juvenile blue fish: species discrimination by pcr-rflp

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.
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
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
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)
Position/Fragments length (bp)
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
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.
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.
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