Ruin Lizard Podarcis Sicula Research Articles

The lizard celestial compass detects linearly polarized light in the blue

The present study first examined whether ruin lizards, Podarcis sicula, are able to orientate using plane-polarized light produced by an LCD screen. Ruin lizards were trained and tested indoors, inside a hexagonal Morris water maze positioned under an LCD screen producing white polarized light with a single E-vector, which provided an axial cue. White polarized light did not include wavelengths in the UV. Lizards orientated correctly either when tested with E-vector parallel to the training axis or after 90 deg rotation of the E-vector direction, thus validating the apparatus. Further experiments examined whether there is a preferential region of the light spectrum to perceive the E-vector direction of polarized light. For this purpose, lizards reaching learning criteria under white polarized light were subdivided into four experimental groups. Each group was tested for orientation under a different spectrum of plane-polarized light (red, green, cyan and blue) with equalized photon flux density. Lizards tested under blue polarized light orientated correctly, whereas lizards tested under red polarized light were completely disoriented. Green polarized light was barely discernible by lizards, and thus insufficient for a correct functioning of their compass. When exposed to cyan polarized light, lizard orientation performances were optimal, indistinguishable from lizards detecting blue polarized light. Overall, the present results demonstrate that perception of linear polarization in the blue is necessary - and sufficient - for a proper functioning of the sky polarization compass of ruin lizards. This may be adaptively important, as detection of polarized light in the blue improves functioning of the polarization compass under cloudy skies, i.e. when the alternative celestial compass based on detection of the sun disk is rendered useless because the sun is obscured by clouds.

A sky polarization compass in lizards: the central role of the parietal eye

The present study first examined whether ruin lizards Podarcis sicula are able to orientate using the e-vector direction of polarized light. Ruin lizards were trained and tested indoors, inside a hexagonal Morris water maze, positioned under an artificial light source producing plane polarized light with a single e-vector, which provided an axial cue. Lizards were subjected to axial training by positioning two identical goals in contact with the centre of two opposite side walls of the Morris water maze. Goals were invisible because they were placed just beneath the water surface, and water was rendered opaque. The results showed that the directional choices of lizards meeting learning criteria were bimodally distributed along the training axis, and that after 90 deg rotation of the e-vector direction of polarized light the lizards directional choices rotated correspondingly, producing a bimodal distribution which was perpendicular to the training axis. The present results confirm in ruin lizards results previously obtained in other lizard species showing that these reptiles can use the e-vector direction of polarized light in the form of a sky polarization compass. The second step of the study aimed at answering the still open question of whether functioning of a sky polarization compass would be mediated by the lizard parietal eye. To test this, ruin lizards meeting learning criteria were tested inside the Morris water maze under polarized light after their parietal eyes were painted black. Lizards with black-painted parietal eyes were completely disoriented. Thus, the present data show for the first time that the parietal eye plays a central role in mediating the functioning of a putative sky polarization compass of lizards.

Orientation of lizards in a Morris water-maze: roles of the sun compass and the parietal eye

The present study examined for the first time whether a Morris water-maze can be used to explore compass and other orientation mechanisms in the ruin lizard Podarcis sicula. In the open field, during sunny days, lizards were individually trained to swim from the center of the water maze onto a hidden platform (the goal), positioned at the periphery of the maze in a single compass direction. The goal was invisible because it was placed just beneath the water surface and the water was rendered opaque. The results showed that lizards learn to swim directly towards the hidden goal under the sun in the absence of visual feature cues. We further examined whether the observed orientation response would be due to lizards learning the spatial position of the goal relative to the sun's azimuth, i.e. to the use of a time-compensated sun compass. Lizards reaching learning criteria were subjected to 6 h clock-shift (fast or slow), and tested for goal orientation in the Morris water-maze. Results demonstrated that the learned orientation response is mediated by a time-compensated sun compass. Further investigations provided direct evidence that in ruin lizards an intact parietal eye is required to perform goal orientation under the sun inside a Morris water-maze, and that other brain photoreceptors, like the pineal or deep brain photoreceptors, are not involved in orientation.

