Search

We investigated the relationship between age and body length, and age at sexual maturity of

The land snail taxon Helix straminea Briganti, 1825 has been reintroduced as a valid species in 2014. We provide here a comprehensive account of its taxonomy, distribution, anatomy, phylogeny and karyology in Italy. An overview of the historical views on the validity of the species is presented and faunistic data are reviewed and implemented with new records from Campania and Basilicata. A lectotype is fixed for H. straminea from the syntypes stored in the Muséum d’Histoire Naturelle of Genève, as well as for three other taxa (Helix straminiformis Bourguignat, 1876, Helix yleobia Bourguignat, 1883 and Helix straminea ssp. elongata Bourguignat, 1860). Genital system, radula and karyotype are described for the first time. Molecular analysis of two mitochondrial genes combining GenBank data and the new sequences presented in this paper showed no differentiation between the northern and southern Italian populations. The conservation status of the species and its possible threats are discussed.

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.

The knowledge of demographic traits such as longevity, growth rates and age at sexual maturity is crucial for understanding the structure of a population in its natural environment and implementing appropriate strategies for its management and conservation. Based on counts of growth layer groups in sections of decalcified teeth using the paraffin technique, we estimated the age and growth of 25 individuals of striped dolphin (Stenella coeruleoalba) found dead stranded along the coast of Campania and Calabria (south Italy, central-western Mediterranean) from 2013 to 2018. Seven individuals, with TL of 100–110 cm, were calves under 1 year old. The oldest male and female individuals were 19 and 14 years old, respectively. Growth curve estimated using the Gompertz growth model (GGM) showed that in S. coeruleoalba male growth trajectories are partly in accordance with those reported in other studies on the same species from different Mediterranean areas. The high frequency (28%) of calves strongly suggests that females of this species use the marine area all around the south-western Italian coasts to give birth to their offspring. Furthermore, a comparison with the estimated age of striped dolphins from other Mediterranean marine areas shows that the longevity of the individuals examined in this study is much lower. Our study provides information toward understanding the demographic traits of S. coeruleoalba from Mediterranean Sea. The results reported here can be useful for future research aimed at understanding population structure, mortality patterns and the effects of anthropogenic activity on the survival of this species in this marine area.

Simple Summary The superfamily Helicoidea is a large and diverse group of Eupulmonata. The superfamily has been the subject of several molecular and phylogenetic studies which greatly improved our knowledge on the evolutionary relationships and historical biogeography of many families. In contrast, the available karyological information on Helicoidea still results in an obscure general picture, lacking a homogeneous methodological approach and a consistent taxonomic record. Nevertheless, the available karyological information highlights the occurrence of a significant chromosomal diversity in the superfamily in terms of chromosome number (varying from 2n = 40 to 2n = 62), chromosome morphology and the distribution of different karyological features among different taxonomic groups. Here we performed a molecular and a comparative cytogenetic analysis on of 15 Helicoidea species of three different families. Furthermore, to provide an updated assessment of the chromosomal diversity of the superfamily we reviewed all the available chromosome data. Finally, superimposing all the chromosome data gathered from different sources on the available phylogenetic relationships of the studied taxa, we discuss the overall observed chromosome diversity in Helicoidea and advance a hypothesis on its chromosomal evolution. Abstract 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 2n = 44 to 2n = 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 A3 (CMA3) 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 2n = 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 (2n = 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.

Sexual differentiation (SD) during development results in anatomical, metabolic, and physiological differences that involve not only the gonads, but also a variety of other biological structures, such as the brain, determining differences in morphology, behavior, and response in the breeding season. In many reptiles, whose sex is determined by egg incubation temperature, such as the leopard gecko, Eublepharis macularius, embryos incubated at different temperatures clearly differ in the volume of brain nuclei that modulate behavior. Based on the premise that "the developmental decision of gender does not flow through a single gene", we performed an analysis on E. macularius using three approaches to gain insights into the genes that may be involved in brain SD during the thermosensitive period. Using quantitative RT-PCR, we studied the expression of genes known to be involved in gonadal SD such as WNT4, SOX9, DMRT1, Erα, Erβ, GnRH, P450 aromatase, PRL, and PRL-R. Then, further genes putatively involved in sex dimorphic brain differentiation were sought by differential display (DDRT-PCR) and PCR array. Our findings indicate that embryo exposure to different sex determining temperatures induces differential expression of several genes that are involved not only in gonadal differentiation (PRL-R, Wnt4, Erα, Erβ, p450 aromatase, and DMRT1), but also in neural differentiation (TN-R, Adora2A, and ASCL1) and metabolic pathways (GP1, RPS15, and NADH12). These data suggest that the brains of SDT reptiles might be dimorphic at birth, thus behavioral experiences in postnatal development would act on a structure already committed to male or female.

