Podarcis Sicula Research Articles

The first transcriptome of Italian wall lizard, a new tool to infer about the Island Syndrome.

Some insular lizards show a high degree of differentiation from their conspecific mainland populations, like Licosa island lizards, which are described as affected by Reversed Island Syndrome (RIS). In previous works, we demonstrated that some traits of RIS, as melanization, depend on a differential expression of gene encoding melanocortin receptors. To better understand the basis of syndrome, and providing raw data for future investigations, we generate the first de novo transcriptome of the Italian wall lizard. Comparing mainland and island transcriptomes, we link differences in life-traits to differential gene expression. Our results, taking together testis and brain sequences, generated 275,310 and 269,885 transcripts, 18,434 and 21,606 proteins in Gene Ontology annotation, for mainland and island respectively. Variant calling analysis identified about the same number of SNPs in island and mainland population. Instead, through a differential gene expression analysis we found some putative genes involved in syndrome more expressed in insular samples like Major Histocompatibility Complex class I, Immunoglobulins, Melanocortin 4 receptor, Neuropeptide Y and Proliferating Cell Nuclear Antigen.

Reactive oxygen species and glutathione antioxidants in the testis of the soil biosentinel Podarcis sicula (Rafinesque 1810).

Important toxicological achievements have been made during the last decades using reptiles. We focus our investigation on gonadal reproductive health of the soil biosentinel Podarcis sicula which is very sensitive to endocrine-disrupting chemicals. The aim of this study is to quantitatively detect, by sensitive microassays, reactive oxygen species and the glutathione antioxidants in the testis and investigate if they are differentially expressed before and after remediation of a site of the "Land of Fires" (Campania, Italy) subject to illicit dumping of unknown material. The oxidative stress level was evaluated by electron spin resonance spectroscopy applying a spin-trapping procedure able to detect products of lipid peroxidation, DNA damage and repair by relative mobility shift, and poly(ADP-ribose) polymerase enzymatic activity, respectively, the expression of glutathione peroxidase 4 transcript by real-time quantitative PCR analysis, the antioxidant glutathione S-transferase, a well-assessed pollution index, by enzymatic assay and the total soluble antioxidant capacity. Experimental evidences from the different techniques qualitatively agree, thus confirming the robustness of the combined experimental approach. Collected data, compared to those from a reference unpolluted site constitute evidence that the reproductive health of this lizard is impacted by pollution exposure. Remediation caused significant reduction of reactive oxygen species and downregulation of glutathione peroxidase 4 mRNAs in correspondence of reduced levels of glutathione S-transferase, increase of antioxidant capacity, and repair of DNA integrity. Taken together, our results indicate directions to define new screening approaches in remediation assessment.

Vasoactive intestinal peptide (VIP) localization in the epididymis of two vertebrate species

VIP and its receptors (VPACRs) are largely investigated in vertebrate testis, as well as their functions in the control of spermatogenesis and steroidogenesis. By contrast, a few data are available about the presence and role of VIP in the epididymis. The aim of the present paper was to investigate the localization of VIP and its receptors in the epididymis of two vertebrates: Podarcis sicula, a seasonal reproducer, and Rattus rattus, a continuous reproducer. By immunohystochemical investigation, we demonstrated for the first time that VIP and its receptors are widely represented in the epididymis of Podarcis sand Rattus; in particular in Podarcis, we showed that during the reproductive period, as well as in Rattus, VIP and its receptors are well represented in all the epithelial cells and the connective tissue of the epididymis; by contrast, during the non-reproductive period, VIP and its receptors are represented only in the connective tissue. The possible role of the VIP/VPACR system in the control of reproduction is discussed.

Unravelling the Role of Metallothionein on Development, Reproduction and Detoxification in the Wall Lizard Podarcis sicula.

