behaviour of the seta tips, the so-called spatulae. At the lowest level, on the order of a few angstroms, a molecular interaction potential is used to describe the van der Waals adhesion forces between spatulae and substrate. Coarse-graining techiques are used to bridge the scale between the model levels. To illustrate and validate the proposed gecko seta model, numerical pull-off simulations are shown and compared to experimental data from the literature.     

Passively stuck: death does not affect gecko adhesion strength

Many geckos use adhesive toe pads on the bottom of their digits to attach to surfaces with remarkable strength. Although gecko adhesion has been studied for hundreds of years, gaps exist in our understanding at the whole-animal level. It remains unclear whether the strength and maintenance of adhesion are determined by the animal or are passively intrinsic to the system. Here we show, for the first time, that strong adhesion is produced passively at the whole-animal level. Experiments on both live and recently euthanized tokay geckos (Gekko gecko) revealed that death does not affect the dynamic adhesive force or motion of a gecko foot when pulled along a vertical surface. Using a novel device that applied repeatable and steady-increasing pulling forces to the foot in shear, we found that the adhesive force was similarly high and variable when the animal was alive (mean ± s.d. = 5.4 ± 1.7 N) and within 30 min after death (5.4 ± 2.1 N). However, kinematic analyses showed that live geckos are able to control the degree of toe pad engagement and can rapidly stop strong adhesion by hyperextending the toes. This study offers the first assessment of whole-animal adhesive force under extremely controlled conditions. Our findings reveal that dead geckos maintain the ability to adhere with the same force as living animals, disproving that strong adhesion requires active control.