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Satellite DNA (sat-DNA) was previously described as junk and selfish DNA in the cellular economy, without a clear functional role. However, during the last two decades, evidence has been accumulated about the roles of sat-DNA in different cellular functions and its probable involvement in tumorigenesis and adaptation to environmental changes. In molluscs, studies on sat-DNAs have been performed mainly on bivalve species, especially those of economic interest. Conversely, in Gastropoda (which includes about 80% of the currently described molluscs species), studies on sat-DNA have been largely neglected. In this study, we isolated and characterized a sat-DNA, here named PcH-sat, in the limpet Patella caerulea using the restriction enzyme method, particularly Hae III. Monomeric units of PcH-sat are 179 bp long, AT-rich (58.7%), and with an identity among monomers ranging from 91.6 to 99.8%. Southern blot showed that PcH-sat is conserved in P. depressa and P. ulyssiponensis , while a smeared signal of hybridization was present in the other three investigated limpets ( P. ferruginea , P. rustica and P. vulgata ). Dot blot showed that PcH-sat represents about 10% of the genome of P. caerulea , 5% of that of P. depressa , and 0.3% of that of P. ulyssiponensis . FISH showed that PcH-sat was mainly localized on pericentromeric regions of chromosome pairs 2 and 4-7 of P. caerulea (2n = 18). A database search showed that PcH-sat contains a large segment (of 118 bp) showing high identity with a homologous trait of the Nin-SINE transposable element (TE) of the patellogastropod Lottia gigantea , supporting the hypothesis that TEs are involved in the rising and tandemization processes of sat-DNAs.

Evolutionary shifts in chromosome compositions (karyotypes) are major drivers of lineage and genomic diversification. Fusion of ancestral chromosomes is one hypothesized mechanism for the evolutionary reduction of the total chromosome number, a frequently implied karyotypic shift. Empirical tests of this hypothesis require model systems with variable karyotypes, known chromosome features, and a robust phylogeny. Here we used chameleons, diverse lizards with exceptionally variable karyotypes ($2n=20\text{-}62$), to test whether chromosomal fusions explain the repeated evolution of karyotypes with fewer chromosomes than ancestral karyotypes. Using a multidisciplinary approach including cytogenetic analyses and phylogenetic comparative methods, we found that a model of constant loss through time best explained chromosome evolution across the chameleon phylogeny. Next, we tested whether fusions of microchromosomes into macrochromosomes explained these evolutionary losses using generalized linear models. Multiple comparisons supported microchromosome fusions as the predominant agent of evolutionary loss. We further compared our results to various natural history traits and found no correlations. As such, we infer that the tendency of microchromosomes to fuse was a quality of the ancestral chameleon genome and that the genomic predisposition of ancestors is a more substantive predictor of chromosome change than the ecological, physiological, and biogeographical factors involved in their diversification., (© The Author(s) 2023. Published by Oxford University Press.)

We analyzed the body length, age structure, and age at sexual maturity of the invasive Asian common toad Duttaphrynus melanostictus from different sites in Toamasina, east Madagascar. We used skeletochronology as a proxy for age estimation, while gonads were histologically analyzed to determine the age of sexual maturity. The analysis of pooled age data from three sites investigated in 2016 showed that both sexes were larger, although not older, than those of native populations. For the individuals from Madagascar, the males were significantly smaller and younger (mean ± SD, SVL: 71.4 ± 1.6 mm; age: 1.8 ± 0.7 years) than the females (SVL: 78.42 ± 1.9 mm; age: 2.7 ± 1.3 years), when the data were pooled, but when the data were analyzed separately for each of the three sites, similar results were obtained only for one site. The oldest recorded male and female were 3 and 6 years old, respectively. Gonadal histology showed that the males and females reach sexual maturity after the first and second years of age, respectively. Further studies are needed to understand if the larger size and faster growth rates observed in the invasive population of D. melanostictus in Madagascar are a consequence of more favorable environmental conditions with respect to the native range (e.g., the availability of larger trophic niches, a lack of competitors, and lower predatory pressure), and we suggest to extend the monitoring of these life history traits to understand how they might influence the invasion.

We performed a molecular and phylogenetic analysis and a comparative cytogenetic study with standard karyotyping, silver staining (Ag-NOR) and sequential C-banding + Giemsa, + fluorochromes on several Blaesodactylus samples. The phylogenetic inference retrieved two main clades, the first comprises B. victori , B. microtuberculatus and B. boivini , while the second includes B. sakalava , B. antongilensis and B. ambonihazo . The available samples of B. sakalava form two different clades (here named B. sakalava clade A and clade B), which probably deserve a taxonomic re-evaluation. We found a karyological variability in Blaesodactylus in terms of chromosome number (2n = 40-42), morphology, location of NORs, and heterochromatin distribution pattern. Blaesodactylus antongilensis and B. sakalava clade A and B showed a karyotype of 2n = 40 mostly telocentric chromosomes. Pairs 1 and 6 were metacentric in B. sakalava clade A and B, while pair 1 was composed of subtelocentric/submetacentric elements in B. antongilensis. In contrast, B. boivini displayed a karyotype with 2n = 42 only telocentric chromosomes. NORs were on the first chromosome pair in B. boivini , and on the second pair in B. antongilensis . Adding our data to those available from the literature on evolutionarily related species, we highlight that the chromosome diversification in the genus probably proceeded towards a progressive reduction in the chromosome number and the formation of metacentric elements.

True polyploid organisms have more than two chromosome sets in their somatic and germline cells. Polyploidy is a major evolutionary force and has played a significant role in the early genomic evolution of plants, different invertebrate taxa, chordates, and teleosts. However, the contribution of polyploidy to the generation of new genomic, ecological, and species diversity in tetrapods has traditionally been underestimated. Indeed, polyploidy represents an important pathway of genomic evolution, occurring in most higher-taxa tetrapods and displaying a variety of different forms, genomic configurations, and biological implications. Herein, we report and discuss the available information on the different origins and evolutionary and ecological significance of true polyploidy in tetrapods. Among the main tetrapod lineages, modern amphibians have an unparalleled diversity of polyploids and, until recently, they were considered to be the only vertebrates with closely related diploid and polyploid bisexual species or populations. In reptiles, polyploidy was thought to be restricted to squamates and associated with parthenogenesis. In birds and mammals, true polyploidy has generally been considered absent (non-tolerated). These views are being changed due to an accumulation of new data, and the impact as well as the different evolutionary and ecological implications of polyploidy in tetrapods, deserve a broader evaluation.