Metallothioneins (MTs) are an evolutionary conserved multigene family of proteins whose role was initially identified in binding essential metals. The physiological role of MT, however, has been revealed to be more complex than expected, since not only are MTs able to bind to toxic heavy metals, but many isoforms have shown specialized and alternative functions. Within this uncertainty, the information available on MTs in non-mammalian vertebrates, particularly in neglected tetrapods such as the reptiles, is even more scant. In this review, we provide a summary of the current understanding on metallothionein presence and function in the oviparous lizard Podarcis sicula, highlighting the results obtained by studying MT gene expression in most representative adult and embryonic tissues. The results demonstrate that in adults, cadmium induces MT transcription in a dose- and tissue-specific manner. Thus, the MT mRNAs appear, at least in some cases, to be an unsuitable tool for detecting environmental ion contamination. In early embryos, maternal RNAs sustain developmental needs for MT protein until organogenesis is well on its way. At this time, transcription starts, but again in a tissue- and organ-specific manner, suggesting an involvement in alternative roles. In conclusion, the spatiotemporal distribution of transcripts in adults and embryos definitively confirms that MT has deserved the title of elusive protein.

Phylogenetic relationships of Podarcis siculus (Rafinesque-Schmaltz, 1810) and Podarcis tauricus (Pallas, 1814) in Turkey, based on mitochondrial DNA

The Italian wall lizard and the Balkan wall lizard have a series of taxonomic revisions. However, their phylogenetic relationships still remain uncertain in Turkey. In the present study, we have assessed taxonomic relationships, both of Podarcis siculus and Podarcis tauricus through estimation of phylogenetic relationships among 43 and 42 specimens, respectively, using mtDNA (16 S rRNA and cytb) from great main populations in Turkey. The genetic distances among the populations of P. siculus in Turkey were very low and they were ranged from 0.2 to 1.6% in 16 S rRNA while they were ranged from 0.0% to 3.3% in cytb. On the other hand, the p-distances among the populations of P. tauricus were ranged from 0.0 to 0.6% in 16 S rRNA while they were 0.2% cytb in Turkey. Finally, most of the topologically identical trees of phylogenetic analyses and p-distances showed that monophyly was found in extant populations of P. siculus and P. tauricus. The nominate subspecies, P. s. siculus and P. t. tauricus are representatives of these lizards in Turkey.

PL10 DEAD-Box Protein is Expressed during Germ Cell Differentiation in the Reptile Podarcis sicula (Family Lacertidae).

Among genes involved in the regulation of germ cell differentiation, those of DDX4/Vasa and the Ded1/DDX3 subfamilies encode for DEAD-box ATP-dependent RNA helicases, proteins involved in many mechanisms related to RNA processing. For the first time in reptiles, using specific antibodies at confocal microscopy, we analysed the localization pattern of a Ded1/DDX3 subfamily member in testis and ovary of Podarcis sicula (Ps-PL10) during the reproductive cycle. In testis, Ps-PL10 is expressed in the cytoplasm of spermatocytes and it is not detected in spermatogonia. Differently from Ps-VASA, in round spermatids, Ps-PL10 is not segregated in the chromatoid body but it accumulates in the cytoplasm of residual bodies, and mature spermatozoa are unstained. These observations suggest that in males, Ps-PL10 (1) is involved in spermatogenesis and (2) is then eliminated with residual bodies. In the ovary, Ps-PL10 is present with granules in the cytoplasm of early meiotic cells of the germinal bed (GB), while it is not present in oogonia and somatic cells of the GB stroma. In follicular cells of ovarian follicles, Ps-PL10 expression starts after their fusion with the oocyte. Numerous Ps-PL10 spots are visible in pyriform (nurse-like) cells concomitantly with the protein accumulation in the cytoplasm of differentiating oocyte. In pyriform cells, Ps-PL10 spots are present in the cytoplasm and nuclei, as observed for Ps-VASA, and in the nucleoli, suggesting for Ps-PL10 a role in rRNA processing and in the transport of molecules from the nucleus to cytoplasm and from nurse cells to the oocyte.

