Anisakis spp. Larvae in Deboned, in-Oil Fillets Made of Anchovies (Engraulis encrasicolus) and Sardines (Sardina pilchardus) Sold in EU Retailers.
ABSTRACT: Sardina pilchardus and Engraulis encrasicolus are considered the principal target species for commercial fishing in Europe and are widely consumed as semipreserved products. Although they are considered shelf-stable products, if treatment is not correctly applied, their consumption may represent a public health risk in regard to anisakiasis and allergic reactions. Little is known about the prevalence of Anisakis spp. in ripened products. This study aimed to evaluate the presence of Anisakis spp. larvae in deboned, in-oil anchovy and sardine fillets marketed in the EU to assess the influence of processing techniques on the prevalence of larvae. Ninety semipreserved anchovy and sardine products deriving from the Mediterranean Sea or Atlantic Ocean were collected from different EU retailers and examined using chloropeptic digestion to evaluate the presence of larvae and identify them. Thirty nonviable Anisakid larvae-A. pegreffii (30%) and A. simplex (70%)-were found. The frequency of larvae was higher in anchovies (28.8%). The low frequency of parasites found proved that processing technologies can influence the presence of larvae in final products, but it is important that visual inspection is performed only by trained people. The sources of raw materials should be considered in the production flow chart.
Project description:The third-stage larvae (L3) of Anisakis simplex are the most important source of hidden allergens in seafood products. However, there exist no commercial methods for detecting Anisakis proteins in food. Furthermore, only a few methods have been validated for the detection of A. simplex in thermally processed food. The aims of our study are (i) the development and validation of high-sensitivity chemiluminescent (CL) immunoassays for the detection of A. simplex proteins in processed seafood, (ii) and A. simplex antigen detection in common seafood products from Polish markets. We developed and validated CL sandwich ELISA (S-ELISA) and CL competitive ELISA (C-ELISA) methods for A. simplex proteins detection in food, with respective detection limits of 0.5 and 5 ng/mL. The usefulness of the assays for detecting A. simplex proteins in highly processed food was evaluated by examination of autoclaved canned fish spiked with A. simplex larvae (1-8 larvae/200 g). Commercial real-time PCR was unable to detect A. simplex in autoclaved samples at all levels of enrichment with Anisakis larvae. CL-S-ELISA was used to test various types of seafood products from Polish markets. Among all tested products (n = 259), 28% were positive. A. simplex antigens were found mostly (n = 39) in smoked fish products: mackerel, herring, cod, and hake. Other positive samples were found in marinated herrings, canned cod livers, canned mackerels, and surimi sticks. In tuna, Atlantic argentine, anchovy, sardine, sprat, and squid products, A. simplex antigens were not detected. This study provides novel effective tools for the detection of A. simplex proteins in processed food and highlights the potential allergic hazards for Anisakis-sensitized Polish consumers of seafood.
Project description:BACKGROUND:The European sardine (Sardina pilchardus Walbaum, 1792) is culturally and economically important throughout its distribution. Monitoring studies of sardine populations report an alarming decrease in stocks due to overfishing and environmental change, which has resulted in historically low captures along the Iberian Atlantic coast. Important biological and ecological features such as population diversity, structure, and migratory patterns can be addressed with the development and use of genomics resources. FINDINGS:The genome of a single female individual was sequenced using Illumina HiSeq X Ten 10x Genomics linked reads, generating 113.8 gigabase pairs of data. Three draft genomes were assembled: 2 haploid genomes with a total size of 935 megabase pairs (N50 103 kilobase pairs) each, and a consensus genome of total size 950 megabase pairs (N50 97 kilobase pairs). The genome completeness assessment captured 84% of Actinopterygii Benchmarking Universal Single-Copy Orthologs. To obtain a more complete analysis, the transcriptomes of 11 tissues were sequenced to aid the functional annotation of the genome, resulting in 40,777 genes predicted. Variant calling on nearly half of the haplotype genome resulted in the identification of >2.3 million phased single-nucleotide polymorphisms with heterozygous loci. CONCLUSIONS:A draft genome was obtained, despite a high level of sequence repeats and heterozygosity, which are expected genome characteristics of a wild sardine. The reference sardine genome and respective variant data will be a cornerstone resource of ongoing population genomics studies to be integrated into future sardine stock assessment modelling to better manage this valuable resource.
