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Twenty-five species of cetaceans have been reported throughout the Mediterranean Sea, eight of them are commonly distributed in the whole basin and are regularly found beached or adrift in the sea. Stranded animals are frequently found in poor conservation status, preventing reliable identification; identification is thus often based solely on morphological features. Therewith, molecular tools are especially useful to provide taxonomic identification. In this work, a four-enzymes PCR-RFLP in silico protocol, based on a fragment of the mitochondrial gene cytb, has been designed for cetacean species occurring in the Mediterranean Sea. Moreover, beached or floating specimen samples belonging to the eight common species have been tested in the laboratory, providing evidence that this approach represents a reliable, cost- and time-effective tool for their specific identification.

Non-indigenous freshwater bivalves negatively affect invaded ecosystems through different mechanisms, including inter-specific competition for trophic resources. Here, we investigated in Lake Trasimeno (Central Italy) the diet of the invasive Dreissena polymorpha and the native Anodonta anatina. δ15N and δ13C stable isotopes were measured in winter and summer in bivalves, phytoplankton, and sedimentary organic matter (SOM); the relative dietary contributions of the two resources were determined using Bayesian mixing models. To elucidate the different carbon and nitrogen pools characterizing the study site, isotopic analyses were extended to zooplankton and to representatives of the benthic flora and macroinvertebrate fauna. Independently from the season, the two bivalves showed a limited trophic overlap, as mixing models indicated for D. polymorpha a diet based primarily on phytoplankton, while A. anatina relied mainly on SOM. Dietary differences were less marked in summer, when comparable isotopic values characterized phytoplankton and SOM. In winter, conversely, the trophic differentiation between the two species was more evident, and corresponded with a significant enrichment in SOM δ13C values, likely due to a substantial contribution of carbon deriving from decaying macrophytes. Whether differences in ecological and behavioral traits alone can explain the observed trophic segregation between the two species, or if they have actively shifted their diet to reduce competition for food is discussed. We conclude emphasizing the need of an advanced resolution of the influence of non-indigenous species on the flux of energy and matter in invaded lentic systems, including Lake Trasimeno.

In animals, doubly uniparental inheritance (DUI) is a major exception to the common strict maternal inheritance of mitochondria. To date, DUI has only been found in many bivalve species, but its distribution is still unclear. Given the great species richness of the class, much effort is needed to further investigate the occurrence of DUI in unsampled species. A compelling evidence of DUI is generally the presence of a sex-linked heteroplasmy, where two divergent mitochondrial lineages are found: One is isolated from the male germline, and the other one is isolated from the female germline and, normally, from the soma of both sexes. In the present study, we investigated the sex-linked heteroplasmy in the razor clam Solen marginatus Pulteney, 1799 and in the lagoon cockle Cerastoderma glaucum (Bruguière, 1789) using two mitochondrial markers (cox1 and rrnL). We found evidence of DUI in the species S.marginatus, with a divergence up to 21% for the rrnL gene, but not in C.glaucum. Moreover, our phylogenetic reconstruction includes all the available data for heterodont species with sex-linked heteroplasmy and suggests multiple origins of DUI in this subclass, as well as the presence of DUI in other species of the genus Solen.

Small Heat Shock Proteins (sHSP) are molecular chaperones that play an essential role in maintaining protein homeostasis and promoting cell survival. In this work, for the first time, multiple cDNAs encoding for small Hsp27 and Hsp30, designed, respectively, as PbHsp27-(1-2) and PbHsp30-(1-5), were cloned and characterized in the amphibian Pelophylax bergeri, which is a suitable model for studying biological responses to environmental perturbations. Domain architecture analysis showed that PbHsp27 and PbHsp30 cDNAs displayed the typical signature motifs of the sHSP family such as the conserved α-crystallin domain flanked by variable N-terminal and C-terminal regions. Phylogenetic analysis revealed that PbHsp27 and PbHsp30 clustered, respectively, with Hsp27 and Hsp30 members of other vertebrates, but more closely with amphibians. Overall PbHsp27 and PbHsp30 transcriptional activity, analyzed by qRT-PCR, evidenced that, in ex vivo skin exposed to thermal shock and cadmium treatment, PbHsp27 and PbHsp30 mRNAs were inducible and regulated differently. This study provides the basis for future research on the potential use of PbHsp27 and PbHsp30 as biomarkers of proteotoxic stress in amphibians.

