Italian Journal of Food Safety 2018; volume 7:6893

A case study on the labeling of FishStat Plus data sources, the total fishery bottarga produced in Sardinia production of mullets from the Eastern Correspondence: Pierluigi Piras, Scuola di Central Atlantic was over 30 thousand Dottorato di Ricerca in Produzione e from ovaries of grey mullets tonnes in 2010 (Harrison, 2016). They are Sicurezza degli Alimenti di Origine Animale, (Mugil cephalus and Mugil members of the order Mugiliformes, repre- Dipartimento di Biologia Animale, Università degli Studi di Sassari, via Vienna sented by a single family, and including capurrii) caught in Eastern 2, 07100 Sassari, Italy. Central Atlantic coasts officially 62 species belonging to 14 genera Tel. +39.339.2056525 - Fax: +39.079.229458. (Thomson, 1997). Recently, (Whitfield et E-mail: [email protected] al., 2012) it was proposed to recognize 20 Pierluigi Piras,1 Francesco Sardu,2 mullet genera including 70 species (11 of Key words: Grey mullets, Species identifica- Domenico Meloni,3 which belong to the genus Mugil). tion, Fish products, Bottarga. Maria Vittoria Riina,4 Systematics of Mugilidae are still much 4 4 debated and based primarily on morpholog- Acknowledgments: the Authors thank Chiara Beltramo, Pier Luigi Acutis Giuseppe Damele, whose professional colla- 1 ical characters, but those classically used in Veterinary Public Health and Food boration in executing fish photographs has species identification are remarkably simi- Security Service of the Region of been extremely helpful. lar within this family (Durand et al., 2012) Sardinia, Cagliari; 2Oristano Local and make quite challenging species identifi- Contributions: the authors contributed equally. Socio-Sanitary Area of Sardinia Public cation (Gonzales-Castro and Ghasemzadeh, Company for Health Protection, 2016). Using a mitochondrial gene-based Conflict of interest: the authors declare no 3 Oristano; Department of Veterinary phylogeny as criterion, Mugilidae potential conflict of interest. Medicine of the Sassari University, classification was recently proposed (Xia et Funding: none. Sassari; 4Genetics and al., 2016). While not disputing such Immunobiochemistry Laboratory of the author’s molecular results, Harrison (2016) Received for publication: 10 July 2017. Zooprophylactic Institute of Turin, Italy found that they disagree with morphologi- Revision received: 29 August 2017. cal analyses, and the nomenclature does not Acceptedonly for publication: 7 September 2017. seem to be properly used in commercial and research fisheries communities and by other This work is licensed under a Creative applied fieldworkers who might not be Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). Abstract familiar with the systematic literature. Traditionally, the features of diagnosticuse The aim of this case study is to show ©Copyright P. Piras et al., 2018 value for Mugilidae include (Thomson, how traditional and molecular methods can Licensee PAGEPress, Italy 1997): the presence or absence of an adi- Italian Journal of Food Safety 2018; 7:6893 be employed to identify the Mugilidae pose eyelid (this character is commonly doi:10.4081/ijfs.2018.6893 species currently used in Sardinia (Italy) to employed to differentiate the genus Mugil produce the traditional bottarga for the pro- from remaining genera of the family), the cessing of their ovaries. A total of six spec- origin of the various fins and the number of imens of Mugil cephalus (n=3) and Mugil the Storia naturale di Sardegna (Cetti, fin rays, the linear morphometric measure- capurrii (n=3) were subjected to external 1777), in which it is reported that several ments of body proportions using the tradi- morphology and meristic measurements. species of mullets are found in the tional measures employed on fishes Subsequently, tissue samples of white mus- Sardinian Sea, but that: ... as far as I am (Harrison, 2016). cle and ovaries from three individuals per concerned, having observed these mullets, I Mugil cephalus (Linnaeus, 1758) is dif- species were underwent PCR-sequencing have not found any difference indicating fused worldwide and according to FAO is assay of mitochondrial DNA cytochrome that they could be considered different marketed as flathead grey mullet and MUF oxidase subunit I (COI). The external mor- species. The morphological distinction (3-Alpha Species Codes). Mugil cephalus is phology and meristic characters showed a between the various species belonging to an important species for both aquaculture sufficient level of reliability in the identifi- the Mugilidae has always been a difficult and fisheries, and in many regions of the cation between the two species. At theNon-commercial same task because of the extreme homogeneity of world it is captured during the spawning time, the molecular techniques showed the the family. In particular, referring to the sys- migration to harvest the egg roe which is discriminatory power and confirmed the tematic of Mugilidae, in the eleventh vol- salted and