Transposable elements (TEs) constitute a considerable fraction of eukaryote genomes representing a major source of genetic variability. We describe two DNA sequences isolated in the lizard Zootoca vivipara , here named Zv516 and Zv817. Both sequences are single-copy nuclear sequences, including a truncation of two transposable elements (TEs), SINE Squam1 in Zv516 and a Tc1/Mariner-like DNA transposon in Zv817. FISH analyses with Zv516 showed the occurrence of interspersed signals of the SINE Squam1 sequence on all chromosomes of Z. vivipara and quantitative dot blot indicated that this TE is present with about 4700 copies in the Z. vivipara genome. FISH and dot blot with Zv817 did not produce clear hybridization signals. Bioinformatic analysis showed the presence of active SINE Squam 1 copies in the genome of different lacertids, in different mRNAs, and intronic and coding regions of various genes. The Tc1/Mariner-like DNA transposon occurs in all reptiles, excluding Sphenodon and Archosauria. Zv817 includes a trait of 284 bp, representing an amniote ultra-conserved element (UCE). Using amniote UCE homologous sequences from available whole genome sequences of major amniote taxonomic groups, we performed a phylogenetic analysis which retrieved Prototheria as the sister group of Metatheria and Eutheria. Within diapsids, Testudines are the sister group to Aves + Crocodylia (Archosauria), and Sphenodon is the sister group to Squamata. Furthermore, large trait regions flanking the UCE are conserved at family level.

In this paper, the combination of two algorithms, a cell counting algorithm and a velocity algorithm based on a Digital Particle Image Velocimetry (DPIV) method, is presented to study the collective behavior of micro-particles in response to hydrodynamic stimuli. A wide experimental campaign was conducted using micro-particles of different natures and diameters (from 5 to 16 μ m ), such as living cells and silica beads. The biological fluids were injected at the inlet of a micro-channel with an external oscillating flow, and the process was monitored in an investigated area, simultaneously, through a CCD camera and a photo-detector. The proposed data analysis procedure is based on the DPIV-based algorithm to extrapolate the micro-particles velocities and a custom counting algorithm to obtain the instantaneous micro-particles number. The counting algorithm was easily integrated with the DPIV-based algorithm, to automatically run the analysis to different videos and to post-process the results in time and frequency domain. The performed experiments highlight the difference in the micro-particles hydrodynamic responses to external stimuli and the possibility to associate them with the micro-particles physical properties. Furthermore, in order to overcome the hardware and software requirements for the development of a real-time approach, it was also investigated the possibility to detect the flows by photo-detector signals as an alternative to camera acquisition. The photo-detector signals were compared with the velocity trends as a proof of concept for further simplification and speed-up of the data acquisition and analysis. The algorithm flexibility underlines the potential of the proposed methodology to be suitable for real-time detection in embedded systems., (© 2023 Author(s).)

We investigated the relationship between age and body length, and age at sexual maturity of Physeter macrocephalus individuals stranded along the Italian coast. Our molecular analysis shows that all our samples belong to the C.001.002 haplotype, shared between Atlantic and Mediterranean populations. We show that males attain sexual maturity at 10 years, similar to those from other marine areas. However, considering the same body length class, Mediterranean males are older than Atlantic ones. Our finding of a Mediterranean pregnant female of only 6.5 m in length and an assessed age of 24-26 years is particularly noteworthy, considering that females reach sexual maturity at about 9 years and 9 m of total length in other regions. Comparing our results with the literature data, we highlight the positive correlation between lifespan, adult body length and weight of males from the Mediterranean and Atlantic Ocean. Regardless of whether the relatively small size of Mediterranean specimens is a consequence of an inbreeding depression or an adaptation to less favorable trophic conditions, we recommend to closely monitor this population from a conservation perspective. In fact, its low genetic diversity likely corresponds to a relatively limited ability to respond to environmental changes compared with other populations.

We provide here the first karyotype description of eight Uroplatus species and a characterization of their chromosomal diversity. We performed a molecular taxonomic assessment of several Uroplatus samples using the mitochondrial 12S marker and a comparative cytogenetic analysis with standard karyotyping, silver staining (Ag-NOR) and sequential C-banding + Giemsa, +Chromomycin A3 (CMA 3 ), +4',6-diamidino-2-phenylindole (DAPI). We found chromosomal variability in terms of chromosome number (2n = 34-38), heterochromatin composition and number and localization of loci or Nucleolar Organizer Regions (NORs) (alternatively on the 2nd, 6th, 10th or 16th pair). Chromosome morphology is almost constant, with karyotypes composed of acrocentric chromosomes, gradually decreasing in length. C-banding evidenced a general low content of heterochromatin, mostly localized on pericentromeric and telomeric regions. Centromeric bands varied among the species studied, resulting in CMA 3 positive and DAPI negative or positive to both fluorochromes. We also provide evidence of a first putative heteromorphic sex chromosome system in the genus. In fact, in U. alluaudi the 10th pair was highly heteromorphic, with a metacentric, largely heterochromatic W chromosome, which was much bigger than the Z. We propose an evolutionary scenario of chromosome reduction from 2n = 38 to 2n = 34, by means of translocations of microchromosomes on larger chromosomes (often involving the NOR-bearing microchromosomes). Adding our data to those available from the literature, we show that similar processes characterized the evolutionary radiation of a larger gecko clade. Finally, we hypothesize that sex chromosome diversification occurred independently in different genera.