The production of cupped oysters is an important component of European aquaculture. Most of the production relies on the cultivation of the Pacific oyster Crassostrea gigas, although the Portuguese oyster Crassostrea angulata represents a valuable product with both cultural and economic relevance, especially in Portugal. The authors of the present study investigated the genetic diversity of Portuguese oyster populations of the Sado estuary, both from natural oyster beds and aquaculture facilities, through cox1 gene fragment sequencing. Then, a comparison with a wide dataset of cupped oyster sequences obtained from GenBank (up to now the widest available dataset in literature for the Portuguese oyster) was performed. Genetic data obtained from this work confirmed that the Pacific oyster does not occur in the natural oyster beds of the Sado estuary but showed that the species occasionally occurs in the oyster hatcheries. Moreover, the results showed that despite the founder effect and the bottleneck events that the Sado populations have experienced, they still exhibit high haplotype diversity. Risks are arising for the conservation of the Portuguese oyster reference populations of the Sado estuary due to the occurrence of the Pacific oyster in the local hatcheries. Therefore, researchers, local authorities, and oyster producers should work together to avoid the loss of this valuable resource.

Amphibian parasites of the genus Amphibiocystidium are members of the class Ichthyosporea (=Mesomycetozoea), within the order Dermocystida. Most of the species in the Dermocystida fail to grow in ordinary culture media, so their life cycle has only been partially constructed by studies in host tissues. However, to date, there have been few reports on the life cycle of Amphibiocystidium parasites with respect to the developmental life stages of both Dermocystidium and Rhinosporidium parasites. In this study, we provide light and electron microscopic findings of developmental phenotypes of Amphibiocystidium sp., a parasite previously characterized in the Italian stream frog (Rana italica), which has caused an ongoing infection in a natural population of Central Italy. These phenotypes exhibited distinct morphological characteristics that were similar to A. ranae from the skin of R. temporaria, but showed histochemical properties particularly comparable with those of maturing phenotypes of Rhinosporidium seeberi, and compatible with fungal-like parasites. Therefore, for Amphibiocystidium sp. phenotypes, we suggest adopting the terminology used for maturing stages of R. seeberi, such as juvenile sporangia, early mature sporangia and mature sporangia. The characterization of these developmental stages will be useful to increase the understanding of the life cycle of parasites of the genus Amphibiocystidium and of the interactions with their amphibian hosts.

Esox flaviae represents the native esocid species of the Italian peninsula at present potentially highly threatened by the diffusion of exotic E. lucius. Here, we present a novel mtDNA (N = 272) and microsatellite (N = 275) dataset including 13 test and 3 reference samples, aimed to delineate the distribution of the native as well as the exotic species and to unravel potential introgressive hybridisation between the two species in Northern Italy. We highlight a complex mosaic distribution of both species, with contrasting occurrence even between neighbouring sites. Significant genetic substructure is still observed within E. flaviae, while the dispersal of the invader seems to be promoted by restocking actions. In addition, we prove the existence of introgressive hybridisation between native and exotic pikes. Here, gender-biased hybridisation is suggested, with native E. flaviae constituting the predominant ‘mother species’ in the hybridisation process. Finally, we underline the need for a revision of fisheries management regulations, for which a nation-wide and exhaustive genetic screening in the near future should build the scientific basis.

Mesomycetozoean-induced infections (order Dermocystida, genusAmphibiocystidium) in European and North American amphibians are causing alarm. To date, the pathogenicity of these parasites in field conditions has been poorly studied, and demographic consequences on amphibian populations have not been explored. In this study, anAmphibiocystidiumsp. infection is reported in a natural population of the Italian stream frog (Rana italica) of Central Italy, over a 7-year period from 2008 to 2014. Light and electron microscope examinations, as well as partial 18S rDNA sequence analysis were used to characterize the parasite. Moreover, a capture-mark-recapture study was conducted to assess the frog demographics in response to infection. Negative effects of amphibiocystidiosis on individual survival and population fitness were absent throughout the sampling period, despite the high estimates of disease prevalence. This might have been due to resistance and/or tolerance strategies developed by the frogs in response to the persistence ofAmphibiocystidiuminfection in this system. We hypothesized that in the examinedR. italicapopulation, amphibiocystidiosis is an ongoing endemic/epidemic infection. However, ecological and host-specific factors, interacting in a synergistic fashion, might be responsible for variations in the susceptibility toAmphibiocystidiuminfection of both conspecific populations and heterospecific individuals ofR. italica.