Project description:It is well known that temporal fluctuations in small populations deeply influence evolutionary potential. Less well known is whether fluctuations can influence the evolutionary potentials of species with large census sizes. Here, we estimated genetic population parameters from as survey of polymorphic microsatellite DNA loci in archived otoliths from Adriatic European anchovy (Engraulis encrasicolus), a fish with large census sizes that supports numerous local fisheries. Stocks have fluctuated greatly over the past few decades, and the Adriatic fishery collapsed in 1987. Our results show a significant reduction of mean genetic parameters as a consequence of the population collapse. In addition, estimates of effective population size (Ne) are much smaller than those expected in a fishes with large population census sizes (Nc). Estimates of Ne indicate low effective population sizes, even before the population collapse. The ratio Ne/Ne ranged between 10-6 and 10-8, indicating a large discrepancy between the anchovy gene pool and population census size. Therefore, anchovy populations may be more vulnerable to fishery effort and environmental change than previously thought.
Project description:MicroRNAs are endogenous highly conserved short (~?21 nucleotides) non-coding RNA molecules that play key roles in post-transcriptional gene regulation by translational inhibition or by target mRNA cleavage. In this report, using high stringent computational-based methods, a total of 101 putative miRNAs were identified from European sardine fish (Sardina pilchardus Walbaum, 1792). All the precursors of identified sardine miRNAs formed stable stem-loop structures and displayed high minimum free energy index (MFEI) values. For the experimental validation of the computationally predicted miRNAs, a tissue-specific quantitative study of eight randomly selected putative sardine miRNAs (spi-miR9, spi-miR26, spi-miR128, spi-miR129, spi-miR132, spi-miR212, spi-miR219, and spi-miR338) was performed in brain and liver and all the selected miRNAs were found to be overexpressed in brain tissue. Moreover, using RNAhybrid, a total of 83 potential target proteins of the characterized sardine miRNAs were identified those are involved in transcription, cellular development, defense mechanism, and various signaling pathways. To the best of our knowledge, this is the first report of sardine microRNAs and their targets.
Project description:<h4>Background</h4>Marine pelagic fishes exhibit rather complex patterns of genetic differentiation, which are the result of both historical processes and present day gene flow. Comparative multi-locus analyses based on both nuclear and mitochondrial genetic markers are probably the most efficient and informative approach to discerning the relative role of historical events and life-history traits in shaping genetic heterogeneity. The European sardine (Sardina pilchardus) is a small pelagic fish with a relatively high migratory capability that is expected to show low levels of genetic differentiation among populations. Previous genetic studies based on meristic and mitochondrial control region haplotype frequency data supported the existence of two sardine subspecies (S. p. pilchardus and S. p. sardina).<h4>Results</h4>We investigated genetic structure of sardine among nine locations in the Atlantic Ocean and Mediterranean Sea using allelic size variation of eight specific microsatellite loci. Bayesian clustering and assignment tests, maximum likelihood estimates of migration rates, as well as classical genetic-variance-based methods (hierarchical AMOVA test and RST pairwise comparisons) supported a single evolutionary unit for sardines. These analyses only detected weak but significant genetic differentiation, which followed an isolation-by-distance pattern according to Mantel test.<h4>Conclusion</h4>We suggest that the discordant genetic structuring patterns inferred based on mitochondrial and microsatellite data might indicate that the two different classes of molecular markers may be reflecting different and complementary aspects of the evolutionary history of sardine. Mitochondrial data might be reflecting past isolation of sardine populations into two distinct groupings during Pleistocene whereas microsatellite data reveal the existence of present day gene flow among populations, and a pattern of isolation by distance.
Project description:Clupeiformes, such as sardines and herrings, represent an important share of worldwide fisheries. Among those, the European sardine (Sardina pilchardus, Walbaum 1792) exhibits significant commercial relevance. While the last decade showed a steady and sharp decline in capture levels, recent advances in culture husbandry represent promising research avenues. Yet, the complete absence of genomic resources from sardine imposes a severe bottleneck to understand its physiological and ecological requirements. We generated 69 Gbp of paired-end reads using Illumina HiSeq X Ten and assembled a draft genome assembly with an N50 scaffold length of 25,579 bp and BUSCO completeness of 82.1% (Actinopterygii). The estimated size of the genome ranges between 655 and 850 Mb. Additionally, we generated a relatively high-level liver transcriptome. To deliver a proof of principle of the value of this dataset, we established the presence and function of enzymes (Elovl2, Elovl5, and Fads2) that have pivotal roles in the biosynthesis of long chain polyunsaturated fatty acids, essential nutrients particularly abundant in oily fish such as sardines. Our study provides the first omics dataset from a valuable economic marine teleost species, the European sardine, representing an essential resource for their effective conservation, management, and sustainable exploitation.