dried prior to be marketed as a correct species identification in all the sam- delicatessen (Livi et al., 2011). Linnaeus, ume of the Histoire naturelle des poissons, pling units. DNA barcoding may be an referring briefly to this species in his Valenciennes (1836) reported that ...in gen- effective aid to traditional taxonomy and Systema Naturae (1758), stated: Botargo eral, the great similarity between species of can facilitate accurate species identification italorum ex hujus ovis. In Italy, the botargo this genus makes their distinction one of the among the Mugilidae. or mullet roe, is traditionally produced from most difficult tasks in Ichthyology. flathead grey mullet’s egg roe, and market- Describing the Mugil cephalus, the same ed as bottarga. This name derives from the author reported that …precisely aligned Arabic word batārikh, which in turn, with the second dorsal fin is the anal, pre- Introduction derives from the greek-byzantine term senting eight soft rays. Despite its global Mugilidae are coastal fishes found in ootàrichon meaning dried and salted fish spread in both hemispheres, Mugil cephalus temperate, subtropical and tropical regions eggs. In the Sardinian language (Italy), is has a discontinuous distribution. As it will within marine, brackish and freshwater called butàriga, preserving a strong asso- be reported later, the key features regarding habitats worldwide and represent an impor- nance with the original Arabic word. its taxonomic status have been raised in tant food source in several Mediterranean Moreover, the technological process of grey many genetic studies, most of which sug- and Atlantic countries. According to FAO’s mullets bottarga was formerly described in gest that this scientific name includes a

[page 6] [Italian Journal of Food Safety 2018; 7:6893] Applied Study species complex (Whitfield et al., 2012) or, phological characters remain the corner- hensive molecular systematic account of the in other words, that Mugil cephalus, once stone of taxonomy. However, there are dif- Mugilidae using nucleotide sequence varia- presented as an example of globally distrib- ficulties in relying primarily on morphology tion at three mitochondrial loci (16S rRNA, uted species, is now shown to harbor sever- when attempting to identify fishes from cytochrome b and cytochrome oxidase I) al cryptic species (Durand et al., 2012). isolated portions or processed products from fifty-five species. Mugil capurrii (Perugia, 1892) inhabits (Ward et al., 2009). In this regard, it has Currently, the use of a universally only the Eastern Central Atlantic and been long recognized that nucleic acids accepted short DNA sequence for identifi- according to FAO is marketed as leaping sequence diversity, whether assessed direct- cation of species (DNA barcoding or African mullet and MUO (3-Alpha Species ly or indirectly through protein analysis, can Barcode) has been proposed for many Codes). It was initially described as having be used to discriminate species. By using groups of animals, both invertebrate and the second dorsal fin behind the anal. At a these methods, the identification of Eastern vertebrate, and a fragment of the mitochon- later time, Tortonese (1963) highlighted one Central Atlantic Mugilidae species has been drial gene cytochrome c oxidase subunit I of the key features allowing to differentiate firstly proposed (Trape et al., 2009) by PCR (COI), is the most used sequence of DNA it in respect to the Mugil cephalus, since his restriction fragment length polymorphism barcoding. In fact, within-species variation anal fin invariably presented nine soft rays. (RFLP) analysis of mitochondrial 16S ribo- for this gene is low if compared with Other authors (Trewavas and Ingham, somal RNA region. The grey mullets from between-species variation. Therefore, 1972) published a key to the mullets African waters and in particular from south- species are regularly identified by a particu- species, with the purpose of supplementing ern Mauritania to Senegalese coast are also lar sequence or by a tight cluster of very the section on the Mugilidae in the fishes increasingly represented in international similar sequences. The effectiveness of this checklist of the Northeastern Atlantic and trade of fishing products. Ovaries of large gene region for species-level identifications Mediterranean (valid as to genera only species such as Mugil cephalus are specifi- was recently validated for application within the region). The following key was cally exported and due to its commercial across all fish species (Ward et al., 2009), reported: cleft of mouth as wide as or wider importance there is an increasing risk of including Mugilidae family (Durand et al., than long = Mugil cephalus; cleft of mouth fraud by substitution (Trape et al., 2009). 