Rock partridge (Alectoris graeca) is included in Annex I of the EU Birds Directive as Species of Conservation Concern (SPEC 1), because of the continuing decline in the area of occupancy. In this context, ex situ conservation programs and production systems of captive Rock partridges suited to the wild are necessary to ensure that insurance, reintroduction or restocking goals are met. Anyway, morphological changes occurring in captive-reared animals indicate the loss of animal’s ability to survive in natural environment. In this study, we used the offspring of wild Apennine rock partridges (Alectoris graeca graeca). The young birds were divided into two groups, the first (G1) was housed in enriched and wild-like environment, the second (G2) was subjected to intensive-rearing conditions. Starting at 14 days post-hatching (DPH) and continuing up to 98 DPH, body mass (BM), tarsus length (TL), tarsus depth (TD), tarsus width (TW), head width (HW), head length (HL), wing length (WL) and beak length (BL), were measured fortnightly. Birds in G1 showed shorter heads (F = 10.50, p = 0.000) and thinner tarsi (F = 9.80, p = 0.000) at 84 DPH. At 98 DPH also the TD measurement (F = 8.71, p = 0.001) became significantly lower in G1 then in G2. Birds in G1 showed morphological disadvantages (shorter heads and less developed limbs) as a possible result of limited physical and cognitive exercise in simple artificial environment. Our results suggest that intensive rearing does not contribute to relevant morphological disadvantages within 70 DPH. Accordingly, birds for restocking or reintroduction should be translocated within this time limit into enriched and wild-like environment where they can exercise before the release into the wild. A first figure of morphological measurements for Apennine Rock partridge is reported.

The Manila clam Ruditapes philippinarum – synonym Venerupis philippinarum (Adams and Reeve, 1850) is now one of the top 5 most commercially valuable bivalve species worldwide. Originally from the Indo-Pacific region, it has been introduced in many countries for fisheries and aquaculture, including estuarine environments along Atlantic and Mediterranean European coasts. Yet despite its commercial value and widespread distribution, the precise origins of stocks remain speculative and the genetic diversity of introduced populations is poorly known. Thus, the aim of this work was to collect mtDNA COI (Cytochrome oxidase I) gene sequences from 5 European countries with Manila clam stocks and compare them with native Asian populations to evaluate their genetic diversity and identify possible routes of invasion. The COI gene sequencing supported a strong founder effect in the European populations with 3 main haplotypes occurring at high frequencies, derived from Japan. However, high haplotype diversity was also observed due to the occurrence of 10 rare haplotypes. This supports hypotheses (i) there have been additional, previous unrecorded, introductions as previously hypothesized by analysis of 16S rDNA, and (ii) there has been a limited loss of genetic diversity in introduced populations, as previously suggested by microsatellite data. This is the first genetic comparison of Manila clam populations introduced in to Europe with native clams. Genetic data herein presented are fundamentally important for the traceability of clam products and stock management programmes and will also inform discussion on the potential resilience of exploited Manila clam populations.

The rock partridge, Alectoris graeca, is a polytypic species declining in Italy mostly due to anthropogenic causes, including the massive releases of the closely related allochthonous chukar partridge Alectoris chukar which produced the formation of hybrids. Molecular approaches are fundamental for the identification of evolutionary units in the perspective of conservation and management, and to correctly select individuals to be used in restocking campaigns. We analyzed a Cytochrome oxidase I (COI) fragment of contemporary and historical A. graeca and A. chukar samples, using duplicated analyses to confirm results and nuclear DNA microsatellites to exclude possible sample cross-contamination. In two contemporary specimens of A. graeca, collected from an anthropogenic hybrid zone, we found evidence of the presence of mtDNA heteroplasmy possibly associated to paternal leakage and suggesting hybridization with captive-bred exotic A. chukar. These results underline significant limitations in the reliability of mtDNA barcoding-based species identification and could have relevant evolutionary and ecological implications that should be accounted for when interpreting data aimed to support conservation actions.

The brook chub, Squalius lucumonis (Bianco, 1983), is an endemic endangered species inhabiting the rivers of Central Italy. Being subject to hybridizations with other cyprinids, particularly the congeneric S. squalus (Bonaparte, 1837), genetic overestimation can emerge when molecular markers are applied and mtDNA barcoding is not sufficient to ensure a correct attribution, due to its maternal inheritance. In this perspective, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach on rDNA is proposed to rapidly discriminate S. lucumonis from nine other cyprinids. For its capacity to identify hybrids of these species with S. lucumonis, this method should be considered as a fundamental tool to be applied prior to the application of other molecular markers in extensive genetic characterizations for phylogenetic, phylogeographic, population and management analyses.