We performed a molecular and cytogenetic analysis on different Mantellinae species and revised the available chromosomal data on this group to provide an updated assessment of its karyological diversity and evolution. Using a fragment of the mitochondrial 16S rRNA, we performed a molecular taxonomic identification of the samples that were used for cytogenetic analyses. A comparative cytogenetic analysis, with Giemsa's staining, Ag-NOR staining and sequential C-banding + Giemsa + CMA + DAPI was performed on eight species: Gephyromantis sp. Ca19, G.striatus (Vences, Glaw, Andreone, Jesu et Schimmenti, 2002), Mantidactylus (Chonomantis) sp. Ca11, M. (Brygoomantis) alutus (Peracca, 1893), M. (Hylobatrachus) cowanii (Boulenger, 1882), Spinomantispropeaglavei "North" (Methuen et Hewitt, 1913), S.phantasticus (Glaw et Vences, 1997) and S. sp. Ca3. Gephyromantisstriatus , M. (Brygoomantis) alutus and Spinomantispropeaglavei "North" have a karyotype of 2n = 24 chromosomes while the other species show 2n = 26 chromosomes. Among the analysed species we detected differences in the number and position of telocentric elements, location of NOR loci (alternatively on the 6 th , 7 th or 10 th pair) and in the distribution of heterochromatin, which shows species-specific patterns. Merging our data with those previously available, we propose a karyotype of 2n = 26 with all biarmed elements and loci of NORs on the 6 th chromosome pair as the ancestral state in the whole family Mantellidae. From this putative ancestral condition, a reduction of chromosome number through similar tandem fusions (from 2n = 26 to 2n = 24) occurred independently in Mantidactylus Boulenger, 1895 (subgenus Brygoomantis Dubois, 1992), Spinomantis Dubois, 1992 and Gephyromantis Methuen, 1920. Similarly, a relocation of NORs, from the putative primitive configuration on the 6 th chromosome, occurred independently in Gephyromantis , Blommersia Dubois, 1992, Guibemantis Dubois, 1992, Mantella Boulenger, 1882 and Spinomantis . Chromosome inversions of primitive biarmed elements likely generated a variable number of telocentric elements in Mantellanigricans Guibé, 1978 and a different number of taxa of Gephyromantis (subgenera Duboimantis Glaw et Vences, 2006 and Laurentomantis Dubois, 1980) and Mantidactylus (subgenera Brygoomantis , Chonomantis Glaw et Vences, 1994, Hylobatrachus Laurent, 1943 and Ochthomantis Glaw et Vences, 1994)., (Marcello Mezzasalma, Franco Andreone, Gaetano Odierna, Fabio Maria Guarino, Angelica Crottini.)

We performed a molecular and a comparative cytogenetic analysis on different Helicoidea species and a review of all the available chromosome data on the superfamily to provide an updated assessment of its karyological diversity. Standard karyotyping, banding techniques, and Fluorescence in situ hybridization of Nucleolus Organizer Region loci (NOR-FISH) were performed on fifteen species of three families: two Geomitridae, four Hygromiidae and nine Helicidae. The karyotypes of the studied species varied from 2 n = 44 to 2 n = 60, highlighting a high karyological diversity. NORs were on a single chromosome pair in Cernuella virgata and on multiple pairs in four Helicidae, representing ancestral and derived conditions, respectively. Heterochromatic C-bands were found on pericentromeric regions of few chromosomes, being Q- and 4',6-diamidino-2-phenylindole (DAPI) negative. NOR-associated heterochromatin was C-banding and chromomycin A 3 (CMA 3 ) positive. Considering the available karyological evidence on Helicoidea and superimposing the chromosome data gathered from different sources on available phylogenetic inferences, we describe a karyotype of 2 n = 60 with all biarmed elements as the ancestral state in the superfamily. From this condition, an accumulation of chromosome translocations led to karyotypes with a lower chromosome number (2 n = 50-44). This process occurred independently in different lineages, while an augment of the chromosome number was detectable in Polygyridae. Chromosome inversions were also relevant chromosome rearrangements in Helicoidea, leading to the formation of telocentric elements in karyotypes with a relatively low chromosome count.

Lizards represent unique model organisms in the study of sex determination and sex chromosome evolution. Among tetrapods, they are characterized by an unparalleled diversity of sex determination systems, including temperature-dependent sex determination (TSD) and genetic sex determination (GSD) under either male or female heterogamety. Sex chromosome systems are also extremely variable in lizards. They include simple (XY and ZW) and multiple (X 1 X 2 Y and Z 1 Z 2 W) sex chromosome systems and encompass all the different hypothesized stages of diversification of heterogametic chromosomes, from homomorphic to heteromorphic and completely heterochromatic sex chromosomes. The co-occurrence of TSD, GSD and different sex chromosome systems also characterizes different lizard taxa, which represent ideal models to study the emergence and the evolutionary drivers of sex reversal and sex chromosome turnover. In this review, we present a synthesis of general genome and karyotype features of non-snakes squamates and discuss the main theories and evidences on the evolution and diversification of their different sex determination and sex chromosome systems. We here provide a systematic assessment of the available data on lizard sex chromosome systems and an overview of the main cytogenetic and molecular methods used for their identification, using a qualitative and quantitative approach.

We performed the first cytogenetic analysis on five out of the seven species of the genus Lyciasalamandra , including seven subspecies, and representatives of its sister genus Salamandra. All the studied species have a similar karyotype of 2n = 24, mostly composed of biarmed elements. C-bands were observed on all chromosomes, at centromeric, telomeric and interstitial position. We found a peculiar taxon-specific NOR configuration, including either heteromorphic and homomorphic NORs on distinct regions of different chromosomes. Lyciasalamandra a. antalyana and L . helverseni showed two homomorphic NORs (pairs 8 and 2, respectively), while heteromorphic NORs were found in L. billae (pairs 6, 12), L. flavimembris (pairs 2, 12), L. l. luschani (pairs 2, 12), L. l. basoglui (pairs 6, 12), L. l. finikensis (pairs 2, 6) and S. lanzai (pairs 8, 10). Homomorphic NORs with an additional supernumerary site were shown by S . s . salamandra (pairs 2, 8) and S. s . gigliolii (pairs 2, 10). This unexpected highly variable NOR configuration is probably derived from multiple independent NOR translocations and paracentric inversions and correlated to lineage divergence in Lyciasalamandra. These results support the taxonomic validity of the studied taxa and are consistent with a hypothesized scenario of synchronous evolution in the genus.