Metallothionein expression and synthesis in the testis of the lizard Podarcis sicula under natural conditions and following estrogenic exposure.

Metallothionein (MT) is the main protein involved in the homeostasis of metallic micronutrients and in cellular defence against heavy metals and reactive oxygen species. Found in almost all vertebrate tissues, MT presence and localization in the testis has been controversial. In the present study, by using in situ hybridization and immunohistochemical analysis we assessed the localization of both MT transcript and protein in Podarcis sicula testes during two different phases of the reproductive cycle: the autumnal resumption and the springearly summer mating period. In addition, with the same methodological approach, we verified the effect of estradiol-1711and nonylphenol, a potent xenoestrogen, on MT expression and synthesis. These results, the first collected in a non-mammalian oviparous vertebrate, demonstrated that the expression profile of MT mRNA and protein changes during the reproductive cycle. In the fall resumption, MT transcripts are absent in spermatogonia and present in all the other cells of tubules, including spermatozoa; vice versa, the MT protein is evident only in spermatozoa and somatic cells. In the mating period, both MT transcripts and proteins are present in spermatogonia, spermatocytes and spermatids, whereas in the spermatozoa only the proteins are detected, thus suggesting that the MTs translated in the earlier germinal stages are stored up to spermatozoa. Results also demonstrated that in lizard testis the expression of MT gene undergoes a cell-specific regulation after estrogenic exposure; the possible role and the mechanism by which this regulation occurs have been discussed.

Quantitative abilities in a reptile (Podarcis sicula).

The ability to identify the largest amount of prey available is fundamental for optimizing foraging behaviour in several species. To date, this cognitive skill has been observed in all vertebrate groups except reptiles. In this study we investigated the spontaneous ability of ruin lizards to select the larger amount of food items. In Experiment 1, lizards proved able to select the larger food item when presented with two alternatives differing in size (0.25, 0.50, 0.67 and 0.75 ratio). In Experiment 2 lizards presented with two groups of food items (1 versus 4, 2 versus 4, 2 versus 3 and 3 versus 4 items) were unable to select the larger group in any contrast. The lack of discrimination in the presence of multiple items represents an exception in numerical cognition studies, raising the question as to whether reptiles' quantitative abilities are different from those of other vertebrate groups.

Estrogen-dependent, extrahepatic synthesis of vitellogenin in male vertebrates: A mini-review

In the last years, the hormonal balance is threatened by the interferences of substances with hormone-like action (endocrine disruptor chemicals, EDCs) that may harm animal reproduction. Most EDCs are resistant to environmental degradation and are considered ubiquitous contaminants. EDCs may have synthetic or natural origins. Pesticides used in intensive agriculture contain large amounts of chemicals with estrogenic properties, such as the alkylphenol nonylphenol (NP). Besides, animal feeding operations are important sources of natural estrogen metabolites introduced into the environment through manure application in organic farming. In both cases, EDCs can reach animals, including humans particularly at risk due to their position in the food chain. This is the reason for which it is important to use terrestrial vertebrates as sentinels in soil biomonitoring programmes. Today, the most validated biomarker of estrogenic exposure is the expression in male liver of the vitellogenin (VTG), an estrogen-dependent glycolipophosphoprotein naturally expressed only in the liver of oviparous females during the reproductive season. This report summarizes the data available on the EDC-dependent expression and the synthesis of VTG in male vertebrates, highlighting our latest studies that demonstrate the ability of testis and epididymis of the lacertid Podarcis sicula to synthesize VTG following estrogenic exposure. These findings provide, for the first time, evidence on an extrahepatic expression and synthesis of VTG in a terrestrial vertebrate and lay the groundwork for a new value of the VTG as a biomarker of environmental contamination. In addition, the results open a new scenario on the role of VTG in cells other than oocytes.

Effects of cadmium on the glial architecture in lizard brain.

