“Frauds and fish species authentication: study of the complete mitochondrial genome of some Sparidae species to provide specific barcode markers”

The growing global diffusion of seafood for human consumption requires always more accurate sanitary and quality controls. Among the globally marketed fish, the species belonging to the family Sparidae are excellent food-fishes of high economic value. The Sparidae family comprises about 38 genera and 159 species, some of which are highly appreciated as seafood. In Italy, the fish species of commercial interest are defined by the law (DM n. 19105 del 22 Settembre 2017). Among them, 41species belong to the Sparidae family. The identification of these species is difficult even when external characters are preserved due to their morphological similarity. Species identification becomes even harder to achieve after industry processing, when distinctive external traits are removed. Nevertheless, despite their similarity, sparid species have different organoleptic quality that corresponds to variable prices in fish markets. Consequently, substitution of commercially important Sparidae species is common. The sparid specie with highest commercial value, Dentex, is often replaced with less expensive species or lower quality alternatives. All this premised, Sparidae species identification requires immediate resolutions. Research on fish mitochondrial DNA (mtDNA, mitogenome) has led to substantial advances in the fields of species authentication and population biology. The main species’ specific DNA sequences used as markers for fish species identification belong to the mitochondrial genes encoding ribosomal 16S and 12S subunits, cytochrome b (Cytb), and cytochrome c oxidase I (COI). However, current research shows that mitochondrial DNA markers perform well for certain species but may be less discriminating for others. Therefore, they cannot be used indiscriminately for the identification of all fish species. This situation has led to the formulation of the proposal to study and analyze the complete mtDNA sequence with the aim to identify mitochondrial markers or multiple marker approaches with higher and more specific discrimination capacity. At present, mtDNA genomes of Sparidae fishes are not conspicuously represented in the scientific literature, which makes difficult to understand if currently used genetic markers are the most effective for sparid species identification. This thesis project had the aim to study and analyze Sparids’ complete mtDNA sequence to identify mitochondrial markers or a multiple marker approach to achieve a higher and more specific discrimination capacity. We focused on the genera Dentex, Pagellus and Pagrus, listed in the Ministerial Decree

Modulation of the One Health Approach to Tackle Brucellosis in Buffaloes and Cattle in Two Italian Territories with Different Characteristics

The new European Union animal health law and its rule concerning brucellosis in cattle and buffaloes The authors examine the latest European Union (EU) rules concerning eradication and surveillance of brucellosis and animal infectious diseases of EU concern. The Italian rules concerning brucellosis in cattle and buffaloes Italy is included in the EU co-financed compulsory eradication and surveillance programmes for brucellosis in cattle and in buffaloes in the frame of the EU and the related Italian laws, which allowed reaching the Brucellosis Free status without vaccination (former "Officially Brucellosis Free - OBF" status) in the majority of the northern and middle Italian Regions and in some middle and southern Provinces included in Regions where the infection persists. Epidemiology of brucellosis in the EU and in Italy In the Italian Province of Caserta, the highest prevalence of brucellosis in buffaloes is reported; in 2017-2021, a total of 314 outbreaks occurred, in which 39,163 heads tested positive. Here, brucellosis is threatening not only human health and the widespread buffaloes breeding but also the important satellite activities concerning the Protected Designation of Origin (P.O.D.) cheese “Mozzarella di Bufala Campana". The authors also discuss the reemerged brucellosis in cattle in the Molise Region, which despite bordering the Province of Caserta, shows different hydrographic, orographic, and breeding characteristics. In Molise, the reemerged brucellosis had a very different epidemiological course, which allowed it to limit the adoption of One Health measures. The One Health approach to tackle brucellosis in buffaloes In order to tackle brucellosis in Campania Region and in its Province of Caserta, the One Health approach has been predisposed through strict control of animal health, human health, and the environment. The adopted model could be exportable to territories having similar characteristics

The Sparidae mitochondrial genomes comparison may provide alternative barcode markers

The molecular markers used for differentiating fish species to detect fraudulent substitutions in prepared and transformed fishery products are mitochondrial (mt) DNA sequence fragments. The most used belong to the genes encoding for ribosomal 16S and 12S subunits, cytochrome b (cytb), and cytochrome c oxidase I (COI). The genetic variability of the complete mt genome of the perciform fishes of the family Sparidae has never been investigated before and remains elusive. Prompted by the aim to identify new specie-specific genetic markers to use against frauds, we analyzed and compared the complete mitogenome of thirteen Sparidae species, including four newly sequenced ones. We searched for mtDNA regions with high interspecific variability (barcodes) flanked by sequences with high sequence conservation (primer). Results showed that the nucleotide sequence variability in NAD group genes (NAD1 39%, NAD2 50%, NAD3 39%, NAD4L 39%, NAD4 43%, NAD5 41%, NAD6 44%) was much higher than in the molecular markers used for species identification, i.e. cyt b (36%), COI (32%) and ribosomal 16S (24%) and 12S (21%). Further, NAD group genes showed a very high discrimination capacity, suggesting their utilization as alternative DNA barcodes for the Sparidae fishes. In particular, the NAD5 gene allows to seek for regions with high nucleotide variability flanked by conserved areas appropriate to design Sparidae-specific primers. This study highlights the importance of complete mtDNA genome comparisons of commercially valuable fish species to identify regions with high (barcode) and low (primer) interspecific nucleotide variation, to be used for species identification of fishery products