Chameleons display high karyological diversity in chromosome number (from 2n = 20 to 62), morphology, heterochromatin distribution and location of specific chromosomal markers, making them unique study models in evolutionary cytogenetics. However, most available cytogenetic data are limited to the description of the chromosome number and morphology. Concerning sex chromosomes, our knowledge is limited to ZZ/ZW and Z1Z1Z2Z2/Z1Z2W systems in the genus Furcifer and the isolation of sex-linked, male-specific, sequences in Chamaeleo calyptratus, but the putative XY chromosomes have still to be identified in Chamaeleo and the conservation of male heterogamety in the genus needs confirmation from other species. In this study we performed a molecular and a cytogenetic analysis on C. chamaeleon, using standard, banding methods and molecular cytogenetics to provide a throughout karyological characterization of the species and to identify and locate the putative XY chromosomes. We confirm that the chromosome formula of the species is 2n = 24, with 12 metacentric macrochromosomes, 12 microchromosomes and NORs on the second chromosome pair. Heterochromatin was detected as weak C-bands on centromeric regions, differently from what was previously reported for C. calyptratus. Fluorescence in situ hybridization (FISH) showed the occurrence of interspersed telomeric signals on most macrochromosomes, suggesting that ancient chromosome fusions may have led to a reduction of the chromosome number. Using a combination of molecular and FISH analyses, we proved that male specific Restriction site-Associated DNA sequences (RADseq) isolated in C. calyptratus are conserved in C. chamaeleon and located the putative XY chromosomes on the second chromosome pair. We also identified different transposable elements in the focal taxa, which are highly interspersed on most chromosome pairs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

The data presented in this paper stand as supplementary information of the associated article "Karyological characterization of the common chameleon ( Chamaeleo chamaeleon ) provides insights on the evolution and diversification of sex chromosomes in Chamaeleonidae" [1]. This work provides (i) raw experimental data on the karyology of the common chameleon Chamaeleo chamaeleon and (ii) the results of bioinformatic analysis on sex-specific and repeated DNA sequences found in the same species. The karyological information here presented includes traditional staining method (Giemsa staining) and sequential C-banding + fluorochromes performed on Tunisian samples of the species. The sequence data include the alignments of the isolated DNA sequences with homologous sequences found in squamate Short Read Archives (SRAs) and the results of searches in public nucleic acid databases., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article., (© 2020 Published by Elsevier Inc.)

Background: Endemic fluorosis induced by high concentrations of fluoride in groundwater and soils is a major health problem in several countries, particularly in volcanic areas. Aim: To evaluate the occurrence of dental fluorosis resulting from exposure to high levels of environmental fluoride in 79 AD Herculaneum and close Vesuvius towns. Subjects and methods: The occurrence of dental fluorosis from teeth of the Herculaneum victims of the 79 AD eruption and some individuals from Pompeii (14-37 AD) and Nocera Inferiore (Salerno, IV sec. AD) was detected by means of Particle Induced Gamma-ray Emission technique (PIGE). Results: A clinical and analytical scenario of dental fluorosis resulted from the extreme high fluorine tooth content detected in teeth from Herculaneum and the Vesuvius area inhabitants. The adoption of PIGE technique has proved to be particularly effective in showing moderate as well as milder forms of dental fluorosis, otherwise not clearly detectable by clinical and histological analysis. Conclusions: Morphological, histological and elemental analysis of teeth of the 79 AD Herculaneum population show that in this area fluorosis occurred since Roman times.

The common lizard (Zootoca vivipara) displays characteristic cytogenetic, reproductive, molecular, and biogeographic variability. This species comprises oviparous and viviparous populations with disjunct distribution and sex chromosome polymorphisms, from simple ZZ/ZW to complex Z1Z1Z2Z2/Z1Z2W systems with different morphologies of the W chromosome. In this study, we used the primers SINE A and SINE B and a newly designed primer pair to (1) obtain information on the presence and distribution of transposable elements (TEs) in 8 squamate families and (2) assess the chromosomal location of SINE Squam elements in Z. vivipara. PCR amplification with SINE A and SINE B produced single or multiple products in different Z. vivipara populations, subsequently used to design the SINE-Zv primers. Using the newly designed SINE-Zv primers, we identified 2 sequences of about 700 and 300 bp (SINE-Zv 700 and SINE-Zv 300) in all the investigated populations of Z. vivipara. Fluorescence in situ hybridizations showed a preferential localization of SINE-Zv sequences in the peritelomeric regions of almost all chromosomes, with the exception of the W. Both sequences contained a distinct segment of SINE Squam2. SINE-Zv 700 appeared to be restricted to Z. vivipara, while SINE-Zv 300 contained a partial Gypsy sequence that is highly conserved among Squamata and showed high identity values (72-93%) with several transcripts from different species. Using the same primers, we also highlighted the presence of another highly conserved Gypsy-like fragment in snakes which displayed significant similarity with the stomatin-like protein 2 of colubrids. Our results suggest that SINEs and the Gypsy-like elements are widely distributed among squamates and may have played an active role in their genomic evolution and differentiation., (© 2019 S. Karger AG, Basel.)