The glial cells are positioned to be the first cells of the brain parenchyma to face molecules crossing the blood-brain barrier with a relevant neuroprotective role from cytotoxic action of heavy metals on the nervous system. Cadmium is a highly toxic metal and its levels in the environment are increasing due to industrial activities. This element can pass the blood-brain barrier and have neurotoxic activity. For this reason we have studied the effects of cadmium on the glial architecture in the lizard Podarcis siculus, a significant bioindicator of chemical exposure due to its persistence in a variety of habitats. The study was performed on two groups of lizards. The first group of P. siculus was exposed to an acute treatment by a single i.p. injection (2 mg/kg-BW) of CdCl2 and sacrificed after 2, 7 and 16 days. The second one was used as control. The histology of the brain was studied by Hematoxylin/Eosin and Cresyl/Violet stains while the glial structures were analyzed by immunodetection of the glial fibrillary acidic protein (GFAP), the most widely accepted marker for astroglial cells. Evident morphological alterations of the brain were observed at 7 and 16 days from the injection, when we revealed also a decrease of the GFAP-immunopositive structures in particular in the rhombencephalic ventricle, telencephalon and optic tectum. These results show that in the lizards an acute exposure to cadmium provokes morphological cellular alterations in the brain but also a decrement of the expression of GFAP marker with possible consequent damage of glial cells functions.

A combination of long term fragmentation and glacial persistence drove the evolutionary history of the Italian wall lizard Podarcis siculus

BackgroundThe current distribution of genetic diversity is the result of a vast array of microevolutionary processes, including short-term demographic and ecological mechanisms and long-term allopatric isolation in response to Quaternary climatic fluctuations. We investigated past processes that drove the population differentiation and spatial genetic distribution of the Italian wall lizard Podarcis siculus by means of sequences of mitochondrial cytb (n = 277 from 115 localities) and nuclear mc1r and β-fibint7genes (n = 262 and n = 91, respectively) from all its distribution range. The pattern emerging from the genetic data was compared with current and past (last glacial maximum) species distribution modeling (SDM).ResultsWe identified seven deeply divergent parapatric clades which presumably remained isolated in different refugia scattered mainly throughout the Tyrrhenian coast. Conversely, the Adriatic coast showed only two haplogroups with low genetic variability. These results appear to agree with the SDM prediction at the last glacial maximum (LGM) indicating a narrow area of habitat suitability along the Tyrrhenian coast and much lower suitability along the Adriatic one. However, the considerable land exposure of the Adriatic coastline favored a glacial colonization of the Balkan Peninsula.ConclusionsOur population-level historical demography showed a common trend consistent with glacial expansions and regional persistence during the last glacial maximum. This complex genetic signature appears to be inconsistent with the expectation of the expansion-contraction model and post-LGM (re)colonizations from southern refugia. Hence it is one of an increasing number of cases in which these assumptions are not met, indicating that long-term fragmentation and pre-LGM events such as glacial persistence were more prominent in shaping genetic variation in this temperate species.

Thermal ecology of Podarcis siculus (Rafinesque-Schmalz, 1810) in Menorca (Balearic Islands, Spain)

We studied the thermal ecology of an introduced population of the Italian wall lizard, Podarcis siculus , in Menorca (Balearic Islands, Spain). We measured field body temperatures of adult lizards, as well as air and substrate temperatures at their capture places, during spring and summer. We assessed the relations between body and air temperatures, and between body and substrate temperatures, for both seasons. We studied the preferred temperature range of P. siculus in a laboratory thermal gradient. In addition, we recorded the operative temperatures of the habitat of the Italian wall lizard during summer. Then, we calculated the three indexes of behavioural thermoregulation for summer: thermal quality of the habitat, accuracy of thermoregulation, and effectiveness of thermoregulation. As expected, our results show that Italian wall lizards achieved significantly higher body temperatures during summer than during spring. Body temperatures were not significantly related to air temperatures in spring, but the correlation was significant in summer. In addition, body temperatures were not significantly related to substrate temperatures for any season. The preferred temperature range of the species was similar for males and females: 28.40-31.57 °C. Introduced Italian wall lizards of Menorca are effective thermoregulators, with an effectiveness of 0.82 during summer.