Frauds and fish species authentication: Study of the complete mitochondrial genome of some Sparidae to provide specific barcode markers

Sparids have different organoleptic properties that correlate with a wide variety of retail prices in the market. Components of the observed morphology are rarely sufficient for full identification of these fish species, whose authentication requires specialist knowledge. Genetic diversity or variation and their measurements enable molecular methods as one of the most suggested remedies for aliud pro alio frauds. Genetic approaches have the potential for reducing costs and providing correct identification for a large number of market products. Mitochondrial (mt) DNA sequences (16S and 12S ribosome subunits, cytochrome b-CYTB, and cytochrome c oxidase I-COI) have been widely used for fish species identification. Yet, these mtDNA regions perform well for certain species but are less discriminating for others. Here, we report the first study of the whole mtDNA of the perciform fishes of the family Sparidae with the aim to select more efficient barcoding markers for taxonomical discrimination against frauds. For species-level sequence information, we analyzed and compared the whole sequence of thirteen Sparidae mitogenomes, nine publicly available and four recently sequenced ones. In particular, we searched for effective DNA barcode markers for the correct identification of sparid species by looking for interspecific variable regions flanked by conserved sequences for PCR primer design. We found that only four mtDNA genes are devoid of insertions or deletions, which can complicate the process of sequence alignment. Among them, NAD genes show encouraging utility in discriminating closely related sparid species owing to nucleotide sequence variability compared with classical barcodes for species. Discrimination capacity of NAD genes suggests their application as alternative mtDNA tools for the identification of Sparidae fishes. In particular, NAD5 fragments with high interspecific nucleotide sequence divergence were amplified and appear flawless for Sparidae species identification

Comparative mitogenomic analysis of Sparids and evaluation of a new potential DNA barcoding marker for Dentex dentex.

Dentex dentex is one of the most commercially caught Sparidae species in the Mediterranean Sea and Atlantic Ocean. It is very appreciated in European markets and consequently more subjected to species substitution frauds [1]. The currently mitochondrial (mt) DNA sequences used for fish species identification in prepared and processed products are cytochrome b-CYTB, cytochrome c oxidase I-COI, 16S and 12S genes. Recent researches showed that the study of the whole mtDNA allows to identify new, effective and specie-specific barcode markers [2]. In particular, NAD5 gene has high discrimination capacity for Sparidae species. However, the use of all these genes needs amplification and a sequencing process [2,3]. Therefore, a valuable species identification requires many laboratory steps and is time consuming. The aim of this research was to analyze and compare the whole mtDNA sequence of Sparidae species to find a barcoding marker useful to identify the sparid species D. dentex, avoiding the sequencing step. Thirteen Sparidae complete mitogenomes were compared in this study. They were aligned by UGENE software. Hamming Distance algorithm was used to evaluate in percent the genetic dissimilarity among species and genes. Overall mean p-genetic distance analyses were conducted using the Maximum Composite Likelihood model. The nucleotide sequence variability was determined by aligning gene-by-gene sequences of Sparidae species using MEGA 6.0. Primers were designed by eye after multiple alignment of the Sparidae complete mtDNA sequences using BioEdit Sequence Alignment Editor. Primers efficiency for D. dentex identification was tested using PCR reaction. Results of Hamming Distance, nucleotide sequence variability and p-genetic distance analysis showed the potentiality of NAD2 gene as barcode marker for sparids. The PCR reaction confirmed the discrimination capacity of NAD2 gene. In particular, the amplification of the selected NAD2 fragment was possible only for the species D. dentex. In conclusion, NAD2 gene showed high interspecific nucleotide dissimilarity to provide unambiguous results for D. dentex species authentication without sequencing, reducing time, costs and efficiency. In fact, species identification results can be obtained in a few hours of lab work. Therefore, competent national authorities responsible for monitoring and enforcing could improve and make full use of DNA-testing methods in order to deter operators from false labelling of seafood. In agreement with Regulation (EU) 1379/2013, this study contributes to the molecular traceability of fishery products. [1] Katavic et al. Growth performance of pink dentex as compared to four other sparids reared in marine cages in Croatia. Short Communication. Aquaculture International, 8:455–461, 2000. [2] Ceruso et al. Frauds and fish species authentication: study of the complete mitochondrial genome of some Sparidae to provide specific barcode markers, Food Control, accepted for publication, 2019. [3] Armani et al. DNA and Mini-DNA barcoding for the identification of Porgies species (family Sparidae) of commercial interest on the international market. Food Control, 50: 589-596, 2015