In this work, we performed a biogeographic analysis with Bayesian binary MCMC (BBM) statistical dispersal-vicariance analysis (S-DIVA) and species distribution models (SDM) on three phylogenetically closely related Mediterranean whipsnakes (Hierophis gemonensis, H. carbonarius, H. viridiflavus), to investigate the pathways of their geographical diversification and locate putative refugial areas in the last glacial maximum (LGM). Our analysis suggests that the diversification processes between the studied species overall followed an east-west route, from eastern Greece to the Iberian Peninsula and continental France, highlighting a significant role of dispersal and vicariance processes at both inter- and intraspecific levels. In particular, the main lineage-splitting events between H. gemonensis, H. carbonarius and H. viridiflavus coincide with two events of vicariance, involving respectively eastern Greece and eastern Italy, and eastern Italy and western Italy, Iberian Peninsula and continental France. SDM analyses highlight the occurrence of multiple putative glacial refugia in the Balkans, Italy and southern France, which represent well the occurrence of distinct "refugia within refugia" in the main Mediterranean peninsulas. Furthermore, our results suggest how these refugia may have played an important role during the Quaternary climatic oscillations in shaping the current haplotype distribution of European whipsnakes., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Lepidosaurs are frequently described as having highly kinetic skulls, and different forms of cranial kinesis have been described as being characteristic of their radiation. The model of amphikinesis proposed by Frazzetta, J Morphol 1962; 111:287-319, which was long considered a synapomorphy of the large suborder Sauria, is now much debated given its uncertain distribution among the various lizard taxa and the lack of data about its morphological correlates. In this article, we analyze the anatomical correlates of different forms of cranial kinesis, with particular regard to the putative saurian amphikinesis, describing the possible diverse skull movements of several species of European gekkotans (Hemidactylus turcicus, Mediodactylus kotschyi, and Tarentola mauritanica) and lacertids (Lacerta agilis, L. bilineata, Podarcis muralis, P. siculus, and Teira dugesii). Using serial and whole-mount histology, we found clear differences between gekkotans and lacertids in the structure of several cranial joints underlining the existence of two degrees of intracranial mobility. The lacertid species possess the anatomical features for streptostyly (quadrate joints) and metakinesis (parietal-supraoccipital and parabasisphenoid-pterygoid joints) and lack the anatomical correlates for mesokinesis (mobility of frontal-parietal and palatine-pterygoid joints) and amphikinesis (coupled mesokinesis, metakinesis, and streptostyly). In contrast, geckos present all the anatomical correlates for amphikinesis as described by the traditional quadratic crank model. Finally, we present a comprehensive summary of the different forms of squamate cranial kinesis, advancing two alternative hypotheses about the evolutionary origin of amphikinesis., (Copyright © 2013 Wiley Periodicals, Inc.)

Ovarian tissue cryopreservation is a promising technique for fertility preservation in young female cancer patients and efforts have been made to improve its effectiveness. During cooling and thawing, sodium ions significantly contribute to the 'solute effect' that plays a major role in disrupting cell membranes. Choline ions, which do not cross the cell membrane, should not contribute to the intracellular solute load. The present study assessed the effects of sodium substitution with choline in slow-cooling freezing media on human ovarian cortical strip cryopreservation. A total of 629 follicles (fresh control n=266; cryopreserved n=363), collected from ovarian biopsies of 11 women (22-40years) during laparoscopic surgery, were studied by light microscopy, immunohistochemistry and transmission electron microscopy to evaluate their morphology, apoptosis and ultrastructure. The results demonstrate that choline substitution leads to: (i) an improved preservation of oocytes and follicular cells; (ii) the recovery of a higher percentage of grade-1 follicles negative for p53, p21 and Apaf-1 apoptotic markers; (iii) a reduced mitochondrial damage as observed at an ultrastructural level; and (iv) a better preservation of ovarian tissue stroma. In conclusion, the use of choline-based media may represent a valuable tool to improve human ovarian tissue cryopreservation. Ovarian tissue cryopreservation is a promising fertility preservation approach for cancer patients before undergoing treatments that irreversibly reduce the ovarian reserve. Autotransplantation of ovarian cortical strips has resulted in viable offspring in animal models and human. Worldwide, 20 live births have been reported thus far following autotransplantation of frozen-thawed ovarian tissue. However, currently the success rate of this technology is far from being satisfactory. This could be due to inappropriate cryopreservation procedures that might impair the physiology of ovarian follicles. Sodium ions contained in freezing media significantly contribute to the 'solute effect' that plays a major role in disrupting cell membranes. Choline ions, which do not cross the cell membrane, would not be expected to contribute to the intracellular solute load. In the present study we assessed the effects of sodium substitution with choline in slow-cooling freezing media on human ovarian cortical strip cryopreservation. A total of 629 follicles, collected from ovarian biopsies of 11 women (aged 22-40years) during laparoscopic surgery, have been studied by light microscopy, immunohistochemistry and transmission electron microscopy to evaluate their morphology, apoptosis and ultrastructure. Results demonstrated that choline substitution allowed: (i) a better preservation of oocytes and follicular cells; (ii) the recovery of an higher percentage of healthy follicles negative for apoptotic markers; (iii) a lower mitochondria ultrastructural damage; and (iv) a better preservation of ovarian tissue stroma. In conclusion, the use of choline-based media could represent a valuable tool to cryopreserve human ovarian tissue for fertility preservation., (Copyright © 2013 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

A karyological analysis on six Italian populations the slow worm (Anguis veronensis Pollini, 1818) was performed and their genetic differentiation at the mitochondrial 16S rRNA gene fragment from a Spanish sample has been assessed. The Italian populations were karyologically uniform, all showing 2n=44 elements, of which 20 were macrochromosomes and 24 microchromosomes. Comparison with literature data on Central European populations showed a difference on the morphology of the 10(th) chromosome pair: submetacentric in Italian populations and telocentric in the Central European ones. Our analysis showed the presence of a fragile site on chromosomes of this pair, suggesting its propensity for structural rearrangements. Analysis of the 16S rRNA gene fragment showed uniformity among Italian populations (uncorrected genetic distance of 0.4%), and their genetic distinctness from the Spanish individual (uncorrected genetic distance of 4.2%). Our results confirm the existence of two different Anguis fragilis Linnaeus, 1758 lineages, each one characterized by a different cytotype.

