European plaice (Pleuronectes platessa) is quite frequently substituted with other flatfish, especially with Yellow fin sole (Limanda aspera), which differs not only in meat quality but also particularly in its origin and manipulation chain. We propose an integrated approach of laboratory and in silico mtDNA PCR-RFLP procedures generating a set of restriction patterns easily resolvable in agarose gel, able to discriminate with certainty P. platessa from other 20 flatfish species. The herein proposed procedure is an economical and valid tool in detecting mislabelled seafoods. Frecuentemente, la solla europea (Pleuronectes platessa) es sustituida por otros peces planos, utilizandose especialmente la limanda (Limanda aspera). Esta se distingue de la primera no solo por la calidad de su carne, sino tambien por su origen y por su cadena de manejo. En el presente articulo, los autores proponen realizar un enfoque integrado de procedimientos de laboratorio y de in silico mtDNA PCR-RFLP, a partir de los cuales se genera un conjunto de patrones de restriccion facilmente resolubles en gel de agarosa. Dicho enfoque puede discriminar con seguridad entre P. platessa y otras veinte especies de peces planos. El procedimiento que aqui se propone representa un metodo economico y valido para la deteccion de pescados mal etiquetados.

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.

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 these species aiming to phylogeographical approaches and conservation strategies.

Development perturbations may affect the regular phenotype and are commonly measured through fluctuat-ing asymmetry (FA) levels. Short-term climatic variations, that modify the temperature, can influence chemical and physical water characteristics. Fishes have been used as model organisms for studying stress-induced changes in body symmetry, since they are ectothermic, good bioindicators, easy to find and having economic relevance. Northern pike being a holoarctic, big, edible, top predator is one of the most economically important freshwater fish for recreational and commercial fisheries and freshwater ecosystems management. The isolated population of Lake Trasimeno (Central Italy)—in good health conditions and that can be considered one of the genetically best conserved of Italy—, was chosen as model. FA, seven mi-crosatellite loci and early developmental stages were investigated in order to correlate the developmental stability of this population to its genetic variability and to environmental perturbations. The results obtained underlined a positive correlation (>>0.40) between FA indexes and temperature; the non-parametric Kruskall- Wallis test showed significant differences in FA levels for some FA indexes and parameters. Over-all results underlined that FA increased in individuals grown at a temperature above 8°C as compared with those grown at 5°C or at lower temperatures. Both positive and negative correlations between FA parameters and chemical and physical water characteristics were shown. The comparison of genetic and FA data under-lined a low correlation between microsatellite and FA pairwise distances, nevertheless a positive and signifi-cant correlation emerged for some FA measurements and microsatellite data. In particular, only Elu87 locus showed a statistical significant correlation versus total FA. Finally, as expected, results indicated that the in-cubation time was temperature-dependent; the ODT was in the range 8–10°C and lower and higher tempera-tures caused drastic embryo mortality. These results showed robust correlations, both positive and negative, between some FA parameters and chemical and physical characters and were in agreement with the assump-tion that temperature variations as well as pH, conductibility and chloride variation, may increase molecular perturbations and, subsequently, the global developmental noise during development. These data suggest that FA could be considered a measure of animal welfare. The relative breeding easiness of this species may be a valid tool for the estimation of controlled environmental stress influences, not only of thermal origin, and a valid information basis for studies on wild populations. Furthermore, it has long been debated whether FA levels depend upon genetic variability, the particular molecular marker notwithstanding, and whether it is possible to use one or more molecular markers to better understand FA data. The Mantel tests performed in this study showed very interesting correlations between FA and the investigated microsatellites. For the lack of a linkage map for the investigated microsatellite loci, it is presently impossible to establish the relation-ships between the FA parameter and the microsatellite loci that the Mantel test defines as correlated.

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.

Genetic analyses allowed management and conservative programmes specifically designed for biodiversity conservation purposes. To date, no information is available for the Ne definition of any Italian northern pike population, because of their wide, highly fragmented, distribution. Low gene flow levels may have caused some of the health population problems emerging from professional fisheries data, underlining a marked reduction of these populations. This paper reports the first estimate of genetic Ne of the Lake Trasimeno's northern pike, by means of microsatellites data, over the last four decades compared with five other significant Italian populations. Ne values were evaluated through specific software and θ estimator. The Ne results confirm that, despite a marked reduction between 1966 and 1997 and a less than optimal status, the Trasimeno population shows a positive trend in its health status, presumably due to proper management programmes. Considerations on the biological conservation and management of this pike population are also reported.

