Javier Quinteiro

Challenges in the identification of species of canned fish

Abstract The identification of fish species becomes a problem when the usual identifying characteristics are removed on processing and only a portion of flesh is available. When the flesh is raw or cooked under normal conditions, the species is readily established by electrophoresis of the muscle proteins. The procedure cannot be used for heat-sterilised canned fish as the proteins are severely denatured. DNA is also degraded but techniques are now available for targeting and amplifying species-specific fragments. The amplified products can then be analysed by a range of techniques some of which are suitable for food control laboratories.

Conservation of the S10-spc-α Locus within Otherwise Highly Plastic Genomes Provides Phylogenetic Insight into the Genus Leptospira

S10-spc-α is a 17.5 kb cluster of 32 genes encoding ribosomal proteins. This locus has an unusual composition and organization in Leptospira interrogans. We demonstrate the highly conserved nature of this region among diverse Leptospira and show its utility as a phylogenetically informative region. Comparative analyses were performed by PCR using primer sets covering the whole locus. Correctly sized fragments were obtained by PCR from all L. interrogans strains tested for each primer set indicating that this locus is well conserved in this species. Few differences were detected in amplification profiles between different pathogenic species, indicating that the S10-spc-α locus is conserved among pathogenic Leptospira. In contrast, PCR analysis of this locus using DNA from saprophytic Leptospira species and species with an intermediate pathogenic capacity generated varied results. Sequence alignment of the S10-spc-α locus from two pathogenic species, L. interrogans and L. borgpetersenii, with the corresponding locus from the saprophyte L. biflexa serovar Patoc showed that genetic organization of this locus is well conserved within Leptospira. Multilocus sequence typing (MLST) of four conserved regions resulted in the construction of well-defined phylogenetic trees that help resolve questions about the interrelationships of pathogenic Leptospira. Based on the results of secY sequence analysis, we found that reliable species identification of pathogenic Leptospira is possible by comparative analysis of a 245 bp region commonly used as a target for diagnostic PCR for leptospirosis. Comparative analysis of Leptospira strains revealed that strain H6 previously classified as L. inadai actually belongs to the pathogenic species L. interrogans and that L. meyeri strain ICF phylogenetically co-localized with the pathogenic clusters. These findings demonstrate that the S10-spc-α locus is highly conserved throughout the genus and may be more useful in comparing evolution of the genus than loci studied previously.

Fish species identification in canned tuna by PCR-SSCP: validation by a collaborative study and investigation of intra-species variability of the DNA-patterns

Analysis of single strand conformation polymorphism (SSCP) of an amplicon (123 bp) obtained by polymerase chain reaction (PCR) of the mitochondrial cytochrome b gene was used to identify the fish species in canned tuna. Single-stranded DNA (ssDNA) was separated by polyacrylamide gel electrophoresis, and visualised by silver staining. The reliability of the method was tested by a collaborative study in which eight European laboratories participated. Seven unknown samples (five from individual species and two mixtures of two tuna species) of canned tuna had to be identified by comparison with reference material. From a total of 72 cases, 65 (90.3%) were assigned correctly. Intra-species variability of SSCIP patterns was found in the case of Katsuwonus pelamis and Sarda sarda. As specimens from various fishing grounds gave two or three different patterns of ssDNA, the possibility of some variability of the DNA patterns has to be considered in SSCP analysis of these species.

Identification of European Hake Species (Merluccius merluccius) Using Real-Time PCR

A rapid and precise method for identifying European hake (Merluccius merluccius) based on TaqMan technology is presented. The method can be applied to fresh, frozen, and processed fish products to detect the fraudulent or unintentional mislabeling of this species. Specific primers and a minor groove binding (MGB) TaqMan probe were designed for this purpose based on partial sequences of the mitochondrial DNA control region. Combinations of primers and probe concentrations that gave the lowest Ct value and the highest final fluorescence value were selected to carry out efficiency, specificity, and cross-reactivity assays. The method was successfully tested on 31 commercial hake samples. A Ct value of about 16 was obtained when Merluccius merluccius was present; however, the fluorescence signal was not detected most of the time (Ct value 40) or presented significantly higher Ct values (38.2 +/- 0.96) for the nonhake species.

Development of a multiplex PCR–ELISA method for the genetic authentication of Thunnus species and Katsuwonus pelamis in food products

In the present work a PCR-ELISA technique for the authentication of Thunnus species was developed. This method is composed by four systems that can be used in a hierarchical way allowing the identification of several scombroids species; or each individual system independently. The hierarchical strategy, proposes a first step, to assign one sample to the Thunnus genus. Next, if the result is positive, several tests can be applied to assign the sample to some particular species of the Thunnus genus. In the case that the result is negative (absence of Thunnus species), it is possible to verify if Katsuwonus pelamis is included in the sample. The method even allows the detection of mixtures of these species in relatively low amounts (up to 1%). Finally, this method was applied to 11 commercial samples to verify the labelling status of tuna products in the market, detecting that 18% were mislabelling.