Glycoconjugates secreted by the pedal system of the rayed limpet, Patella caerulea, were characterised in situ by histochemical and lectin-histochemical methods in individuals collected around the annual cycle, in November, March, and June. Stainings with periodic acid-Schiff (PAS), Alcian blue pH 2.5 (AB pH 2.5), Alcian blue pH 1.0 (AB pH 1.0), high-iron diamine-Alcian blue pH 2.5 and lectin binding assays with 9 lectins (Con A, WGA, succinylated-WGA, PNA, DBA, SBA, AAA, UEA-I, LTA) were performed. Four secreting cell types were observed in the sole, one in the peripheric region, and two in the sidewall. Glycoconjugate composition varied among cell types and also in one and the same cell type throughout the year. β-Elimination followed by PAS and AB pH 2.5 stainings indicated that most saccharidic chains were O-linked to the protein backbone. Secretion by sole and peripheric region was acidic, carboxylated and/or sulfated, whereas that of the sidewall was neutral. Glucosaminylated and 1,4-fucosylated residuals were predominant in the cell types along the year, 1,2-fucosylated residuals being observed only in the sidewall cells in June. Mannosylated and/or glycosylated residuals were observed in all cells mostly in November. Galactosylated/galactosaminylated residuals were present mostly in the sidewall cells and in the sole subepidermal mucocytes in June. Mannosylated and/or glycosylated residuals in November are probably linked to gonad maturation or to higher locomotion and foraging activity, whereas galactosaminylation in the sole cells and 1,2-fucosylation and glucosaminylation in the sidewall cells in June are linked to a prolonged stationary state, increasing water adsorption to counteract dehydration and/or to modulate microbial interactions., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Background: The study of ancient skeletal pathologies can be adopted as a key tool in assessing and tracing several diseases from past to present times. Skeletal fluorosis, a chronic metabolic bone and joint disease causing excessive ossification and joint ankylosis, has been only rarely considered in differential diagnoses of palaeopathological lesions. Even today its early stages are misdiagnosed in endemic areas., Methodology/principal Findings: Endemic fluorosis induced by high concentrations of fluoride in water and soils is a major health problem in several countries, particularly in volcanic areas. Here we describe for the first time the features of endemic fluorosis in the Herculaneum victims of the 79 AD eruption, resulting from long-term exposure to high levels of environmental fluoride which still occur today., Conclusions/significance: Our observations on morphological, radiological, histological and chemical skeletal and dental features of this ancient population now suggest that in this area fluorosis was already endemic in Roman times. This evidence merged with currently available epidemiologic data reveal for the Vesuvius area population a permanent fluoride health hazard, whose public health and socio-economic impact is currently underestimated. The present guidelines for fluoridated tap water might be reconsidered accordingly, particularly around Mt Vesuvius and in other fluoride hazard areas with high natural fluoride levels.

Longevity and age at sexual maturity were estimated in two anurans from the arid Isalo Massif (southern-central Madagascar), the blue-legged frog (Mantella expectata) and the rainbow frog (Scaphiophryne gottlebei). Phalanges from 69 individuals of M. expectata and 38 individuals of S. gottlebei were analyzed, using the skeletochronological method, in samples collected during two periods: January-February and November-December 2004. The male gonads of both species were also analyzed in order to better correlate reproductive activity with phenology. The phalangeal diaphysis in adults of both species was composed of two concentric bone layers: an innermost endosteal bone, which was less developed or sometimes lacking in S. gottlebei, and an outermost and broader layer of periosteal bone. Lines of arrested growth (LAGs) were observed in both species, although their recognition was more problematic and their distinctiveness much less evident in S. gottlebei. The results presented here indicate that M. expectata and S. gottlebei have a short life span and attain sexual maturity within the first active season after metamorphosis. Maximum longevity was 3 years in M. expectata and 2 years in S. gottlebei. In S. gottlebei the adult body size is likely attained during the same season in which metamorphosis occurs, but then breeding occurs only after the first latency period. Thus, mature individuals have only one LAG, corresponding to 1 year. The low number of individuals with two LAGs suggests that most animals die before the second latency period., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Background: Many small vertebrates on islands grow larger, mature later, lay smaller clutches/litters, and are less sexually dimorphic and aggressive than their mainland relatives. This set of observations is referred to as the 'Island Syndrome'. The syndrome is linked to high population density on islands. We predicted that when population density is low and/or fluctuating insular vertebrates may evolve correlated trait shifts running opposite to the Island Syndrome, which we collectively refer to as the 'reversed island syndrome' (RIS) hypothesis. On the proximate level, we hypothesized that RIS is caused by increased activity levels in melanocortin receptors. Melanocortins are postranslational products of the proopiomelanocortin gene, which controls pleiotropically pigmentation, aggressiveness, sexual activity, and food intake in vertebrates., Results: We tested the RIS hypothesis performing a number of behavioral, genetic, and ontogenetic tests on a blue colored insular variant of the Italian Wall lizard Podarcis sicula, living on a small island off the Southern Italian coast. The population density of this blue-colored variant was generally low and highly fluctuating from one year to the next.In keeping with our predictions, insular lizards were more aggressive and sexually dimorphic than their mainland relatives. Insular males had wide, peramorphic heads. The growth rate of insular females was slower than growth rates of mainland individuals of both sexes, and of insular males. Consequently, size and shape dimorphism are higher on the Island. As predicted, melanocortin receptors were much more active in individuals of the insular population. Insular lizards have a higher food intake rate than mainland individuals, which is consistent with the increased activity of melanocortin receptors. This may be adaptive in an unpredictable environment such as Licosa Island. Insular lizards of both sexes spent less time basking than their mainland relatives. We suspect this is a by-product (spandrel) of the positive selection for increased activity of melanocortins receptors., Conclusions: We contend that when population density is either low or fluctuating annually as a result of environmental unpredictability, it may be advantageous to individuals to behave more aggressively, to raise their rate of food intake, and allocate more energy into reproduction.

Background: The evaluation of mortality of pyroclastic surges and flows (PDCs) produced by explosive eruptions is a major goal in risk assessment and mitigation, particularly in distal reaches of flows that are often heavily urbanized. Pompeii and the nearby archaeological sites preserve the most complete set of evidence of the 79 AD catastrophic eruption recording its effects on structures and people., Methodology/principal Findings: Here we investigate the causes of mortality in PDCs at Pompeii and surroundings on the bases of a multidisciplinary volcanological and bio-anthropological study. Field and laboratory study of the eruption products and victims merged with numerical simulations and experiments indicate that heat was the main cause of death of people, heretofore supposed to have died by ash suffocation. Our results show that exposure to at least 250 degrees C hot surges at a distance of 10 kilometres from the vent was sufficient to cause instant death, even if people were sheltered within buildings. Despite the fact that impact force and exposure time to dusty gas declined toward PDCs periphery up to the survival conditions, lethal temperatures were maintained up to the PDCs extreme depositional limits., Conclusions/significance: This evidence indicates that the risk in flow marginal zones could be underestimated by simply assuming that very thin distal deposits, resulting from PDCs with poor total particle load, correspond to negligible effects. Therefore our findings are essential for hazard plans development and for actions aimed to risk mitigation at Vesuvius and other explosive volcanoes.