Influence of Temperature on the Liver Circadian Clock in the Ruin Lizard Podarcis sicula

Reptiles represent an interesting animal model to investigate the influence of temperature on molecular circadian clocks. The ruin lizard Podarcis sicula lives in a continental climate and it is subjected to wide range of environmental temperatures during the course of the year. As consequence, ruin lizard daily activity pattern includes either the hibernation or periods of inactivity determined by hypothermia. Here we showed the rhythmic expression of two clock genes, lPer2 and lClock, in the liver of active lizards exposed to summer photo-thermoperiodic conditions. Interestingly, the exposition of lizards to hypothermic conditions, typical of winter season, induced a strong dampening of clock genes mRNA rhythmicity with a coincident decrease of levels. We also examined the qualitative and quantitative distribution of lPER2 and lCLOCK protein in different cellular compartments during the 24-h cycle. In the liver of active lizards both proteins showed a rhythmic expression profile in all cellular compartments. After 3 days at 6 degrees C, some temporal fluctuations of the lCLOCK and lPER2 are still detectable, although, with some marked modifications in respect to the values detected in the liver of active lizards. Besides demonstrating the influence of low temperature on the lizard liver circadian oscillators, present results could provide new essential information for comparative studies on the influence of temperature on the circadian system across vertebrate classes.

Hypothermia modulates circadian clock gene expression in lizard peripheral tissues

The molecular mechanisms whereby the circadian clock responds to temperature changes are poorly understood. The ruin lizard Podarcis sicula has historically proven to be a valuable vertebrate model for exploring the influence of temperature on circadian physiology. It is an ectotherm that naturally experiences an impressive range of temperatures during the course of the year. However, no tools have been available to dissect the molecular basis of the clock in this organism. Here, we report the cloning of three lizard clock gene homologs (Period2, Cryptochrome1, and Clock) that have a close phylogenetic relationship with avian clock genes. These genes are expressed in many tissues and show a rhythmic expression profile at 29 degrees C in light-dark and constant darkness lighting conditions, with phases comparable to their mammalian and avian counterparts. Interestingly, we show that at low temperatures (6 degrees C), cycling clock gene expression is attenuated in peripheral clocks with a characteristic increase in basal expression levels. We speculate that this represents a conserved vertebrate clock gene response to low temperatures. Furthermore, these results bring new insight into the issue of whether circadian clock function is compatible with hypothermia.

Identification of circadian brain photoreceptors mediating photic entrainment of behavioural rhythms in lizards.

We have shown previously that in ruin lizards (Podarcis sicula) the ablation of all known photoreceptive structures (lateral eyes, pineal and parietal eye) in the same individual animal does not prevent entrainment of their circadian locomotor rhythms to light. The present study was aimed at identifying the circadian brain photoreceptors mediating entrainment. For this purpose, we looked for opsin expression in the brain by means of immunocytochemistry. Using anti-cone-opsin antiserum CERN 874 we have localized photoreceptors in the periventricular area of hypothalamus, near the third cerebral ventricle. We also cloned a brain opsin cDNA that, on the basis of the deduced amino acid sequence, appears to belong to the RH2 class of cone-opsins. We named the cloned cone-opsin Ps-RH2. To examine whether brain cone-opsins mediate photic entrainment of circadian locomotor rhythms, we performed post-transcriptional inactivation experiments by injecting an expression eukaryotic vector transcribing the antisense cone-opsin Ps-RH2 mRNA in the third cerebral ventricle of pinealectomized-retinectomized lizards previously entrained to a light-dark (LD) cycle. Injections of the antisense construct abolished photic entrainment of circadian locomotor rhythms of pinealectomized-retinectomized lizards to the LD cycle for 6-9 days. CERN 874 completely failed to label cells within the periventricular area of hypothalamus of brains injected with antisense construct. Thus, abolishment of photic entrainment is due to inactivation of endogenous brain cone-opsins mRNA. The present results demonstrate for the first time in a vertebrate that brain cone-opsins are part of a true circadian brain photoreceptor participating in photic entrainment of behavioural rhythms.