The faunistic data on the recent land molluscs of Vesuvius National Park (Naples, Southern Italy) and neighbouring areas sampled during field surveys from 2005 to 2006 are reported here. The data were obtained through a careful research on museum collections and literature and were supplemented with observations from field surveys. Up to now, only 23 land snails species were known from the Vesuvius National Park through bibliographical data. The faunistic research, carried out in 25 stations of different environment typologies, allowed us to discriminate 31 species: 15 of these species are reported here for the first time for the study area. The presence of 16 species was confirmed by other field investigations. Other 19 taxa are reported in the literature, but not confirmed in our research - Helix turriculatella O. G. Costa, 1839 (obsolete nomenclature), Trochoidea trochoides Poiret, 1789, Hygromia cinctella (Draparnaud, 1801), Campylaea planospira (Lamarck, 1822), Vitrea sp. (cf. contracta (Westerlund, 1871)), Cecilioides acicula (O. F. Müller, 1774), Cochlicella acuta (O. F. Müller, 1774), Theba pisana O. F. Müller, 1774). Another 12 taxa are highly doubtful, because of possibilities for confusion with species living in other European or Italian regions, as well as previous misidentifications (Deroceras agreste (Linnaeus, 1758), Monacha gregaria (Rossmässler, 1839), Cochlicella barbara (Linnaeus, 1758), Monachoides incarnatus (O. F. Müller, 1774),Cernuella (Cernuella) cisalpina (Rossmässler, 1837), Cernuella aradasii (Pirajno, 1842), Oxychilus cellarius (O. F. Müller, 1774), O. alliarius (J. S. Miller, 1822), Zonitoides nitidus (O.F. Müller, 1774), Hyalina pseudohydatina, Ferussacia lubrica, Aegopinella nitens (Michaud, 1831); it was impossible in these cases to refer to their current specific name. Eobania vermiculata (O. F. Müller, 1774), Rumina decollata (Linnaeus, 1758), Papillifera papillaris (O. F. Müller, 1774) appear to be the most common species. Interesting are the records of Marmorana muralis (O.F. Müller, 1774), Helix cf. ligata (O.F. Müller, 1774) and Charpentiera gibbula gibbula (Rossmässler, 1836). Charpentieria gibbula honii (Tiberi, 1878) needs genetic investigations in order to confirm its taxonomic status. One allochthonous species (Paralaoma servilis (Shuttleworth, 1852)) was surveyed for the first time in the study area. A Red List of Threatened Species is proposed and the species are classified according to the IUCN criteria (Version 2019-2.1). The malacofauna of Vesuvius - Somma Mountain Complex is composed by 38 land mollusc species and represents 36.2% of the total species present in Campania (about 105 species, personal data) and 12.8% of the Italian fauna (about 530 species of land molluscs extrapolated from the list of Bank (2017). It is assumed that the poor biodiversity of the Vesuvian land malacofauna can be attributed essentially to the scarcity of calcareous substrates and the absence of permanent water sources.

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.

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.

Due to their particular phylogenetic position and biological characteristics, squamate reptiles and, in particular, snakes are becoming an increasingly important model for fields such as evolutionary biology, molecular ecology and adaptation. Recently, during a study to analyze the evolutionary history of European whip snakes, we found a LINE1 (L1)-like sequence (GenBank accession no. LM644476), herein called TRL1L, and while there are data on the abundance of L1 in snakes, their genomic and chromosome localization is still largely unexplored. We therefore performed a study to obtain information on TRL1L abundance, distribution and conservation in snake species, belonging to the Colubridae, Lamprophiidae and Viperidae families, using quantitative dot-blot and fluorescence in situ hybridization (FISH). TRL1L showed a high identity with homologous segments of L1s of lizards the Anolis carolinensis and Lacerta agilis and the zebrafish Danio rerio. The discovered sequences are truncated L1 elements which occur with a low copy number, about 0.1% of the genome of the species studied. This evidence suggests that L1 retroposons have a similar landscape in lizard and snake genomes, probably because similar processes limited L1 distribution in their genomes. TRL1L showed a non-random chromosome distribution pattern. It was scarcely located on autosomes and on the euchromatic W chromosome of Cerastes vipera, while mostly found on the heterochromatic W chromosome of Hierophis carbonarius and Elaphe quatuorlineata. Our data support the hypothesis that a ‘purifying selection’ against the accumulation of L1 elements takes place in recombining regions and highlight the possible role of these elements in the differentiation processes of the snake heterochromatic W chromosome. Interestingly, the preferential distribution of TRL1L on the heterochromatic W chromosomes of the studied snakes appears to be similar to that observed in mammals for L1 accumulation on differentiated Y chromosomes. This finding suggests that a convergent process may have taken place in the differentiation of vertebrate heterochromatic sex chromosomes

A cytogenetic study was carried out by means of standard and NOR-FISH staining methods on the chromosomes of four Alopiinae door snails, two species of the Delimini tribe (Siciliaria paestana and Papillifera bidens) and two species of Medorini tribe (Leucostigma candidescens and Medora sp.). The two Delimini species and Medora sp. showed similar karyotypes, concerning the number of chromosomes, 2n = 62, and the first metacentric pair of chromosomes clearly bigger than the other pairs, but differed in the number of the telocentric pairs: none in P. bidens, four in S. paestana and, even, 21 in Medora sp. Leucostigma candidescens had 2n = 48, all biarmed chromosomes. Loci of NORs were on a single medium small chromosome pair in all the four studied species. The obtained results evidence a relevant degree of intergeneric karyotypic diversity in Alopiinae, concerning both chromosome number and/or shape. The data allow us to make a hypothesis on the evolutionary chromosome trends for this door snail subfamily.