The present paper shows the results of chromosome banding and rDNA-FISH study performed on several specimens of different populations of Patella caerulea, Patella rustica and Patella ulyssiponensis. The taxonomic attribution of specimens was ascertained by the molecular phylogenetic analysis of the mitochondrial 16S rRNA gene. P. caerulea and P. rustica had 2n = 18 chromosomes with first seven of biarmed pairs and the remaining two uniarmed pairs. P. ulyssiponensis had 2n = 16 with all biarmed chromosomes. Ag-NOR loci were on the short arms of the first metacentric pair in the three studied limpets, whereas they showed a different pattern of heterochromatin distribution and composition. A chromosome mosaicism was observed in several P. caerulea specimens, which exhibited an unpaired metacentric element and loss of a telocentric pair. The obtained results suggest that in the genus Patella specific diversification was accompanied by variations in heterochromatin distribution and composition and reduction of chromosome number by Robertsonian centric fusion.

There are always more evidences indicating that 17beta-estradiol (E2) is to be necessary for normal male fertility. Here we report the expression of the most ubiquitously expressed member of the akt family of genes, akt1, in the lizard (Podarcis s. sicula) testis. We have used a nonmammalian vertebrate model (the lizard P. s. sicula) to investigate the regulation of the serine/threonine kinase Akt activity, implicated in the control of cell proliferation, survival, and metabolism, in the testis during the annual sexual cycle and to study whether E2 exerts a role in the spermatogenesis through Akt-1 activity. Immunocytochemistry analysis show that Akt-1 proteins are present in the spermatogonia (SPG), and spermatocytes (SPC), and spermatids (SPT). The annual E2 profile shows a progressive increase during the active spermatogenesis (from April to June) and a peak in the month of August (spermatogonial mitosis). In parallel, Akt-1 (molecular weight 60 kDa) are highly phosphorylated during the period of active spermatogenesis and in post-refractory period (August) compared with the winter stasis (from November to March). Present results demonstrate that E2 treatment induces the activation of Akt-1, and this effect is counteracted by the anti-estrogen ICI 182-780., (Copyright (c) 2005 Wiley-Liss, Inc.)

Longevity and age at sexual maturity in an Italian population of Rana latastei were studied by skeletochronology performed on the phalanges. Frogs collected in 1998 and 1999 by drift fences and pitfall traps were marked by toe-clipping. After marking, individuals were released and the cut phalanges were processed for skeletochronological analysis. The maximum age so far recorded was 3 years in males and 4 years in females. The smallest male and female that were sexually mature on the basis of histological analysis of the gonads were 36 and 35 mm snout vent length (SVL), respectively. In both sexes, most individuals were estimated to breed shortly after emergence from their first overwintering. Among the European Brown Frogs, Rana latastei appears to be one of the shortest-lived and one of the first to reach sexual maturity.

We recently showed that interleukin-1 (IL-1) is secreted by the placenta of a species of squamate reptile, the three-toed skink, Chalcides chalcides. In this study, we used immunohistochemical techniques to investigate the expression of IL-1 (in the two isoforms, IL-1alpha and IL-1beta) and its specific membrane receptor IL-1 RtI in uterine oviduct during the peri-implantation period. We found that both IL-1 and its receptor were expressed in uterine tissues before and after ovulation (in the pre-ovulatory stage, even before the yolk had formed in the ovary). However, while IL-1alpha was mostly localized in the uterine mesenchyme tissue, IL-1beta and IL-1RtI were present in the uterine epithelium. Our data provide a further comparison between the reproduction of mammals and squamate reptiles.

Glycosylation of the foeto-maternal interface of the skink Chalcides chalcides has been examined at various stages of gestation using lectin histochemistry. Specimens of incubatory chamber or placenta from early, mid-, late- and near-term pregnancy were fixed and embedded in epoxy resin. Areas of foeto-maternal apposition were probed with a panel of biotinylated lectins followed by an avidin-peroxidase revealing system to identify various classes of glycan at the interface. Both the external epithelium of unspecialized bilaminar omphalopleure, which forms by early pregnancy, and chorioallantoic membrane which develops by mid-pregnancy, were composed of two phenotypes, one of which secreted a wide range of glycans including high mannose and complex N-glycan, N-acetyl glucosamine, lactosamine and galactosamine, which became less prominent from mid-pregnancy onwards. The uterine epithelium also contained a well-developed secretory apparatus producing a similar range of glycans and there were indications that glycosylated secretions were taken up by the overlying chorioallantois. Foetal vasculature was well developed while maternal vessels appeared more contracted, and both were richly sialylated like their therian equivalents. Our findings indicate that this reptile has evolved a true epitheliochorial placenta with many aspects in common with its therian counterparts but also with unique features of its own.

The H beta 58 gene, whose disruption in mice causes reabsorption of the embryo at 9.5 days post-conception, is believed to be essential for development of the placenta. Although the H beta 58 gene is well conserved in some Amniota, nothing is known about its presence in reptiles, some species of which have developed a chorioallantoic placenta. In this work, we investigated the expression of H beta 58 mRNA and protein in the three-toed skink, Chalcides chalcides. H beta 58 protein expression was found in the uterine epithelium beginning from the peri-ovulatory stage. However, it increased strongly at the moment of placental formation, when a high level of expression of mRNA and protein was also observed in the extra-embryonic membranes. The expression of H beta 58 mRNA and protein was maintained, although to a lesser degree, in the placenta during late pregnancy. It was also present in the early embryo. Finally, cloning and sequencing of a gene fragment revealed strong homology of the reptile gene with that of mammals. The high degree of conservation of the gene in amniote vertebrates and its presence in a viviparous squamate reptile (as in mammals) indicates an important role of this gene in the chorioallantoic placenta formation and development., (Copyright 2001 Harcourt Publishers Ltd.)