2017). Accurate and unambiguous identifi- longer than wide = Mugil capurrii. In this The molecular techniques could help food cationonly of fish and fish products, from eggs regard, in the FAO species identification inspection in order to reinforce labeling reg- to adults, is in fact important in many sheets for fishery purposes of the Eastern ulations. Furthermore, the demand for mul- aspects: it would enable retail substitutions Central Atlantic, fishing areas 34 and, in let roe in many parts of the world has grown of species to be detected, assist in managing part, 47 (Fischer et al., 1981), the same considerably in recent decades and elevated fisheries for long-term sustainability, and approach for the species belonging to the the status of grey mullet to be calleduse grey improve ecosystem research and conserva- same genus has been confirmed. Thomson gold by fishermen (Whitfield et al., 2012). tion (Ward et al., 2009). (1997) reported the diagnostic characters of Several nuclear and mitochondrial DNA The aim of this case study was to show the Mugilidae of the world and highlighted regions have been successfully used for the how both the traditional and new molecular that Mugil cephalus presents robust body, identification of fish species, even though tools can be employed to identify/discrimi- rounded profile and the pectoral fin reach- the reliability of such techniques, relies on nate these two species in order to guarantee ing the posterior rim of eye or slightly DNA regions that are highly conserved the proper commercial labeling of mullet behind when laid forward. On the contrary, within the same species and sufficiently roe bottarga produced in Sardinia (Italy). Mugil capurrii presents slender body, point- variable between species. In previous ed head and a mouth corner on vertical from studies, analysis of random amplified poly- the anterior rim of eye, in other words, the morphic DNA (RAPD) and proteins, cou- lip of upper jaw reaching vertical from ante- pled with both, of above mentioned salted Case Report rior field of iris. In addition, the pectoral fin and semi-dried grey mullet ovary product Only a small part of the mullet egg roes does not reach the eye when laid forward. known as bottarga di muggine from differ- processed in Sardinia (Italy) originates from Even though their weakness, these diagnos- ent geographical origins did not differenti- fishing in its coastal waters and lagoons. On tic characters between these two grey mul- ate samples (Barra et al., 2008). In other the other hand, the ability of Sardinian pro- lets, have recently been confirmedNon-commercial in the studies, mitochondrial DNA sequencing ducers to transform grey mullets ovaries FAO species identification guide of the liv- was used to identify and distinguish differ- imported from different parts of the world ing marine resources of the Eastern Central ent species of commercially important in an excellent product is well-known. As a Atlantic (Harrison, 2016). At present, they Mediterranean grey mullets. These finger- matter of fact, originality of this product is are routinely used as distinctive characters prints were used to identify the species of strongly linked to local raw materials. in ichthyology practice. New methodolo- several samples of bottarga from Mugil Otherwise, it is well-known that the metic- gies have been developed in the past few genus which, as previously reported, is a ulous choice of good raw material of non- decades, which improved the accurate dis- well-established traditional product of local origin and the use of consolidated pro- crimination of species, for example the geo- Sardinia, Italy (Murgia et al., 2002). The cessing techniques as traditional salting and metric morphometrics (González-Castro demand of consumers to know the origin of drying parameters, leads to a finished prod- and Ghasemzadeh, 2016) and the sequenc- food is significantly increasing and novel uct bearing the peculiar characters of bot- ing of nuclear and mitochondrial genes. It is methods of understanding and assessing targa di muggine, even though appropriate clear that historical methods of identifying consumer trust have been studied to meet labelling in terms of ingredients and trace- fishes, even for practical reasons, are this demand. In previous studies, partial ability should be guaranteed. Nowadays, predominantly based on visible morpholo- cytochrome b gene sequences were resolved regular supply of frozen raw material, guar- gy. Although modern taxonomy regularly for Mugil cephalus specimens sampled antees production all year round. The final employs many other traits, including inter- from fourteen different geographic sites products acquire the origin of the country or nal anatomy, physiology, behavior, geogra- (Livi et al., 2011). Other authors (Durand et territory where, pursuant to article 60 of phy and lately isozymes and genes, mor- al., 2012) have provided an initial compre- Regulation (EU) No 952/2013, they under-