Within the scope of a project on the characterization of satellite DNAs in polar mollusks, the Antarctic chiton Nuttallochitonmirandus (Thiele, 1906) was analyzed. Two novel families of tandemly repeated DNAs, namely NmH and NmP, are described in their structure and chromosomal localization, and, furthermore, their presence was analyzed in related species. Data reported here display a particular variability in the structural organization of DNA satellites within this species. Processes driving satellite evolution, which are likely responsible for the intriguing variability of the identified satellite DNAs, are discussed.

Microdissection, DOP-PCR amplification and microcloning were used to study the large Y chromosome of Chionodraco hamatus, an Antarctic fish belonging to the Notothenioidei, the dominant component of the Southern Ocean fauna. The species has evolved a multiple sex chromosome system with digametic males showing an X1YX2 karyotype and females an X1X1X2X2 karyotype. Fluorescence in situ hybridization, performed with a painting probe made from microdissected Y chromosomes, allowed a deeper insight on the chromosomal rearrangement, which underpinned the fusion event that generated the Y. Then, we used a DNA library established by microdissection and microcloning of the whole Y chromosome of Chionodraco hamatus for searching sex-linked sequences. One clone provided preliminary information on the presence on the Y chromosome of the CHD1 gene homologue, which is sex-linked in birds but in no other vertebrates. Several clones from the Y-chromosome mini-library contained microsatellites and transposable elements, one of which mapped to the q arm putative fusion region of the Y chromosome. The findings confirm that interspersed repetitive sequences might have fostered chromosome rearrangements and the emergence of the Y chromosome in Chionodraco hamatus. Detection of the CHD1 gene in the Y sex-determining region could be a classical example of convergent evolution in action.

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 10th 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.

Sexual differentiation (SD) during development results in anatomical, metabolic, and physiological differences that involve not only the gonads, but also a variety of other biological structures, such as the brain, determining differences in morphology, behavior, and response in the breeding season. In many reptiles, whose sex is determined by egg incubation temperature, such as the leopard gecko, Eublepharis macularius, embryos incubated at different temperatures clearly differ in the volume of brain nuclei that modulate behavior. Based on the premise that "the developmental decision of gender does not flow through a single gene", we performed an analysis on E. macularius using three approaches to gain insights into the genes that may be involved in brain SD during the thermosensitive period. Using quantitative RT-PCR, we studied the expression of genes known to be involved in gonadal SD such as WNT4, SOX9, DMRT1, Erα, Erβ, GnRH, P450 aromatase, PRL, and PRL-R. Then, further genes putatively involved in sex dimorphic brain differentiation were sought by differential display (DDRT-PCR) and PCR array. Our findings indicate that embryo exposure to different sex determining temperatures induces differential expression of several genes that are involved not only in gonadal differentiation (PRL-R, Wnt4, Erα, Erβ, p450 aromatase, and DMRT1), but also in neural differentiation (TN-R, Adora2A, and ASCL1) and metabolic pathways (GP1, RPS15, and NADH12). These data suggest that the brains of SDT reptiles might be dimorphic at birth, thus behavioral experiences in postnatal development would act on a structure already committed to male or female.

A karyological study conducted by both conventional and banding staining methods (Ag-NOR-, CMA3, and C-banding) evidenced a peculiar heterochromatin pattern on chromosomes of Moldovan specimens of the agile frog, Rana dalmatina. As is normal for all agile frogs, the Moldovan population presented 2n = 26 chromosomes, with NOR loci on the short arms of the third chromosome pair, but differed in showing heavy centromeric, CMA3 positive C-bands on the seventh chromosome pair, and light centromeric, DAPI positive C-bands on five chromosome pairs. In contrast, Moldovan specimens showed no significant difference in two segments of the 16S mitochondrial rRNA gene and in the S1 satellite DNA sequence and organization, in comparison with the GenBank deposited 16S rDNA and S1 DNA satellite sequences of R. dalmatina. Molecular similarity and chromosomal differences between agile frogs from Moldovan and extra-Moldovan populations are discussed.

Within the scope of a project on the characterization of satellite DNAs in polar mollusks, the Antarctic chiton Nuttallochitonmirandus (Thiele, 1906) was analyzed. Two novel families of tandemly repeated DNAs, namely NmH and NmP, are described in their structure and chromosomal localization, and, furthermore, their presence was analyzed in related species. Data reported here display a particular variability in the structural organization of DNA satellites within this species. Processes driving satellite evolution, which are likely responsible for the intriguing variability of the identified satellite DNAs, are discussed.