A new model of surgical injury for the induction and development of stenosis in common rat carotids is described. This model differs from balloon angioplasty or vein graft systems currently applied on animals to develop stenosis, since it involves the entire vessel wall layers and mimics the injury occurring during arterial grafts, endarterectomy or organ transplantation. At different times following arterial damage, the pattern of expression of genes already known to be involved in the proliferation, differentiation, and apoptosis of smooth muscle cells (c-myc, Angiotensin II receptor 1, Bcl-2 and Bax alpha), as well as of Rb and Rb2 genes, whose pattern of expression after arterial injury has not yet been reported, was analyzed by semi-quantitative reverse transcription-polymerase chain reaction technique. Histological and histochemical analysis on carotid sections shows the morphological changes which occurred 30 days after surgical injury in the vessel wall. Molecular and histological data demonstrate that this model of surgical injury induces neointimal proliferation in about 30% of rats. In about 70% of the remaining rats, it induces the processes responsible for negative remodelling, namely the significant accumulation of extracellular matrix and fibers and disorganization of arterial tunics. This model is therefore available for further studies on the expression of genes involved in the arterial stenotic process, as well as for testing drugs aimed at limiting this recurrent pathophysiological phenomenon., (Copyright 2001 Wiley-Liss, Inc.)

Neuroanatomical distribution of FMRFamide-like immunoreactivity was investigated in the brain and olfactory system of the viviparous skink, Chalcides chalcides. In the adult brain FMRFamide immunoreactive (ir) perikarya were observed in the diagonal band of Broca, medial septal nucleus, accumbens nucleus, bed nucleus of the anterior commissure, periventricular hypothalamic nucleus, lateral forebrain bundle, and lateral preoptic, subcommissural, suprachiasmatic and lateral hypothalamic areas. This pattern was seen in both male and female brains. Though all major brain areas showed FMRFamide-ir innervation, the densest ir fiber network was observed in the hypothalamus. During development, ir elements were observed for the first time in embryos at mid-pregnancy. FMRFamide perikarya were located along the ventral surface of the vomeronasal nerve, in the olfactory peduncle mediobasally, as well as in the anterior olfactory nucleus and olfactory tubercle. Furthermore, some ir neurons were observed in the rhombencephalic reticular substance; however, the ir fiber network was poorly developed. Later in development FMRFamide-ir neurons appeared also in the bed nucleus of the anterior commissure as well as the rhombencephalic nucleus of solitary tract and the dorsal motor nucleus of vagus nerve. In juveniles, the distribution profile of FMRFamide immunoreactivity was substantially similar to that of the adults, with a less widespread neuronal distribution and a more developed fiber network. Ontogenetic presence of FMRFamide immunoreactivity in the nasal area has been linked to the presence of a nervus terminalis in this reptile., (Copyright 2001 S. Karger AG, Basel.)

We describe histochemical techniques for detecting DNA within the osteocytic lacunae of ancient bones. The bones examined were fragments of femurs from two human individuals found in the Pompeian C. I. Polybius house and fragments of metacarpals from two horses (Equus sp.) found in the Pompeian "Casti Amanti" house. Both buildings were buried by the 79 A. D. Vesuvius eruption. Fragments of femurs from a modern horse, a modern swine and a modern amphibian also were studied as controls. Some bone sections were stained with two different DNA-specific fluorochromes, 4'-'6-diamidino-2-phenylindole (DAPI) and chromomycin A3 (CMA), while others were stained by the Feulgen reaction. All of the techniques gave a positive reaction within the osteocytic lacunae. Histological analysis of the undecalcified, ground and unstained sections agreed well with results of bone sections stained with either the fluorochromes or the Feulgen reaction. Bones showing good histology also were positive by our DNA-specific stain. Histochemical and histological analyses correlated well with the success of DNA extraction and amplification. Using conventional DNA-specific histochemical techniques in conjunction with histological analysis can be useful in the study of DNA extracted from ancient bone remains while reducing both the amount of time and cost.

Thirteen skeletons found in the Caius Iulius Polybius house, which has been the object of intensive study since its discovery in Pompeii 250 years ago, have provided an opportunity to study either bone diagenesis by histological investigation or ancient DNA by polymerase chain reaction analysis. DNA analysis was done by amplifying both X- and Y-chromosomes amelogenin loci and Y-specific alphoid repeat locus. The von Willebrand factor (vWF) microsatellite locus on chromosome 12 was also analyzed for personal identification in two individuals showing alleles with 10/11 and 12/12 TCTA repeats, respectively. Technical problems were the scarcity of DNA content from osteocytes, DNA molecule fragmentation, microbial contamination which change bone structure, contaminating human DNA which results from mishandling, and frequent presence of Taq DNA polymerase inhibiting molecules like polyphenols and heavy metals. The results suggest that the remains contain endogenous human DNA that can be amplified and analyzed. The amplifiability of DNA corresponds to the bone preservation and dynamics of the burial conditions subsequent to the 79 A.D. eruption.

The structure of the corpus luteum and the steroidogenic activity of the corpus luteum and placenta in the viviparous reptile Chalcides chalcides have been investigated. The corpus luteum has a compact structure, almost without internal vascularized connective septa. It begins to degenerate after the middle of pregnancy, when plasma progesterone (P) remains high. The sections of the corpora lutea taken during early pregnancy showed an intense 3beta-HSDH reaction, whereas the sections taken in late pregnancy gave weak reactions localized exclusively in the peripheral luteal cells. In contrast, sections of placentae taken at the beginning and in the middle of pregnancy always gave negative 3beta-HSDH reactions, whereas those of late pregnancy were always strongly positive, localized in the maternal component of the placenta. In vitro, the corpora lutea from early pregnancy secreted significant amounts of P, whereas appreciable amounts of P were not detected in incubates of early pregnancy placentae. Near the time of delivery, P levels decreased in the culture medium of the corpora lutea, but increased in that of the placentae. The addition of pregnenolone (a precursor of P biosynthesis) to the culture medium caused an increase in the luteal and placental P levels, whereas the addition of trilostane (an inhibitor of 3beta-HSDH) reduced them. The placenta of C. chalcides is suggested to have an endocrine function and to replace the corpus luteum in the production of P when the gland degenerates in late pregnancy., (Copyright 1998 Academic Press.)