Daily and circadian rhythms of neurotransmitters and related compounds in the hypothalamic suprachiasmatic nuclei of a diurnal vertebrate

By using immunocytochemistry we tested whether neurotransmitters, and enzymes specific to neurotransmitters synthesis are rhythmically expressed in the suprachiasmatic nuclei of the hypothalamus of Ruin lizards Podarcis sicula either kept in light–dark cycles or constant darkness. Within the suprachiasmatic nuclei, prominent 24 h rhythms under 12:12 light–dark cycles were found for vasoactive intestinal polypeptide (VIP) and for tyrosine hydroxylase (TH). Peaks of both VIP and TH fell in the light phase of the cycle. Rhythmic expression of TH persisted under constant temperature and darkness, demonstrating the existence of circadian rhythms of TH in the suprachiasmatic nuclei. No rhythmic expression of neurotransmitters and related compounds was found in the periventricular nuclei, the supraoptic nuclei, and the rest of the hypothalamus. Our data are the first demonstration of rhythmic expression of neurotransmitters and related compounds in the suprachiasmatic nuclei of a non-mammalian vertebrate. The demonstration of a diurnal peak of VIP in a diurnal reptile—vs. nocturnal peak of VIP typical of nocturnal mammals—provides new information for comparative studies on the circadian physiology of the suprachiasmatic nuclei across vertebrate classes and their adaptation strategies to different temporal niches.

Photoperiod affects amplitude but not duration of in vitro melatonin production in the ruin lizard (Podarcis sicula).

The pineal gland and its major output signal melatonin have been demonstrated to play a central role in the seasonal organization of the ruin lizard Podarcis sicula. Seasonal variations in the amplitude of the nocturnal melatonin signal, with high values in spring as compared to low values in summer and autumn, have been found in vivo. The authors examined whether the pineal gland of the ruin lizard contains autonomous circadian oscillators controlling melatonin synthesis and whether previously described seasonal variations of in vivo melatonin production can also be found in isolated cultured pineal glands obtained from ruin lizards in summer and winter. In vitro melatonin release from isolated pineal glands of the ruin lizard persisted for 4 days in constant conditions. Cultured explanted pineal glands obtained from animals in winter and summer showed similar circadian rhythms of melatonin release, characterized by damping of the amplitude of the melatonin rhythm. Although different photoperiodic conditions were imposed on ruin lizards before explantation of pineal glands, the authors did not find any indication for corresponding differences in the duration of elevated melatonin in vitro. Differences were found in the amplitude of in vitro melatonin production in light/dark conditions and, to a lesser degree, in constant conditions. The presence of a circadian melatonin rhythm in vitro in winter, although such a rhythm is absent in vivo in winter, suggests that pineal melatonin production is influenced by an extrapineal oscillator in the intact animal that may either positively or negatively modulate melatonin production in summer and winter, respectively.

Toward a seasonal model of the circadian system: the case of Ruin lizards.

The pineal gland plays a role in the seasonal re-organization of the circadian system that occurs in Ruin lizard Podarcis sicula. In fact, either pinealectomy or melatonin implants induce an immediate transition from the circadian locomotor pattern typical of summer to the circadian locomotor pattern typical of spring or autumn. Furthermore, the degree of involvement of melatonin and the pineal in circadian organization changes dramatically with season: 1. The effects of pinealectomy, as well as those of melatonin implants on circadian locomotor rhythms are strong in summer and weak in the other seasons; 2. Daily melatonin injections entrain locomotor rhythms in summer, but not in other seasons; 3. A phase response curve for melatonin was generated in summer, but not in winter; 4. Daily injections of luzindole, an antagonist for melatonin receptors alter locomotor rhythms in summer, but not in other seasons; 5. A robust pineal-dependent circadian rhythm of circulating melatonin exists in summer, but not in spring or in autumn. This confirms that the central role of the pineal in determining circadian organization in summer and the marginal role of the pineal in either spring or autumn are respectively due to appearance (summer) or disappearance (spring, autumn) of a circadian melatonin signal in the circulation.