RICONOSCIMENTO DI ALCUNE SPECIE DI PLEURONECTIFORMES MEDIANTE ANALISI PROTEOMICA

L’ordine dei Pleuronectiformes comprende numerose specie ittiche, note come “Pesci piatti”. Si tratta di specie morfologicamente simili tra loro, ma con caratteristiche organolettiche e nutrizionali molto diverse e, quindi, differente valore commerciale. Quando queste specie sono poste sul mercato sottoforma di prodotti preparati o trasformati, è difficile risalire alla specie d’appartenenza mediante il semplice esame visivo. L’analisi del DNA mitocondriale è il metodo più diretto per l’identificazione di specie. Tuttavia, qualora le specie ittiche presentino un elevato grado di omologia del genoma, come nel caso dei Pleuronectiformes, il riconoscimento è particolarmente complesso, poiché i frammenti indagati si differenziano per poche mutazioni puntiformi. In questo caso la proteomica, ovvero lo studio del completo corredo proteico espresso da una cellula o da un tessuto di un individuo, si rivela complementare alla genomica. A tale scopo, è stato analizzato e confrontato il proteoma di sei specie di appartenenti all’ordine dei Pleuronectiformes. • Metodi Lo studio è stato condotto su sei specie di Pleuronectiformes, con diverso pregio commerciale e diversa origine geografica. Le specie oggetto di indagine sono state le seguenti: 1. Solea vulgaris (Sogliola) 2. Solea senegalensis (Sogliola atlantica) 3. Solea lascaris (Sogliola dal porro) 4. Solea kleinii (Sogliola turca) 5. Microchirus variegatus (Sogliola fasciata) 6. Synaptura cadenati (Sogliola oceanica) Le proteine sarcoplasmatiche sono state analizzate mediante elettroforesi monodimensionale (SDS-PAGE) ed elettroforesi bidimensionale (2-DE). Per l’SDS-PAGE sono state utilizzate 100 µg di proteine. La IEF degli estratti è stata effettuata su 300 µg di proteine, utilizzando strip di gel di 7 cm di lunghezza in un range di pH 3-10 non lineare. La seconda dimensione è stata eseguita su un gel al 12,5% di acrilammide, con amperaggio costante di 25 mA per gel. La colorazione delle mappe proteiche è stata effettuata attraverso l'utilizzo di Bio-Safe Coomassie (Biorad). • Risultati L’elettroforesi monodimensionale degli estratti proteici ha consentito di rilevare un elevato grado di polimorfismo tra le sei specie appartenenti all’ordine dei Pleuronectiformes. Le 2-DE hanno permesso di evidenziare spot diversi per qualità e quantità tra le specie in analisi. • Conclusioni L’acquisizione e l’analisi del pattern proteico degli esemplari appartenenti all’ordine dei Pleuronectiformes indagati si è confermato uno strumento valido per discriminare tra le specie. E’ stato possibile individuare spot specifici per ciascuna delle specie oggetto di indagine. Evoluzioni di questo studio sono in corso per identificare le proteine specie-specifiche mediante tecniche di spettrometria di massa e per risalire al gene che codifica tali proteine. In questo modo sarebbe possibile individuare tratti del genoma dei Pleuronectiformes che presentino un maggiore polimorfismo di specie rispetto ai tratti finora utilizzati in letteratura ai fini identificativi di specie. Tale approccio consentirebbe di disegnare primers specifici che permettano il riconoscimento anche in prodotti trasformati mediante l’utilizzo di tecniche molecolari di routine

Development of a method to extract and amplify the complete mitogenome of some sparidae species

Previous studies showed that fish mitochondrial DNA (mtDNA) is set up by a closed circular molecule of 16-17 kilobases (kb), comprising 2 ribosomal RNA genes (rRNA), 22 transfer RNA genes (tRNA), 13 protein-coding genes and 2 non-coding regions. The analysis of single mtDNA genes, such as Cytb, COI, 16S and 12S, or short segment of them, has been widely used against species substitution in both fresh and processed fish products. The analysis of the complete mitochondrial genome of fishery products allows to better study and characterise fish species. The aim of this research was to extract and amplify the complete mtDNA of some fish species of commercial interest belonging to the Sparidae family. The studied species were Dentex dentex, Dentex gibbosus, Dentex nufar, Pagellus acarne and Pagellus erythrinus. The entire mitogenome was obtained by gene amplification using long polymerase chain reactions. The analysis of the complete mitochondrial sequences will allow to gain further insights on these species and to find polymorphic sites that assess the degree of genetic variability of the species belonging to the family Sparidae