The two freshwater sponges Ephydatia fluviatilis and Ephydatia mülleri 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. mülleri. 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.

A rapid, nondestructive, reproducible and cheap DNA extraction method from body mucus and buccal cells of northern pike and brown trout is described. Buccal cells and body mucus were sampled on FTA Cards; the captured DNA was used directly for microsatellite and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses. A complete concordance with control DNA was found. The genotyping error rate for microsatellite ranged from 1.9% to 3.3% for the northern pike and brown trout, respectively. This methodology, using for the first time these materials as a fish DNA source, combines speed of sampling and processing, with a twofold to a threefold time and costs saving.

A set of 11 polymorphic markers (1 cleaved amplified polymorphic sequence (CAPS), 2 sequence-characterized amplified regions (SCARs), and 8 single-nucleotide polymorphism (SNP)-derived markers) was obtained for olive cultivar identification by comparing DNA sequences from different accessions. Marker development was more efficient, using sequences from the database rather than cloning arbitrary DNA fragments. Analyses of the sequences of 3 genes from 11 diverse cultivars revealed an SNP frequency of 1 per 190 base pairs in exons and 1 per 149 base pairs in introns. Most mutations were silent or had little perceptible effect on the polypeptide encoded. The higher incidence of transversions (55%) suggests that methylation is not the major driving force for DNA base changes. Evidence of linkage disequilibrium in 2 pairs of markers has been detected. The set of predominantly SNP-based markers was used to genotype 65 olive samples obtained from Europe and Australia, and was able clearly to discriminate 77% of the cultivars. Samples, putatively of the same cultivar but derived from different sources, were revealed as identical, demonstrating the utility of these markers as tools for resolving nomenclature issues. Genotyping data were used for constructing a dendrogram by UPGMA cluster analysis using the simple matching similarity coefficient. Relationships between cultivars are discussed in relation to the route of olive's spread.

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 14 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 observed and from populations of the Adriatic slope of the Apennines. This population should be employed in managing and breeding programmes finalised to an eco-sustainable restocking of brown trout in the Nera River Basin, with periodic monitoring by genetic analyses.

Seven microsatellite loci were recently described for the mayfly Baetis rhodani, but, at present, no data are available on the population structure of this species using these loci. We used four of these microsatellite loci for a preliminary analysis of the genetic differentiation of the mayfly B. rhodani in six populations from three Italian streams belonging to the same or different, but geographically distant, drainage basins. From these data, genetic polymorphism was observed between the sampled populations and clear differences were identified between pairs of populations; however, no specific population pattern was observed. The high F-ST values suggest a limited dispersal of the species across the sampling sites. On the whole, the results obtained with microsatellite loci, characterized by marked heterozygosity deficiency, confirm the hypothesis of the patchy recruitment from a small number of matings and limited in-stream dispersal, as previously pointed out for some other baetid mayflies by genetic analyses using allozymes and mtDNA. This is the first contribution to a knowledge of the genetic structure of Italian Baetis rhodani populations.

Using light microscopy the morphology, the mitotic index and levels of erythroid cell types were detected from 48 h pike Esox lucius embryos before hatching to adult specimens. At the same developmental stages, the haemoglobins and globin chains expressed were electrophoretically characterized. The erythroid cells of the primitive generation were the most abundant from 48 h before hatching until 15-20 days after hatching, then their number decreased and only rare cells remained in the 3 month-old juvenile specimens. These cells divided and differentiated in the blood and were substituted by the definitive erythrocyte series. As in other vertebrates, the immature cells of the two generations differed in morphological properties and in the synthetized haemoglobin. The circulating erythroid cells of the definitive population cell lineage were, at all differentiation stages, smaller than those of the primitive generation. The definitive erythrocytes appeared in blood smears of 7 days post-hatching larvae, they increased rapidly and at 20 days they represented the predominant red blood cell population in the circulation of young pike. Electrophoretic analysis of haemolysates obtained from different developmental stages indicated the presence of distinct embryonic, larval and adult haemoglobins. The embryonic haemoglobins differed from those of the older larva and juvenile specimens and were detectable within the first week of post-hatching development when only primitive erythrocytes were present in the blood. (c) 2005 The Fisheries Society of the British Isles.