[Italian Journal of Food Safety 2018; 7:6893] [page 7] Applied Study went their substantial processing or work- certificate reporting Mugil cephalus as different species of Mugil. As a matter of ing. However, it is only for the product species of origin and was destined to a pro- fact the sequences of the two samples pre- obtained from ovaries of local Mugil cessing plant located in the Sardinian sented heterozygous mutation sites and the cephalus that the matched geographical province of Oristano. Five sampling units comparison with the databases revealed a name di Sardegna or Sarda can be used, were collected from the whole batch and significant similarity with different species referring in this case to a traditional sent to the Genetics and of Mugil. According to these results, an regional product included in the national list Immunobiochemistry laboratory of the information for attention (iRASFF No provided by Italian Ministerial Decree n. Zooprophylactic Institute of Turin for fur- 2017/327039 of March 28) was notified. 350 of 1999 and confirmed with the in force ther biomolecular analysis. Species identifi- Successively, since the notification of dif- seventeenth revision of 2017. At the end of cation was performed using COI gene as ferent species identified does not represent January 2017, the regional daily newspaper genetic marker and comparing the obtained any health related issues, and then does not L’Unione Sarda published an investigative nucleotide sequences with public DNA fall within the scope of the RASFF system, report on Sardinian products deserving the databases. Genomic DNA was extracted this information for attention was rejected. naming of Protected Designation of Origin from all samples using commercial kit Although this topic essentially concerns (PDO) or Protected Geographic Indication based on spin column containing a silica with fair practices in food trade and protec- (PGI) on their labels, focusing on bottarga. resin (ReliaPrepTM gDNA Tissue Miniprep tion of consumer interests, including food At present, this regional product is not System, Pomega), and a specific PCR reac- labeling, it is included in the scope of under the protected geographical status due tion amplified a portion of COI gene fol- Regulation (EC) No 882/2004 on official to …too little production… or because of lowing the standard protocol described by controls and, pursuant to article 35 of the incapability to …gather the manufactur- Ward et al. (2005). The reaction was run on Regulation (EU) No 1379/2013, as fishery ers around a table…, leading to a …market GeneAmp® PCR System 9700 (Applied products can be offered for sale to the final invaded by mullet egg roes imported from Biosystems, Life Tecnologies), and the consumer or to a mass caterer only if appro- African countries... In order to guarantee amplicons were sequenced according to priate labelling indicates: the commercial the right of consumers to consciously make Sanger’s method on both strands using the designation of the species and its scientific their choice when purchasing bottarga on same PCR primers on ABI Prism 3130 name. the basis of a proper labelling, the compe- Genetic Analyzer (Applied Biosystems, Therefore,only we decided to study in depth tent authorities decided to conduct regular Life Techonolgies). The obtained sequences the manageability aspects regarding the dif- checks focusing on verification of correct- were compared with those deposed both in ferential identification of the two Mugil ness of geographical and species traceabili- GenBank and in BOLD databases. Based on species of the Eastern Central Atlantic ty of all batches of ovaries of grey mullets validation data of the method, the use final coasts (Mugil cephalus and Mugil capurrii), caught in Eastern Central Atlantic coasts. At specimens assignment to species was based upstream as well as downstream of the sup- the beginning of March 2017, a batch of on a minimum similarity value of 98%. Out ply chain. The availability of vouchered 3119 kg of frozen fish eggs from Senegal of the five sampling units, three were iden- sequences of the other Mugil spp. sharing and designated with CN code 0303.9190 of tified as Mugil capurrii and the remaining the aforesaid FAO area (e.g. Mugil bana- Regulation (EU) No 2016/1821, has been two as Mugil spp. In the latter two cases, it nensis, Mugil curema and Mugil curvidens) subjected to regular sampling at the Border was not possible to define the species, but has not been verified because these species Inspection Post of Genova (Italy). The only a genus level determination analyses reach too small adult medium lengths entire batch was accompanied by a health revealed a mixture of eggs deriving from (Harrison, 2016) and, therefore, the ovaries

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Figure 1. Adult specimens of the Mugilidae family: A = Mugil Figure 2. External morphology and usual measures employed in cephalus; B = Mugil capurrii. the Mugilidae (full lateral view and ventral view of head): A = Mugil cephalus; B = Mugil capurrii; SL = standard length; nd D2O = 2 dorsal fin origin; HL = head length; prO = preorbital length; ED = eye diameter; poO = postorbital length; LM = length of mouth; WM = width of mouth.