European (Dermatophagoides pteronyssinus) and American (Dermatophagoides farinae) house dust mite species are considered the most common causes of asthma and allergic symptoms worldwide. Der p 1 protein, one of the main allergens of D. pteronyssinus, is found in high concentration in mites faecal pellets, which can became easily airborne and, when inhaled, can cause perennial rhinitis and bronchial asthma. Here we report the isolation of the Der p 1 gene from an Italian strain of D. pteronyssinus and the PVX-mediated expression of its mature form (I-rDer p 1) in Nicotiana benthamiana plants. Human sera from characterized allergic patients were used for IgE binding inhibition assays to test the immunological reactivity of I-rDer p 1 produced in N. benthamiana plants. The binding properties of in planta produced I-rDer p 1 versus the IgE of patients sera were comparable to those obtained on Der p 1 preparation immobilized on a microarray. In this paper we provide a proof of concept for the production of an immunologically active form of Der p 1 using a plant viral vector. These results pave the way for the development of diagnostic allergy tests based on in planta produced allergens.

Grey partridge and Red-legged partridge are Galliformes needing special conservation strategies. Reintroduction may represent conservation solutions solely with the support of an in-depth genetic and ecologic evaluation, particularly of grey partridge, of which an Italian subspecies was described. Protocols for conservative DNA isolation are fundamental to study breeders and wild samples. For these reasons, two DNA extraction protocols on different kinds of feathers (P1–P6), oral and anal swabs were tested. DNA suitability was assayed through PCR amplification and capillary sequencing of both 408 bp of Control Region and 658 bp of COI. Both protocols yielded well-amplifiable DNA, they were not time and money consuming and did not require harmful reagents. Sequences of DNA extracted through Wizard Genomic DNA Purification Kit (WGDPK) were better than those based on polyvinylpyrrolidone (PVP); buccal swab, P3 and P5 seemed the best sampling methods, in particular, P5-feather sampling did not affect the flying capacity.

Pikes are fish species belonging to order Esociformes, family Esocidae, genus Esox. Species of the genus Esox are common, large, and economically important for food and fishing activities. Recently, a new species, southern pike E. flaviae, was described for a well-studied area such as Italy, using also two mtDNA markers: cox1 and cytb. A scant number of remnant populations of the species persist in Italy, threatened by habitat loss and degradation and by competition and possible hybridization with E. lucius, massively and recurrently stocked to sustain angling pressure. The availability of new mtDNA markers will possibly contribute to the conservation of the species. Currently, whole mitogenome information for the genus is available only for E. lucius and for E. reichertii. The aim of the present paper is to report novel mitogenomic information for southern pike.

Although the symbionts of non-vertebrate metazoans have received scarce attention, scientific interest has recently increased as these symbionts can spread diseases and alter the diversity of the ecosystem where hosts are introduced. We report here a symbiont newly described by molecular markers and observed on the yabby Cherax destructor Clark, 1936, collected in Italian freshwaters. Preliminary morphological observations were performed and a species identification was proposed using molecular techniques. In particular, a 644-bp long 28S rDNA fragment was successfully sequenced and could be aligned for the entire sample set. The minimum spanning network identified two haplotypes clustered into a single haplogroup (H1) together with Temnosewellia minor Haswell, 1888 reference sequences. Considering the introduction of temnocephalans, this represents the third discovery of alien crayfish symbionts in Italian freshwaters, but the first obtained by molecular data. Outside Europe, there are only few records of alien temnocephalans worldwide, introduced with the crayfishes from Australia. The occurrence of this new temnocephalid species and the great number of potential hosts species inhabiting Italian freshwaters suggest that the Italian peninsula might have many undescribed temnocephalid populations/species.