The status of these two ?twin? species in Italy is extremely different; stone marten is widely spread throughout the entire peninsula, while absent in the islands, pine marten is spread in Sardegna, Sicilia and Isola d'Elba, but its distribution in the peninsula is very fragmented. Such a situation allows presuming a competition between the two species, where the beech marten comes out as the ?winner?. This information points out the possibility of conservation programs to preserve the pine marten and to contain the stone marten. Thus it is of primary the necessity of an unmistakable differential taxonomical diagnosis between the two species. Objective difficulties in the identification and recognition of these Mustelids are already known. Thus a program of genetic characterization of pine and stone marten is being performed, also to prove the existence of effectively discriminating morphological characters. Up to now, 24 individuals were been analyzed from Toscana, Umbria, Marche, Lazio and Basilicata. Taxonomical ascription was been performed according to several morphological and metrical characters such as the coat-color and markings pattern and the length of the baculum . The reliability of this diagnosis has been verified through the genetic analysis performed on tissues sampled from each one individual. Genetic analysis was arranged both with direct sequencing and specific PCR-RFLP processing DNA by specific restriction enzymes (AluI and HaeIII) which cut a diagnostic mtDNA fragment in the beech but not in the pine marten. Morphological characters ascribed 10 individuals to Martes martes and 14 to Martes foina ; genetic results confirm the 10 pine martens, but only 12 out of the 14 stone martens. Therefore 2 individuals morphologically diagnosed as ?stone marten? resulted genetically ?pine marten?. Furthermore, for 6 individuals it was possible to analyze the DNA extracted from the faeces, thus giving the chance to perform a genetic identification through those indices of presence.

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 studied. A fertilized egg raft was divided and placed in four tanks kept at 10degrees, 17degrees, 20degrees C and variable temperature (10degrees to 20degrees C), respectively. Short-time effects of temperature were investigated analyzing fluctuating asymmetry by measuring three meristic and seven morphometric bilateral characters, Perches reared below 17degrees C or at variable temperature were less asymmetric than those reared at 20degrees C. Perches reared at 10degrees 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 HSP72 was detected only at the variable temperature and 20degrees C. The muscle tissue always showed the HSC73 form. Our data suggest that below 20degrees C only some morphological characters were affected, whereas the 20degrees C temperature and, presumably, higher ones, seemed to have both short- and long-term effects.

The presence of immunologically and biochemically related low-molecular-weight phosphotyrosyl protein phosphatase forms in vertebrate brain was investigated. An affinity purified polyclonal 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 elasmobranch 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 molecular mass and biochemical properties of low-molecular-weight phosphotyrosyl protein phosphatase in brain of all the animals studied, suggesting a similar physiological role in vertebrate phylogenesis.

High molecular weight zinc ion-dependent acid p-nitrophenylphosphatase (HMW-ZnAPase) was purified from bovine liver to homogeneity as judged by native and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The partial sequence of the purified enzyme electroblotted on PVDF membrane reveals a 95% sequence homology with human and bovine liver fructose-1,6-bisphosphate aldolase isozyme B (FALD B). FALD B was isolated from bovine liver using an affinity elution from phosphocellulose column. FALD B from bovine liver shows a native and subunit molecular weight that is indistinguishable from that of HMW-ZnAPase. In addition, an affinity purified antiserum raised in rabbits against purified HMW-ZnAPase cross-reacts with bovine liver FALD B and rabbit muscle isozymes. Despite these similarities, HMW-ZnAPase does not show FALD activity and bovine liver FALD does not display any zinc ion-p-nitrophenylphosphatase activity. These results suggested the existence of structural and immunological similarities between bovine liver HMW-ZnAPase and FALD B. Differences in some amino acid residues in enzyme activity indicate that they may be involved in different biochemical functions.

Fluctuating asymmetry (FA), the random deviation from perfect bilateral symmetry, is the basis of an acknowledged method to analyse developmental stability in animals and plants. This study investigates the fluctuating asymmetry in perch (Perca fluviatilis L.) from one natural lake (Trasimeno) and two regulated lakes (Corbara, Piediluco) of central Italy. Seven morphometric (pec toral and ventral fins length, pre-pectoral fin distance, pre-ventral fin distance, pectoral-ventral fins distance, postorbital distance, head length) and six meristic (lateral line scales, pectoral and ventral fins rays, mandibular pores, upper and lower gill rakers) bilateral characters were selected to detect fluctuating asymmetry. Total and standard length, weight, sex and age were employed to investigate growth of perch. On the basis of FA levels a comparison of the three lakes was then made. Statistical analysis shows a relationship between fluctuating asymmetry levels and growth, and the possibility to order the lakes according to FA levels. Therefore, FA can be considered a tool to detect stress occurring during development.