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cannot be used in the technological process units of muscle and ovary. The molecular of bottarga production. In order to compare method showed the discriminatory capabil- with local specimens of Mugil cephalus, ity in terms of verification of species identi- one of the main Sardinian importer was fication and compliance to the requirements asked to integrate the next buying of grey for the labelling of fishery products. Taking mullets ovaries from Mauritania, with a into account the different importance of the monospecific batch of frozen adult speci- two approaches in forensic sciences, they mens of Mugil capurrii. Comparable Mugil should be considered as complementary in capurri and Mugil cephalus specimens with the routinely practice, both by fisheries the same standard length of 40 cm were operators and by competent authorities, as it subjected to external morphology and is happening for example, in the field of meristic measurements (Figure 1). integrative taxonomy of fishes as the Subsequently, the same specimens under- Mugilidae family. This integrate approach went PCR-sequencing assays as previously combines morphological and meristic anal- described for frozen fish eggs, isolating ysis with newer disciplines, such as molec- samples both from fragments of white mus- ular genetics (González-Castro and cle samples and ovaries of three individuals Ghasemzadeh, 2016). per species. Biomolecular tests showed that three samples of muscle belonged to Mugil cephalus, while the others to Mugil capurii. Same results were obtained from the analy- Conclusions ses of the correspondent ovaries. According to other authors (Durand et al., 2017), DNA barcoding may be an effec- tive aid to traditional taxonomy, designed to facilitateonly fast and accurate species identifi- Discussion cation, especially among the Mugilidae. The results of the present case study Although these results should be considered highlighted that the external morphology preliminary, we have confirmed that DNA and meristic characters of practicaluse use, if barcoding discriminates against the two correctly applied by well-trained workers, mullet species (Mugil cephalus and Mugil showed sufficient level of reliability in the capurrii), and can identify individually iso- identification between the two Mugilidae lated fish ovary and muscle portions from species. These results are in accordance these species. However, in order to better with the findings of previous authors control the identity of species upstream of (Fischer et al., 1981; Harrison, 2016; the supply chain and in particular the whole Thomson, 1997; Trewavas and Ingham, fish from which the ovaries are extracted, 1972). In particular, the more specific char- practical and up-to-date criteria of external Figure 3. Lateral and ventral view of acters are those related to head length morphology and meristic characters (Figure mouth in Mugil cephalus (A) and Mugil development of labial split (both in profile capurrii (B). 2) are still necessary. This will help in the and ventral vision) and lastly to number of systematic and immediate use in the fishing soft rays of the anal fin (Figure 2). The and selection stages of Mugilidae batches examination of mouth shape and length or, as reported by previous authors (Ward et (Figure 3) in relation to eye margin, togeth- al., 2009), barcoding and morphological er with development of pectoral fin (if con- analysis should go hand-in-hand. veniently tilt forward) as regards to rear- Non-commercialocular space and, in the event of doubt, the count of soft rays of the anal fin (Figure 4), represent confident criteria for effectiveness of a practical diagnosis, or at least a screen- References ing, in mono or polyspecific fishings of Barra A, Garau VL, Dessì S, Sarais G, Mugil spp. on the Eastern Central Atlantic Cereti E, Arlorio M, Coisson JD, coasts by local fishermen. At present, the Cabras P, 2008. Chemical characteriza- systematic control of the species identity tion and DNA tracking of sardinian upstream of the supply chain can be sup- botargo by Mugil cephalus from diffe- ported by the examination (on a sampling rent geographical origins. J Agr Food basis) through molecular analysis aimed to Chem 56:10847-52. determine the presence of genetic markers Cetti F, 1777. Pesci addominali. In: Storia specific for each species. Our results, relat- naturale di Sardegna. Nella stamperia di ed to a blind trial (the samples were Giuseppe Piattoli, Sassari, pp 195-201. delivered to the laboratory not reporting the Durand JD, Shen KN, Chen WJ, Jamandre species) on three female specimens for each BW, Blel H, Diop K, Nirchio M, Garcia Figure 4. Spiny and soft rays of the anal fin of the two species Mugil cephalus and de León FJ, Whitfield AK, Chang CW, in Mugil cephalus (A) and Mugil capurrii (B). Mugil capurrii, confirmed the correct Borsa P, 2012. Systematics of the grey species identification in all the sampling mullets (teleostei: mugiliformes: mugi-

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