There have been a few studies on the negative effects of pollutants on amphibian skin, the first structural barrier that interacts with the environment and its potential contaminants. In this study an ex vivo skin organ culture from the amphibian Pelophylax bergeri was used to evaluate cell stress responses induced by short-term exposure to cadmium (Cd), a toxic heavy metal known to be an environmental hazard to both humans and wildlife. Histopathological studies were carried out on skin explants using light microscopy and changes in the expression of stress proteins, such as Metallothionein (MT) and Heat shock proteins (HSPs), were investigated by Real-time RT-PCR. Results revealed that amphibian skin reacts to Cd-induced stress by activating biological responses such as morphological alterations and dose- and time-dependent induction of Mt and Hsp70 mRNA expression, suggesting their potential role as biomarkers of exposure to Cd. This work provides a basis for a better understanding of the tissue-specific responses of amphibian skin as a target organ to Cd exposure and its in vitro use for testing potentially harmful substances present in the environment.

Genetic diversity was analysed in a putative autochthonous brown trout (Salmo trutta fario L.) population (Monterivoso Stream, Tyrrhenian Apennine Slope) by means of seven microsatellite loci and PCR‐RFLP of two mitochondrial (ND1 and ND5/6) and one nuclear (LDH‐C1∗) locus. Monterivoso data were compared to those obtained analysing populations of the same basin (Nera River) and of the Po basin; Irish populations were used as a source of Atlantic strain brown trout. Haplotypes distributions, heterozygosity, F‐statistic and UPGMA analyses indicated a genetic diversification between these populations, suggesting a widespread alteration of the genetic structure due to repeated stocking with allochthonous material, mainly of Atlantic origin, that has partially polluted the Monterivoso population. This population appeared to be constituted of Mediterranean strain samples and might represent a residue of an indigenous pool: it shows high specificity characteristics and it is genetically separate from the others ...

Northern pike (Esox lucius) is not considered an endangered species in Italy, but since recent studies indicate the decline of this population, conservation and management strategies based on the genetic differentiation of natural northern pike populations are needed. In this paper, genetic diversity was analysed in 10 Italian and 2 East European northern pike populations by means of seven microsatellite loci. Data indicated an appreciable genetic differentiation, in spite of a low genetic variation, and agreed with the low level of genetic polymorphism already observed for this species in North America and North Europe. Results of statistical tests revealed genetic peculiarities of the Italian populations, even though signals of recent contact between populations were found and discussed in relation to anthropic impacts, particularly to the stocking practice. This investigation represents the first approach to the knowledge of the genetic variability of Italian pike populations using microsatellite markers, and reported results could be of interest for future management and conservation programmes of this species in Italy.

Key words: brown trout, conservative DNA extraction, fish fin clip, fish scales, northern pikeNon-destructive protocols for DNA isolationfrom fresh or preserved specimens are fundamen-tal to study endangered or elusive species, breedersor samples sibship and specimens derived frompublic or private collections (Nielsen et al. 1999;Wasko et al. 2003; Hansen and Jensen 2005;Lucentini et al. 2006). Because of the low yieldand poor quality DNA, particular laboratory careand standardizations were needed to allow repro-ducible results. We compared six DNA extractionprocedures from conservative samples: three arecommercial kits [Wizard Genomic DNA Purifi-cation Kit (WGDPK) (Promega); Wizard Mag-netic DNA Purification System for Food(WMDPF) (Promega); NucleoSpin Food (NSF)(Macheray–Nagel)] and three are largelyemployed methodologies [Trizol (Life Technolo-gies); Chelex (Sigma–Aldrich); C-TAB]. For eachmethod, the standard protocol (reported on datasheets or the first published one) and severalvariations were tested varying sample storage andpre-lysis conditions, homogenization procedures,buffer solutions and concentrations, incubationsand resuspension times and temperatures. DNAwas extracted from 200 northern pikes (Esoxlucius L.) and 100 brown trout (Salmo trutta farioL.) (Table 1) from small (£10 mg) fin pieces and5–10 scales stored dried or in absolute alcoholboth at )20 C and at room temperature. DNAextraction was carried out on fresh materials andrepeated within a period of three years (Table 1),and from 34 years old dried scales culled from amuseum collection. Furthermore, DNA wasextracted from liver and muscle of both species,from individuals that had naturally died and usedas controls. Spectrophotometric readings (260 and280 nm) and densitometric evaluations (Image J)of DNAs obtained through different methodolo-gies and protocols (conservative versus controlDNA) were analyzed by means of ANOVA andt-test. DNA suitability was assayed through PCR–RFLP and microsatellite analyses (Lucentini et al.(2006) (Table 2). Microsatellites were run onABI377 DNA sequencer whereas PCR–RFLPswere assayed on 2.5% agarose electrophoresis fortwo mtDNA NADH coding regions (ND-1 ND5/6) (Cronin et al. 1993). DNA stability andamplificability was controlled every three months,for three years, in parallel with control DNA. Toevaluate genotyping errors, null alleles presenceand allelic dropout, all the experiments were rep-licated and statistically treated as suggested(Hoffman and Amos 2005; Roon et al. 2005)through MICRO-CHECKER 2.2.3 (Vanoosterhout et al. 2004). Deviations from theHardy–Weinberg equilibrium were tested byArlequin 2000 and Genepop.Our results indicate that the quality and quan-tity of DNA extracted from fin clips and scalesvaried according to extraction methods (Table 2)and storage conditions. Alcohol preservation at)20 C is fundamental for fin specimens, althoughdried scales conservation at room temperatureallowed good DNA extraction. One percent