Fine structure of the external nasal gland of two lizards (Podarcis sicula campestris and Chalcides chalcides).

In this study, we describe the ultrastructural features of the external nasal gland in two lizards: ruin lizard (Podarcis sicula campestris) and seps (Chalcides chalcides). Two secretory cell types, which differ interspecifically, have been found in the secretory endpieces of the glandular tubules in both species examined. An unusual morphological observation was the presence of paracrystalline structures in the secretory granules of the seromucous cells of the external nasal gland of the seps. These structures may be related to the packaging mechanism of glycoproteins or to their macromolecular structure. They may also reflect segregation of heterogeneous subcomponents within the same secretory granule. The striated cells are located in the distal segment of the glandular tubules, and have the typical ultrastructural features of the cells which in some species of reptiles, but not in these two lizards, are known to be capable of elaborating a hyperosmotic saline solution.

Seasonal Variations of Pineal Involvement in the Circadian Organization of the Ruin Lizard Podarcis Sicula

To establish whether the effects of pinealectomy on circadian locomotor rhythmicity vary with season, we examined, in constant temperature and darkness, the locomotor behaviour of ruin lizards Podarcis sicula collected and subjected to pinealectomy at different times of the year. Changes in the freerunning period in response to pinealectomy were found to be significantly greater in summer than in winter, spring and autumn. Circadian activity time changed significantly in response to pinealectomy only in spring and summer. Furthermore, while pinealectomy was effective in altering the locomotor rhythms of all individual lizards tested in summer, the same surgery was found to leave locomotor rhythmicity of many lizards tested in autumn and winter completely undisturbed. These results demonstrate for the first time in a non-mammalian vertebrate that the pineal gland is centrally involved in determining circadian organization in some seasons and is only marginally involved in others.

Circadian organization in the ruin lizard Podarcis sicula: the role of the suprachiasmatic nuclei of the hypothalamus

The effects of electrolytic lesions to the suprachiasmatic nuclei of the hypothalamus (SCN) on circadian rhythms of locomotor activity were examined in ruin lizards Podarcis sicula maintained in constant darkness and constant temperature (29°C). All lizards (N=15) in which the lesion damaged 80% or more of the SCN became behaviorally arrhythmic. On the contrary, locomotor rhythms persisted in all cases (N=11) when the SCN remained intact and lesions were confined to neighbouring regions of the preoptic area. Taken together with previous work which demonstrates that the pineal and the retinae are not essential for the persistence of circadian locomotor rhythmicity in Podarcis sicula and with recent evidence showing the homology between the SCN of lizards and those of mammals the present results strongly support the view that the SCN of Podarcis sicula contain the primary pacemaker(s) for locomotor rhythms.

Seasonal changes of locomotor activity patterns in ruin lizards Podarcis sicula

Seasonal differences in the locomotor activity pattern of Podarcis sicula held in constant conditions in the laboratory were found to be associated with systematic differences in both the freerunning period of locomotor rhythms (τ) and the circadian activity time (α). In order to establish whether the pineal played a role in the control of seasonal changes in circadian parameters, we examined the effects of pinealectomy in constant conditions on the locomotor behavior of lizards displaying the bimodal activity pattern typical of summer. In most lizards pinealectomy lengthened τ, shortened α and abolished the bimodal activity pattern. Pinealectomy induced a sudden transition from the typical locomotor behavior of summer, characterized by a marked bimodal pattern, short τ and long α, to the typical locomotor behavior of autumn, characterized by a unimodal pattern, a long τ and short α