Expression of caspase 3 in ovarian follicle cells of the lizard Podarcis sicula.

In this study, our aim was to determine whether caspase 3 plays a role, during previtellogenesis, in the ovarian follicular epithelium of the lizard Podarcis sicula. We investigated the presence and localization of proform and active caspase 3 by enzyme assay, Western blotting and immunocytochemistry. In parallel, a fragment of caspase3 was cloned for the first time in this species, sequenced and used for in situ hybridization to localize messengers and analysed by a phylogenetic survey to shed light on its homology with reptilian caspases. Results demonstrated that: (1) the follicle cells expressed a caspase of the 3/7 group and the mRNA for caspase 3 was transcribed in the stem phase and was completely translated during cell differentiation; (2) the proform protein was stored during the differentiated (nurse) stage and activated at the end of previtellogenesis provoking the degeneration of cells; (3) the predicted protein sequence, although partial, had a strong similarity with the known reptilian caspases 3. The epithelial cells of the ovarian follicle, therefore, do not employ caspase 3 during the nurse stage but, instead, prepare for apoptosis long before the process actually begins. The relevance of this strategy is discussed.

Predicted sea-level changes and evolutionary estimates for age of isolation in Central Mediterranean insular lizards

Rates of biological evolution on islands are often presumed to exceed rates on the mainland. We tested this postulation by computing the evolutionary rate of head shape in Italian wall lizard Podarcis siculus, occurring on four islands off the coast of Southern Italy. We calculated the evolutionary rate using a phylogenetic tree whose node ages were derived from Lambeck et al. predicted ages of geographic isolation of the islands. Such ages are based on a relative sea-level change model for the late Pleistocene–Holocene. Through a likelihood optimization procedure, our method allows computing, besides the evolutionary rate, biological estimates of the ages of insular populations, with this indirectly testing Lambeck et al.’s model estimates. We found that the rate of evolution in Podarcis head shapes on islands is not statistically different from the mainland rate, although insular lizards have distinctive head shapes. Overall, the insular phenotype took 1–4000 years to arise (differing among islands). The estimated ages of insular populations are lower than Lambeck et al.’s estimates and fall in the 5- to 6-ka interval.

Alterations in brain morphology and HSP70 expression in lizard embryos exposed to thermal stress

The teratogenic effects of thermal stress were studied in the oviparous Italian wall lizard Podarcis sicula. To this purpose, the eggs were exposed to continuous or temporary cold (15°C) or warm (30°C) stresses and the effects were analysed at the cytological and molecular levels. The results demonstrated the lethality of the continuous regimes and of the warm temporary regime, no matter if given at early (5 days) or late (15 days) stages of development. Temporary cold stress also resulted in lethality, but only if given in the early stage; later, in fact, it resulted in an abnormal development, with marked alterations in the encephalic vesicles, in the eyes and the trunk organs. By in situ hybridization, it was demonstrated that these alterations were often correlated with changes in HSP70 expression. In conclusion, our data indicate that Podarcis embryos have a limited potential to tolerate thermal changes, especially warm ones. The average predicted temperature increase of 2–4°C in the next few decades could therefore represent a real threat for lizard populations living in temperate areas.

P450 aromatase: a key enzyme in the spermatogenesis of the Italian wall lizard, Podarcis sicula.