First global approach: morphological and biological variability in a genetically homogeneous population of the European pilchard, Sardina pilchardus (Walbaum, 1792) in the North Atlantic coast

The European pilchard Sardina pilchardus represents the most commercially relevant fisheries resource in many countries bordering north Atlantic coasts and the Mediterranean Sea, being especially significant along the coast of Morocco. The continuous exploitation of this pelagic species for several decades places Morocco as the leader in sardine production. However, the conditions of exploitation of this resource underwent a great change during the last recent years. In order to identify the populations of the European pilchard sardine (Sardina pilchardus, Walbaum, 1792) in the Atlantic coast of Morocco and Spain, we have combined the truss network data to conduct multivariate analysis with biologic parameters and genetic analysis based on Microsatellite and mitochondrial control region data. Sardine morphometrics data truss variables from 10 samples spanning the Atlantic coast of Morocco were analysed by multivariate analysis. Thirteen morphometric measurements and some biological parameters such as the sex and the age of fishes were made for each individual. Discriminant analysis on size-corrected truss variables and cluster analysis of mean fishes shape using landmark data indicate, that the shape of north Moroccan sardines is distinct from the shape of sardines from south Morocco. However the analysis of the mitochondrial region and four microsatellites loci (Sp2, Sp7, Sp8 and SpI5) demonstrated an homogeneity population in the Moroccan Atlantic coast, with a low but significant genetic differentiation, which followed an isolation-by-distance pattern according to Mantel test.

Quantification of Manila clam ruditapes philippinarum (Adams & Reeve, 1850) larvae based on SYBR green real-time polymerase chain reaction

ABSTRACT Biological and ecological research is seriously handicapped because of difficulties experienced in the reliable detection and quantification of bivalve larvae. This is a critical issue in the case of the Manila clam Ruditapes philippinarum (Adams & Reeve, 1850), a largely invasive and commercially relevant species, with important wild, cultured, and naturalized populations throughout the world. A SYBR Green real-time PCR assay, containing TPHI16S1F and TPHI16S2R primers (specific to R. philippinarum female mtDNA), was designed and tested to provide a rapid and high-throughput PCR-based method for larval quantification. Accurate estimations of larval numbers in spiked plankton samples point to the usefulness of this system. It can be used through a wide range of evaluated variable experimental conditions, such as the presence of closely related bivalve species, stationary-dependent plankton abundance, sampling volumes, and larval size.

Phylogeography of a Marine Insular Endemic in the Atlantic Macaronesia: The Azorean Barnacle, Megabalanus azoricus (Pilsbry, 1916)

The Azorean barnacle, Megabalanus azoricus (Pilsbry, 1916), is a Macaronesian endemic whose obscure taxonomy and the unknown relationships among forms inhabiting isolated Northern Atlantic oceanic islands is investigated by means of molecular analysis herein. Mitochondrial data from the 16S rRNA and COX1 genes support its current species status, tropical ancestry, and the taxonomic homogeneity throughout its distribution range. In contrast, at the intraspecific level and based on control region sequences, we detected an overall low level of genetic diversity and three divergent lineages. The haplogroups α and γ were sampled in the Azores, Madeira, Canary, and Cabo Verde archipelagos; whereas haplogroup β was absent from Cabo Verde. Consequently, population analysis suggested a differentiation of the Cabo Verde population with respect to the genetically homogenous northern archipelagos generated by current oceanographic barriers. Furthermore, haplogroup α, β, and γ demographic expansions occurred during the interglacial periods MIS5 (130 Kya - thousands years ago -), MIS3 (60 Kya), and MIS7 (240 Kya), respectively. The evolutionary origin of these lineages is related to its survival in the stable southern refugia and its demographic expansion dynamics are associated with the glacial-interglacial cycles. This phylogeographic pattern suggests the occurrence of genetic discontinuity informative to the delimitation of an informally defined biogeographic entity, Macaronesia, and its generation by processes that delineate genetic diversity of marine taxa in this area.

Modeling real-time PCR kinetics: Richards reparametrized equation for quantitative estimation of European hake (Merluccius merluccius).

Real-time PCR is the most sensitive method for detection and precise quantification of specific DNA sequences, but it is not usually applied as a quantitative method in seafood. In general, benchmark techniques, mainly cycle threshold (Ct), are the routine method for quantitative estimations, but they are not the most precise approaches for a standard assay. In the present work, amplification data from European hake (Merluccius merluccius) DNA samples were accurately modeled by three sigmoid reparametrized equations, where the lag phase parameter (λc) from the Richards equation with four parameters was demonstrated to be the perfect substitute for Ct for PCR quantification. The concentrations of primers and probes were subsequently optimized by means of that selected kinetic parameter. Finally, the linear correlation among DNA concentration and λc was also confirmed.

Autentificación de lapas del género Fissurella (Mollusca: Vetigastropoda) en la costa chilena, mediante PCR-RFLP

RESUMEN. Las lapas, clasificadas en el genero Fissurella, se explotan desde tiempos prehistoricos en la costa chilena. Las diversas especies de lapas presentan diferencias en la consistencia o dureza del musculo y en cualidades organolepticas, por ello son distintamente valoradas por los consumidores. La ausencia o desconocimiento de los caracteres morfologicos diagnosticos, en algunas presentaciones del recurso, fraudes en el etiquetado o designacion y la sobreexplotacion de este recurso, demanda una metodologia de identificacion de cada especie. Este metodo garantizaria el correcto etiquetado de los productos comerciales y seria de utilidad en la gestion pesquera. Asi, se diseno un protocolo de PCR-RFLP que permite la identificacion de diferentes especies de Fissurella, teniendo en cuenta incongruencias entre la taxonomia actual del genero y los datos geneticos. La digestion de productos de PCR, con dos enzimas de restriccion especificas ( HpyCH4 V y Nla III), genera patrones especificos de restriccion, los cuales permitiran identificar con precision las diversas especies en cualquier fase de su comercializacion y de su ciclo biologico. Palabras clave: lapas, Fissurella, PCR-RFLP, autentificacion, Chile. Authentication of Fissurella species (Mollusca: Vetigastropoda), harvested in the Chilean coast, by PCR-RFLP*