The purification and partial characterization of low molecular weight phosphotyrosine phosphatase (LMW-PTP) was reported for the first time in PC12 cells. In addition, the expression levels during neuronal phenotype induction by nerve growth factor (NGF) and during neurogenesis in chick embryos were investigated. LMW-PTP was purified to homogeneity and showed a single band of about 18 kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis. A native molecular mass of 20.1 kDa was determined by gel filtration on Sephadex G-75 column. The LMW-PTP from PC12 cells displays structural and biochemical characteristics similar to the enzyme isolated for normal tissues. It was specifically immunoprecipitated by an affinity purified antibody directed against the bovine liver enzyme. The enzyme is present in the cytosolic and cytoskeletal cell compartment where is tyrosine phosphorylated. Time course expression of LMW-PTP in PC12 cells was investigated after NGF treatment and showed an increase of about 30% in the basal level of LMW-PTP from 0 to 72 h. These changes were related to the appearance in PC12 cells of neuronal processes and to a decrease in cell proliferation. An increase of the LMW-PTP expression was also observed in vivo during chick embryo neurogenesis from 8-day-old embryos to adult chicks. The protein level, assayed by immunoblotting, increases from 14-day-old embryos to the hatched chicks reaching the adult levels within the first week after birth. These data indicate that the neurogenesis process is accompanied by a physiological increment of LMW-PTP expression in vitro and in vivo.

Eosinophil cationic protein (ECP) is a small, zinc‐containing molecule and one of the most abundant proteins in eosinophil granules of vertebrates. It is characterized by high cytotoxicity for a variety of parasites, bacteria, viruses and mammalian cells and tissues, and is thus recognized as an important component of the innate immune system. The presence of ECP molecules has been demonstrated in serum of major vertebrate taxa from fish to human. Now we report results, obtained using enzyme‐linked immunosorbent assay (ELISA) and western blotting analyses, on the presence of ECP‐like immunoreactive material in blood cells of the freshwater mollusc, Anodonta sp. Other freshwater invertebrates analysed, planarians (Dugesia polychroa) and sponges (Ephydatia fluviatilis) were devoid of any measurable immunoreactivity. The results were compared with those reported for vertebrates.

The effect of short‐ and long‐term exposure to temperature ranges near the optimal developmental values of perch (Perca fluviatilis) during pre‐ and post‐natal development was studied. A fertilized egg raft was divided and placed in four tanks kept at 10°, 17°, 20° C and variable temperature (10° to 20° C), respectively. Short‐time effects of temperature were investigated analyzing fluctuating asymmetry by measuring three meristic and seven morphometric bilateral characters. Perches reared below 17° C or at variable temperature were less asymmetric than those reared at 20° C. Perches reared at 10° C showed a non‐homogeneous trend of the characters analysed. Long‐term effects were investigated by studying the liver and skeletal muscle 70 kDa heat shock proteins (HSP/HSC70) three and six months after hatching. Two HSP isoforms, HSC73 (73 kDa) and HSP72 (72 kDa), were detected in perch liver by immunoblotting. HSC73 was present at the two developmental stages and at all temperature conditions, while...

The presence of immunologically and biochemically related low‐molecular‐weight phosphotyrosyl protein phosphatase forms in vertebrate brain was investigated. An affinity purified polyclon‐al antibody, directed against the bovine liver enzyme, recognizes, in whole chicken brain, an 18‐kDa protein band having the same electrophoretic mobility as the enzyme purified from the same source and from bovine liver used as reference. A similar electrophoretic pattern was found using brain extracts from an amphibian (Rana esculenta), a teleost (Esox lucius) and an elasmo‐branch species (Raya clavata). The immunocytochemical localization in chicken brain showed immunoreactivity in nerve cell cytoplasm and processes. The subcellular localization, carried out using mild homogenization of fresh chicken brain tissue and differential centrifugation, revealed that the enzyme is essentially associated with both cytosolic and nerve‐ending fractions. Using immunoblotting and biochemical methods, were found similar mo...

