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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.

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.

We report an updated and expanded list of Rock Partridge (Alectoris graeca) haplotypes found in wild animals throughout the Apennines of central Italy. Samples were collected and identified during a monitoring program of autochthonous Galliformes and from a private collection. The haplotypes were identified on a longer fragment of the mitochondrial control region (D-loop) based on previously reported haplotypes. This novel evidence, based on a wider sampling area and a higher number of analyzed specimens, will be of relevance in both conservation projects and gamebird breeding for restock, as imposed by the Italian Action Plan. Studying longer fragments can also be useful for phylogeographic analysis.

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.

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.

Gene sequence variation in cultivated olive (Olea europaea L. subsp. europaea var.europaea), the most important oil tree crop of the Mediterranean basin, has been poorlyevaluated up to now. A deep sequence analysis of fragments of four genes, OeACP1,OeACP2, OeLUS and OeSUT1, in 90 cultivars, revealed a wide range of polymorphisms along all recognized allele forms and unexpected allele frequencies and genotypecombinations. High linkage values among most polymorphisms were recorded withineach gene fragment. The great sequence variability corresponded to a low number ofalleles and, surprisingly, to a small fraction of genotype combinations. The distribution,frequency, and combination of the different alleles at each locus is possibly due tonatural and human pressures, such as selection, ancestrality, or fitness. Phylogeneticanalyses of allele sequences showed distant and complex patterns of relationships among cultivated olives, intermixed with other related forms, highlighting an evolutionaryconnection between olive cultivars and the O. europaea subspecies cuspidata andcerasiformis. This study demonstrates how a detailed and complete sequence analysisof a few gene portions and a thorough genotyping on a representative set of cultivarscan clarify important issues related to sequence polymorphisms, reconstructing the phylogeny of alleles, as well as the genotype combinations. The identification ofregions representing blocks of recombination could reveal polymorphisms that representputatively functional markers. Indeed, specific mutations found on the analyzed OeACP1and OeACP2 fragments seem to be correlated to the fruit weight.

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.

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 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.

An apparent paradox is known for crustaceans, rotifers and bryozoans living in inland small water bodies: a potential for wide distribution due to the presence of resting stages is coupled with marked genetic differences between nearby water bodies, with enclave distributions masking clear phylogeographic patterns. According to the monopolisation hypothesis, this is due to the accumulation of resting stages, monopolising each water body. Freshwater sponges could represent a useful system to assess the generality of the mo- nopolisation hypothesis: these organisms i) live in the same habitats as crustaceans, rotifers and bryozoans, ii) produce resting stages that can accumulate, and iii) have indeed a wide distribution. Currently, no studies on spatially explicit genetic differentiation on fresh- water sponges are available. The aim of the present study is to provide additional empirical evidence in support of the generality of the scenario for small aquatic animals with resting stages by analysing genetic diversity at different spatial scales for an additional model system, the freshwater sponge ephydatia fluviatilis (Linnaeus, 1759). We expected that system genetic variability would follow enclave distributions, no clear phylogeographical patterns would be present, and nearby unconnected water bodies would show markedly different populations for this new model too. We analysed the ribosomal internal transcribed spacer regions 5.8S-ITS2-28S, the D3 domain of 28S subunit, the mitochondrial Cytochrome c Oxidase I (COI) and ten specific microsatellite markers of nine Italian and one Hungarian populations. Mitochondrial and nuclear sequences showed no or very low genetic polymorphism, whereas high levels of differentiation among populations and a significant polymorphism were observed using microsatellites. Microsatellite loci also showed a high proportion of private alleles for each population and an overall correlation between geographic and genetic distances among populations. All the expectations from the monopolisation hypothesis seemingly were confirmed for the analysed sponge.