Project description:Increased throughput in sequencing technologies has facilitated the acquisition of detailed genomic information in non-model species. The focus of this research was to discover and validate SNPs derived from the European anchovy (Engraulis encrasicolus) transcriptome, a species with no available reference genome, using next generation sequencing technologies. A cDNA library was constructed from four tissues of ten fish individuals corresponding to three populations of E. encrasicolus, and Roche 454 GS FLX Titanium sequencing yielded 19,367 contigs. Additionally, the European anchovy genome was sequenced for the same ten individuals using an Illumina HiSeq2000. Using a computational pipeline for combining transcriptome and genome information, a total of 18,994 SNPs met the necessary minor allele frequency and depth filters. A series of further stringent filters were applied to identify those SNPs likely to succeed in genotyping assays, and for filtering of those in potential duplicated genome regions. A novel method for detecting potential intron-exon boundaries in areas of putative SNPs has also been applied in silico to improve genotyping success. In all, 2,317 filtered putative transcriptome SNPs suitable for genotyping primer design were identified. From those, a subset of 530 were selected, with the genotyping results showing the highest reported conversion and validation rates (91.3% and 83.2%, respectively) reported to date for a non-model species. This study represents a promising strategy to discover genotypable SNPs in the exome of non-model organisms. The genomic resource generated for E. encrasicolus, both in terms of sequences and novel markers, will be informative for research into this species with applications including traceability studies, population genetic analyses and aquaculture.
Project description:Background:Small fishes play fundamental roles in pelagic ecosystems, channelling energy and nutrients from primary producers to higher trophic levels. They support globally important fisheries in eastern boundary current ecosystems like the Humboldt Current System (HCS) of the SE Pacific (Chile and Peru), where fish catches are the highest in the world (per unit area). This production is associated with coastal upwelling where fisheries target small pelagic fishes including the Peruvian anchovy (Engraulis ringens). The elevated biomass attained by small pelagics is thought to reflect their low trophic position in short/simple food chains. Despite their global importance, large gaps exist in our understanding of the basic ecology of these resources. For instance, there is an ongoing debate regarding the relative importance of phytoplankton versus animal prey in anchovy diet, and ecosystem models typically assign them a trophic position (TP) of ∼2, assuming they largely consume phytoplankton. Recent work based on both relative energetic content and stable isotope analysis (SIA) suggests that this value is too low, with δ 15N values indicating that anchovy TP is ca. 3.5 in the Peruvian HCS. Methods:We characterised the trophic ecology of adult anchovies (n = 30), their putative prey and carnivorous jack mackerel (n = 20) captured from N Chile. SIA (δ 13C and δ 15N) was used to estimate the relative contribution of different putative prey resources. δ 15N was used to estimate population level trophic position. Results:Anchovies showed little variability in δ 13C (-18.7 to -16.1‰) but varied greatly in δ 15N (13.8 to 22.8‰)-individuals formed two groups with low or high δ 15N values. When considered as a single group, mixing models indicated that anchovy diet was largely composed of zooplankton (median contribution: 95% credibility limits), with major contributions of crustacean larvae (0.61: 0.37-0.77) and anchovy (preflexion) larvae (0.15: 0.02-0.34), and the assimilation of phytoplankton was negligible (0.05: 0-0.22). The modal (95% credibility limits) estimate of TP for the pooled anchovy sample was 3.23 (2.93-3.58), overlapping with recent SIA-based estimates from Peru. When the two δ 15N groups were analysed separately, our results indicate that the lower δ 15N group largely assimilated materials from crustacean larvae (0.73: 0.42-0.88), with a TP of 2.91 (2.62-3.23). Mixing models suggested high δ 15N anchovies were cannibalistic, consuming anchovy preflexion larvae (0.55: 0.11-0.74). A carnivorous trophic niche was supported by high TP (3.79: 3.48-4.16), mirroring that of carnivorous juvenile jack mackerel (Trachurus murphyi; 3.80: 3.51-4.14). Our results support recent conclusions regarding high TP values of anchovy from Peru and reveal new insights into their trophic behaviour. These results also highlight the existence of cryptic trophic complexity and ecosystem function in pelagic food webs, classically considered as simple.