The systematic is still unresolved for the genus Squalius (Cyprinidae, Leuciscinae), a rich group of small to large fishes widely distributed throughout Europe. The distinction of one of the Italian narrowly endemic species, Squalius albus (Bonaparte, 1838), described for the area surrounding lake Trasimeno, from the more common and widespread Squalius squalus (Bonaparte, 1837) is doubtful. The application of integrative taxonomy, with DNA taxonomy and quantitative morphometric, using both living and preserved individuals collected from lake Trasimeno before Squalius sp. restocking, allowed us to explicitly test for the identity of the two species in the complex. COI barcoding data, used for phylogenetic reconstructions, underlined that two clades may exist in the complex; nevertheless, DNA taxonomy (ABGD and GMYC) and morphometrics show no statistical support for their identity as separate species. Moreover, during our survey of the genetic diversity of the Italian Squalius, we provided further support for the species status of Squalius lucumonis, and found evidence of the occurrence in Southern Italy of another chub species, Squalius vardarensis (Karaman, 1928), previously known only from the Southern part of the Balkan Peninsula.

Manila clam Ruditapes philippinarum – synonym Venerupis philippinarum (Adams and Reeve, 1850) – is one of the most successful marine invaders, being introduced worldwide for aquaculture and fisheries. Genetic diversity and structure of its populations are largely unknown, especially in the invaded environments. Herein we present molecular genetic data on 12 introduced Manila clam populations in Italy, Spain, and Portugal from both Mediterranean (Adriatic) and Atlantic sampling sites. The phylogenetic information was investigated by the direct sequencing of 16S mitochondrial DNA. Results of mtDNA analyses showed the occurrence of 11 haplotypes for European introduced populations, as a consequence of multiple founder effects from different source populations. Rp hap1 was the most frequent, shared among all populations. The other 10 haplotypes were rare and distributed at local scale, in agreement with what was observed in other invasive bivalves. Biogeographic and phylogenetic analyses based on 16S rDNA of introduced Manila clam populations showed a complex scenario, dominated by multiple pools of individuals coming from different sources, as a consequence of multiple introductions.

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Seven Italian populations of twaite shad Alosa fallax from Northern and Central Italy were investigated to assess genetic diversity by Cytochrome b (Cytb) gene sequencing. The two ecotypes historically referred to landlocked and anadromous populations were investigated for the first time from a genetic point of view, to clarify their phylogenetic relationships. Moreover, results obtained from populations coming from separated Adriatic and Tyrrhenian basins were compared with data assessed in samples of allis shad Alosa alosa from the Atlantic basin. All the Italian samples were recognized at species level as A. fallax, differing for five mutations from A. alosa. The analyses confirmed the occurrence of a single phylogenetic lineage and of a single species within Italian waters, in both landlocked and migratory populations. The minimum spanning network identified six haplotypes for A. fallax and two haplotypes for A. alosa. The neighbour-joining tree and the maximum likelihood on the Cytb gene sequences confirmed two distinct lineages for A. alosa and A. fallax, without evidence of a separation at specific level within the A. fallax group. A weak separation due to incipient population differentiation was detected between anadromous and landlocked Italian populations, supporting the idea of a recent separation. The molecular data herein collected do not support the existence of the already controversial incipient species Alosa agone. Despite this, the two ecotypes could be considered as different management units from a conservation viewpoint.

The Etruscan chub Squalius lucumonis (Bianco, 1983) is an endemic endangered fish inhabiting the lakes and rivers of Central Italy. A microsatellite-enriched genomic library was constructed in order to develop microsatellite loci and eight polymorphic loci were isolated from this species to assess the level of genetic diversity in fifty individuals of S. lucumonis collected in the Umbria Region. The number of alleles ranged from four (Sluc12) to 18 (Sluc4). Observed and expected heterozygosity per locus ranged from 0.05 (Sluc12) to 0.80 (Sluc4; Sluc7 and Sluc11) and from 0.22 (Sluc12) to 0.90 (Sluc4), respectively. Furthermore, these loci were tested on ten other Cyprinidae species and four freshwater fish species belonging to other families. Successful cross-priming amplifications were obtained for several species of Cyprinidae, whereas no positive results were obtained for other species. The availability of the reported microsatellite loci will facilitate population structure investigations of ...