P450 aromatase is a key enzyme in steroidogenesis involved in the conversion of testosterone into 17β-estradiol. We investigated the localization and the expression of P450 aromatase in Podarcis sicula testes during the different phases of the reproductive cycle: summer stasis (July-August), early autumnal resumption (September), middle autumnal resumption (October-November), winter stasis (December-February), spring resumption (March-April) and the reproductive period (May-June). Using immunohistochemistry, we demonstrated that the P450 aromatase is always present in somatic and germ cells of P. sicula testis, particularly in spermatids and spermatozoa, except in early autumnal resumption, when P450 aromatase is evident only within Leydig cells. Using real-time PCR and semi-quantitative blot investigations, we also demonstrated that both mRNA and protein were expressed in all phases, with two peaks of expression occurring in summer and in winter stasis. These highest levels of P450 aromatase are in line with the increase of 17β-estradiol, responsible for the spermatogenesis block typical of this species. Differently, in autumnal resumption, the level of P450 aromatase dramatically decreased, along with 17β-estradiol levels, and testosterone titres increased, responsible for the subsequent renewal of spermatogenesis not followed by spermiation. In spring resumption and in the reproductive period we found intermediate P450 aromatase amounts, low levels of 17β-estradiol and the highest testosterone levels determining the resumption of spermatogenesis needed for reproduction. Our results, the first collected in a non-mammalian vertebrate, indicate a role of P450 aromatase in the control of steroidogenesis and spermatogenesis, particularly in spermiogenesis.

Ectopic synthesis of vitellogenin in testis and epididymis of estrogen-treated lizard Podarcis sicula

In oviparous vertebrates, vitellogenin (VTG) is the major yolk precursor synthesized in the liver of sexually mature females during the reproductive period. In males, the VTG gene is silent, but it may be activated by estradiol-17β (E2) or estrogen-like substances. Until now, extra-hepatic expression and synthesis of VTG after estrogen exposure has been reported only for aquatic vertebrates. This study demonstrates the ability of testis and epididymis of the terrestrial oviparous lacertid Podarcis sicula to synthesize VTG following E2 exposure. The results of in situ hybridization and immunohistochemistry analysis show the presence of both VTG mRNA and protein in these districts besides the known induction in the liver. The possible contemporaneous uptake of the E2-induced hepatic VTG by means of the specific vitellogenin receptor has been also evaluated. Finally, histological analysis shows that the E2-treatment during the mating season impairs spermatogenesis.

Four Cases of Complete Melanistic Italian Wall Lizard [Podarcis siculus (Rafinesque-Schmaltz, 1810)] in Southern Italy and Sicily Island

In this short note are described some reports of a complete melanism in four Italian Wall Lizards ( Podarcis siculus ), recorded in Southern Italy and in Sicily Island. Moreover is shortly described the literature of the countless sightings of melanism in the genus Podarcis .

First records of the Italian wall lizard, Podarcis siculus (Rafinesque-Schmaltz, 1810) (Squamata: Lacertidae) in Albania

The occurrence of the Italian wall lizard (Podarcis siculus) in Albania is confirmed. Our findings are now the southernmost known records of the species on the eastern Adriatic coast, and officially present a new reptile species for the Albanian herpetofauna. The origin of these records is discussed; molecular investigation is needed for future evaluation of P. siculus populations in Albania and the western Balkans.

Role of PACAP on testosterone and 17β-estradiol production in the testis of wall lizard Podarcis sicula

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide that in mammalian testis is involved in the control of testosterone and 17β-estradiol synthesis. A similar involvement was recently postulated in the testis of a nonmammalian vertebrate, the wall lizard Podarcis sicula. Indeed, we reported the presence of PACAP and its receptors throughout the reproductive cycle within both germ and somatic cells. Now, we investigated the effects of PACAP on steroidogenesis in significant periods of Podarcis reproductive cycle: winter stasis, reproductive period and summer stasis. Using different in vitro treatments, in the absence or presence of receptor antagonists, we demonstrated that in P. sicula testis PACAP is involved in the control of testosterone and 17β-estradiol production. In particular we demonstrated that treatment with PACAP induced a testosterone increase only in stasis periods (winter and summer stasis); differently they induced a 17β-estradiol production in all periods analyzed (summer stasis, winter stasis and reproductive period).