Seasonal changes of locomotor activity patterns in ruin lizards Podarcis sicula

Seasonal changes of locomotor activity patterns in ruin lizards Podarcis sicula

Seasonal changes of locomotor activity patterns in ruin lizards Podarcis sicula

The daily pattern of locomotor activity of the ruin lizard Podarcis sicula in its natural environment changes from unimodal in spring (with only one activity peak per day) to bimodal in summer (with two well-separated activity peaks per day) and it becomes unimodal again in autumn. In order to establish whether such seasonal changes in pattern might be at least in part controlled by endogenous temporal programs, lizards were collected at different times of the year and immediately after capture their locomotor behavior was tested in the laboratory under constant temperature (29°C) and in darkness. For some individuals tested in the laboratory the locomotor pattern previously expressed in the field was known. Seasonal differences in pattern have been unequivocally found to have an endogenous component, as most lizards in constant conditions retained the locomotor pattern shown in the field during the same season. Besides, in the bimodal lizards the freerunning period of locomotor rhythms (τ) was significantly shorter and circadian activity time (a) longer than in the unimodal ones. Altogether the data are compatible with the idea that both the interdependent changes of τ and a and the changes in locomotor pattern occurring seasonally in the circadian activity rhythms of P. sicula would depend on changes in the phase relationship between mutually coupled oscillators which drive these rhythms.

Seasonal changes of locomotor activity patterns in ruin lizards Podarcis sicula

Seasonal changes of locomotor activity patterns in ruin lizards Podarcis sicula

The role of extraretinal photoreception in the circadian system of the ruin lizard Podarcis Sicula

1. 1. Combination of parietalectomy, pinealectomy and retinalectomy in the same individual does not prevent Podarcis sicula from obeying Aschoff's rule for diurnal animals. 2. 2. In these operated lizards an increase in intensity of constant illumination (LL) shortens the freerunning period of locomotor rhythms, while a decrease in LL intensity has the opposite effect. 3. 3. These results demonstrate that: (i) extraretinal photoreceptors play a role in mediating parametric effects of light on the circadian system (Aschoff's rule); (ii) extraretinal photoreceptors mediating parametric effects of light lie outside the parietal eye and the pineal.

Vitamin A 2-based visual pigments in fully terrestrial vertebrates

As part of a broad study of the ocular and extraocular photoreceptors of reptiles, we have used high performance liquid chromatography (HPLC) to identify the retinoids present in whole eye extracts of the arboreal lizard Anolis carolinensis and the non-arboreal ruin lizard Podarcis sicula. Unexpectedly, only vitamin A 2-derived chromophore was detected in Anolis, while a mixture of vitamin A 1- and vitamin A 2-derived chromophores was detected in Podarcis. These are the first examples of fully terrestrial vertebrates using vitamin A 2-derived chromophore for visual pigment generation. Furthermore, microspectrophotometric (MSP) data for Anolis show a class of photoreceptor having a visual pigment with maximum absorbance at about 625 nm, some 40 nm further into the red than has been found in any terrestrial vertebrate examined to date.

Melatonin: A coupling device between oscillators in the circadian system of the ruin lizard Podarcis sicula

1. 1. Chronic administration of melatonin (in silastic capsules) lengthened the free-running period of the locomotor rhythm and shortened the circadian activity time in Podarcis sicula held in constant temperature and darkness. 2. 2. Lizards displaying a bimodal pattern of activity invariably became unimodal after melatonin administration. 3. 3. The results support the hypothesis that melatonin acts as a coupling device between circadian oscillators driving the locomotor rhythm in Podarcis sicula.

Body temperatures and exposure to sunshine of ruin lizards Podarcis sicula in central Italy

Abstract Body temperatures (Tbs) of adult ruin lizards, Podarcis sicula, captured near Pisa (Italy) between February and November ranged from 22.4-38.5°C. There were no significant differences between sexes. Mean body temperatures in February-April and November were significantly lower than those recorded from May-October (29.0 and 32.7°C respectively). The overall relationship between Tb and soil temperature at the point of capture (Ts) showed two components. At Ts<28°C there was a highly significant association and 98% of individuals were recorded with Tb>Ts. At Ts> 28°C the association was less clear and Tb<Ts in nearly 30% of lizards. There were diel differences in exposure to sunshine at all seasons; exposure was greatest during the morning after emergence from overnight inactivity and during the late afternoon, least during the mid-day period.