We address the taxonomic position of the southern European individuals of pike, performing a series of tests and comparisons from morphology, DNA taxonomy and population genetics parameters, in order to support the hypothesis that two species of pike, and not only one, exist in Europe. A strong relationship emerged between a northern genotype supported by COI, Cytb, AFLP and specific fragments, and a phenotype with round spot skin colour pattern and a large number of scales in the lateral line, clearly separated from a southern genotype with other skin colour pattern and a low number of scales in the lateral line. DNA taxonomy, based on a coalescent approach (GMYC) from phylogenetic reconstructions on COI and Cytb together with AFLP admixture analysis, supported the existence of two independently evolving entities. Such differences are not simply due to geographic distances, as northern European samples are more similar to Canadian and Chinese samples than the southern Europe ones. Thus, given that the differences between the two groups of European pike are significant at the phenotypic, genotypic and geographical levels, we propose the identification of two pike species: the already known northern pike (Esox lucius) and the southern pike (E. flaviae n.sp.). The correct identification of these two lineages as independent species should give rise to a ban on the introduction of northern pikes in southern Europe for recreational fishing, due to potential problems of hybridisation.

Previous phylogenetic investigations on the mayfly Baetis rhodani Pictet from several European countries, excluding Italy, strongly suggested the presence of cryptic species. Our paper reports a DNA-taxonomy phylogenetic analysis of B. rhodani with additional populations coming from Italian and UK sites, and aims to identify potential cryptic species with a coalescent-based method (GMYC model) and to understand the mechanisms of local coexistence of cryptic species. Twenty-five haplotypes of Italian samples and five haplotypes of UK samples were identified and added to a large European dataset. A total of 11 potential cryptic species have been recognised, and three of them co-occured in one Italian area. Such cryptic species seem to be phylogenetically over-dispersed on the tree and temporally segregated, and the seasonal substitution pattern of cryptic species could explain the apparently widespread distribution of the B. rhodani complex and its ability to adapt to different temperatures and food resources, justifying some of the differences observed in the relationship between water temperature, growth rates and phenology documented from field studies.

The two freshwater sponges Ephydatia fluviatilis and Ephydatia mulleri belong to the widespread Spongillidae family. Their morphological tracts are very similar and can be distinguished mainly on the basis of their gemmuloscleres. However, when gemmules are absent it is essential to have an unambiguous species attribution for a population genetic study based on fresh tissues and historical collections. This article reports a simple Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, applied to DNA extracted from both gemmules and fresh tissues in order to discriminate between the two congeneric E. fluviatilis and E. mulleri. Such a biomolecular method is based on the discriminative enzymes’ digestion of each of the three amplified fragments 5.8S-ITS2-28S, D3 domain of the 28S subunit and COI. Two restriction enzymes were tested for a 620–642 bp fragment of 5.8S-ITS2-28S and for a 342 bp fragment of the D3 domain of the 28S, one restriction enzyme was tested for a 681 bp fragment codifying for COI. Obtained digestion patterns were diagnostic for each of the two species, providing a relatively simple, fast and cheap method for species attribution compared to sequencing.

Myogenesis is mainly sustained by a subpopulation of myogenic cells known as satellite cells (SC). In this paper we studied alpha-smooth muscle (alphaSMA) and alpha-sarcomeric muscle (alphaSRA) actin isoform expression in cultures of human satellite cells (HSC) isolated from skeletal muscle biopsies from a 5-day-old newborn, a 34-year-old young adult and a 71-year-old donor. Myogenicity of cultures was assessed using immunocytochemical detection of desmin and myosin heavy chain. Time-course expression of alphaSRA and alphaSMA were studied with both immunocytochemistry and western blotting procedures. Although alphaSMA was never detected in whole skeletal muscle, both alphaSMA and alphaSRA were detected in proliferating and differentiating HSC derived from donors of all examined ages. The expression level experiments showed that alphaSRA was gradually up-regulated during HSC differentiation, but no significant differences were observed between newborn, young, and elderly HSC cultures. Our data demonstrated that HSC, isolated from subjects of different ages, re-expressed alphaSMA, but its levels and expression pattern varied considerably in the newborn with respect to the young adult and elderly donors. These results are discussed in relation to the myogenic differentiation capability of HSC during human muscle senescence.