Biol. Mar. Mediterr. (2011), 18 (1): 386-387 G. Guerriero, A. Di Finizio, S. Trocchia, N.H. Elnwishy*, G. Ciarcia ECLab (certif. UNI EN ISO 9001:2008), Department of Biological Sciences, University of Naples Federico II - 80134 Naples, Italy. *Biotechnology Research Center, Suez Canal University, Ismailia - 42522 Egypt. JUVENILE BLUE FISH: SPECIES DISCRIMINATION BY PCR-RFLP IL NOVELLAME DI PESCE AZZURRO: DISCRIMINAZIONE DI SPECIE MEDIANTE PCR-RFLP Abstract - A PCR-RFLP analysis of a 630 bp fragment of the mitochondrial 16S rRNA gene was carried out in Sardina pilchardus (Pisces, Clupeidae), Neosalanx tangkahkeii (Pisces, Salangidae), Engraulis encrasicolus (Pisces, Engraulidae). Obtained restriction patterns using MvaI restriction endonuclease allow juvenile blue fish discrimination also in unprocessed commercial samples mistakenly fraudulently listed as “bianchetto”. Key-words: species identification, juvenile blue fish, bianchetto, PCR–RFLP, 16S rRNA, MvaI. Introduction - Along the Italian coastline, many navy during the winter have targeted commercial juvenile Sardina pilchardus, called “bianchetto”. Due to the growing market demand and the overfishing of these resources, species of different commercial value are given up as “bianchetto” (Armani et al., 2011). Analysis of morphological characters is not always able to identify the species and protein patterns analysis has become impossible for the precarious temperature conditions in which they are marketed (Teletchea, 2009). A restriction fragment length polymorphisms (PCR-RFLP) analysis of an about 630 bp mitochondrial 16S rRNA gene fragment was carried out in Sardina pilchardus, Neosalanx tangkahkeii, (a freshwater Chinese specimen sold as “bianchetto”), Engraulis encrasicolus (sort of “blue fish” whose juveniles are often mistakenly considered “bianchetto”), and several fresh samples on the market listed as “bianchetto” for discrimination and exploitation of those Italian fish species resources. Materials and methods - S. pilchardus, and E. encrasicolus (Navy of Torre del Greco, Italy), N. tangkahkeii (Chinese Academy of Fishery Science, Qingta, Beijing, China) adult fresh species were used as reference. Samples listed as “bianchetto” (two preparations) were bought in three different market sites (North-Central-South Italy). PCR amplification of the 630 bp 16S rRNA gene fragment was performed as previously reported (Di Finizio et al., 2007). Virtual restriction maps of 16S rRNA gene sequences were obtained using NEB cutter software (New England Biolabs, MA, USA) on the basis of our previous S. pilchardus, N. tangkahkeii and E. encrasicolus sequences. After the search for species specific restriction sites, expected digestion profiles were calculated to obtain combined restriction patterns informative for species discrimination. PCR products derived from amplification of 16S rRNA gene fragment of whole species and “bianchetto” samples were subjected to restriction digestion without previous purification using MvaI, Bsh 1285I (Fermentas, Milan, Italy), and reaction mixtures specified by the manufacturer. Results - The cleaved fragments obtained from PCR amplicons from 16S rRNA gene digestion of each reference sample E. encrasicolus, N. tangkahkeii and S. pilchardus with MvaI endonuclease resulted in agreement with those expected by theorical analysis of our obtained sequence (Tab. 1) published in GenBank with accession number respectively of EU419752, EU419753, EU419754. After digestion Juvenile blue fish: species discrimination by PCR-RFLP 387 of samples generically referred as “bianchetto”, combined electrophoretic patterns provide evidence of various species (Fig. 1). Samples (B 1÷6) were identified as N. tangkahkeii in B1, B2, B3, B4; E. encrasicolus in B5 and S. pilchardus in B6. Tab. 1 - T heoretical restriction patterns obtained on the basis of sequences of mitochondrial 16S rRNA gene expected after PCR amplification and MvaI digestion in the examined species. Pattern di restrizione teorico ottenuto per il frammento mitocondriale 16S rRNA dopo digestione con l’enzima MvaI per le specie considerate. Fish species (abbreviation) Engraulis encrasicolus (Ee) Neosalanx tangkahkeii (Nt) Sardina pilchardus (Sp) MvaI Cuts 0 1 2 Position/Fragments length (bp) undigested 71/71; 562 181; 288/181; 107; 327 Fig. 1 - PCR–RFLP analyses of the 16S rRNA gene fragment amplified from all “bianchetto” samples (B1-B6) digested with the restriction enzyme MvaI (M) and with Bsh 1285I (H) as endonuclease of control; lane C: undigested amplicon; lane L:100 bp DNA ladder (Fermentas). Analisi RFLP del frammento del gene 16S rRNA dei campioni di bianchetto (B1-B6) digeriti con l’enzima di restrizione MvaI (M) e con Bsh 1285I (H) endonucleasi di controllo; lane C: campione non digerito; lane L:100 bp DNA ladder (Fermentas). Conclusions - The utilized enzyme digests the unpurified 630 bp 16S rRNA gene PCR products in only one step. On a total of six samples examined, four were identified as N. tangkahkeii, and only two (B5 and B6) respectively as blue fish E. encrasicolus and S. pilchardus. Among the other species of juvenile blue fish, E. encrasicolus could be mistakenly marketed as “bianchetto” (Armani et al., 2011); we have not found morphological similar species as Sprattus sprattus (Ss) and Sardinella aurita (Sa) that show a different restriction pattern (Ss: MvaI 139/139; 490 Sa: MvaI 291/291; 439 data unpublished), or Harengula jaguana similar too but not biodistributed in the Mediterranean coasts. This rapid and widely diffused molecular approach indicating only one enzyme, the endonuclease MvaI, allowed an unequivocal discrimination between unprocessed juvenile blue fish economically important species to safeguard. References ARMANI A., CASTIGLIEGO L., TINACCI L., GIANFALDONI D., GUIDI A. (2011) - Molecular characterization of icefish, (Salangidae family), using direct sequencing of mitochondrial cytochrome b gene. Food Control, 22: 888-895. DI FINIZIO A., GUERRIERO G., RUSSO G.L., CIARCIA G. (2007) - Identification of gadoid Species (Pisces, Gadidae) by sequencing and PCR–RFLP analysis of mitochondrial 12S and 16S rRNA gene fragments. Eur. Food. Res. Technol., 225: 337-344. TELETCHEA F. (2009) - Molecular identification methods of fish species: reassessment and possible applications. Rev. Fish Biol. Fisheries, 19: 265-293.