Sardine early survival, physical condition and stress after introduction to captivity

The main factors that affect early survival, physical damage and stress reactions of sardine Sardina pilchardus after live capture and introduction to captivity were examined. A total of 2800 sardines were captured alive from commercial purse seiners in five trials off southern Portugal and monitored for 4 weeks in aquaculture tanks. Survival rates varied considerably between trials (from <20 to >80% after a month), with most deaths occurring in the first 5 days. Sardine early survival was affected by factors related to conditions at sea (catch composition, sea temperature and transportation density), during introduction to captivity (magnitude of thermal shock, land transportation duration and use of antibacterial treatment) and, possibly, their interaction. Physical damage was related to the probability of dying, with fish that died during the first week showing significantly higher scale loss and larger caudal fin erosion that those that were alive in the same period. For all stress variables measured (blood haematocrit, cortisol, glucose and ions in the plasma), the most extreme values were attained during introduction to captivity or in the first hours after. After 2 weeks in captivity, most variables had returned to levels close to those observed at the onset of purse-seine fishing, suggesting that maintenance conditions were adequate to permit a rapid recovery from fishing and transport stress.     

Effect of maternal fat reserves on the fatty acid composition of sardine (Sardina pilchardus) oocytes

We compared the fatty acid (FA) composition of the muscle and gonads of female Iberian sardines with hydrated oocytes collected during the 2002/03 spawning season off southern Portugal (November and February) and off western Portugal (February). Sardine condition and total FA concentration in the muscle decreased between the two sampling dates, while the gonadosomatic index was similar between samples. Total monounsaturated FA concentrations in sardine gonads were different for the three samples while saturated and polyunsaturated FA concentrations were similar. Significant linear relations were found between FA concentrations in female muscle and oocytes, including eicosapentaenoic acid (EPA; 20:5n − 3) and arachidonic acid (AA; 20:4n − 6), both being essential for normal larval development. The concentration of docosahexaenoic acid (DHA; 22:6n − 3) in oocytes was independent on muscle concentration, probably resulting from its selective transfer to the oocytes. The EPA/DHA ratio was highly conserved in sardine tissues, while DHA/AA and EPA/AA ratios varied significantly between samples. These results indicate that the FA content of eggs produced by sardines varies throughout the spawning season, egg FA concentrations decreasing as females lose condition, and FA composition also shows spatial variability. Both types of variability may have a significant impact on egg quality, particularly on the amount of reserves available to larvae affecting their resistance to starvation, and the appropriate FA composition required for normal growth.     

Carotenoids in fish. XXIV. Sardina pilchardus Walb. (Clupeidae)

The author investigated the presence of various carotenoids in Sardina pilchardus Walb. from the coast of Southern Europe.The presence of the following carotenoids has been stated: -carotene, -carotene epoxide, -cryptoxanthin, canthaxanthin, lutein epoxide, zeaxanthin, astaxanthin (free and ester form) and mutatochrome. The dominant carotenoid in all the parts of the body was astaxanthin, especially its ester form. The total content of carotenoid ranged from 10.537 (skin and muscles) to 116.309 µg/g fresh weight (liver).     

Seasonal differences in muscle fibre recruitment of pilchard larvae in the north‐western Mediterranean

The recruitment of slow and fast myotomal muscle fibres with respect to growth in body length in European pilchard larvae Sardina pilchardus [(3·5–13·5 mm standard length ( L _S)] was significantly higher in November 1998 than February 1999. This resulted in a significant seasonal difference in the relationship between fibre number and L _S, particularly for the fast muscle. Mean sea surface temperature was c . 6° C higher in November than February, whereas the mean abundance of potential prey items (copepod nauplii) was comparable between cruises. Laboratory and field data obtained from other clupeid species have indicated the importance of early thermal experience on muscle fibre recruitment patterns. Differences in average sea temperature therefore provide a plausible mechanism for the observed seasonal differences in muscle growth characteristics.     

Purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from European pilchard Sardina pilchardus

The NAD＋-dependent cytosolic glyceralehyde-3-phosphate dehydrogenase （GAPDH; EC 1.2.1.12） was purified from the skeletal muscle of European pilchard Sardina pilchardus and its physicochemical and kinetic properties were investigated. The purification method consisted of two steps, ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography, resulting in an approximately 78-fold increase in specific activity and a final yield of approximately 25%. The Michaelis constants （Kin） for NAD＋ and D-glyceraldehyde-3-phosphate were 92.0 μM and 73.4 μM, respectively. The maximal velocity （Vmax） of the purified enzyme was estimated to be 37.6 U/mg. Under the assay conditions, the optimum pH and temperature were 8.0 and 30 ℃. The molecular weight of the purified enzyme was 37 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yielding a molecular weight of 154 kDa suggested that the enzyme is a homotetramer. Polyclonal antibodies against the purified enzyme were used to recognize the enzyme in different sardine tissues by Western blot analysis. The isoelectric point, obtained by an isoelectric focusing system in polyacrylamide slab gels, revealed only one GAPDH isoform （pI 7.9）.     

Infection of sardine eggs by the protistan endoparasite Ichthyodinium chabelardi off Portugal

In 1997 and 1999 around 30% of the stations off Portugal sampled for sardine eggs Sardina pilchardus included eggs infected with Ichthyodinium chabelardi. A randomization test on the mean nearest neighbour distances of parasitized stations did not reveal evidence of significant spatial clustering. The mean prevalence of infection was c. 0·05 for both years, but the probability of parasite detection increased considerably with egg age. Eggs in their first day of development (before the embryo is formed) were not parasitized; most parasitized eggs were in the cohorts close to hatching. Although the reasons for age-dependent detection are unknown, if all parasitized eggs of the cohort ready to hatch were to die, infection by Ichthyodinium chabelardi would lead to mortality rates similar to those reported for the average daily mortality of sardine eggs and early larvae.     

Isolation and characterization of polymorphic microsatellites for the sardine Sardina pilchardus (Clupeiformes: Clupeidae)

The sardine Sardina pilchardus Walbaum, 1792 is a small pelagic fish species that sustains an economically important fishery in the eastern Atlantic Ocean. Eight perfect microsatellite loci of sardine were characterized from a partial genomic library enriched for CA repeat sequences. All loci revealed high levels of polymorphism, with the number of alleles per locus ranging between 22 and 45, and an average observed heterozygosity of 0.772. The newly described loci can be employed to determine population genetic structure of the sardine, and may aid in fishery management of this species.     

18S rRNA V9 metabarcoding for diet characterization: a critical evaluation with two sympatric zooplanktivorous fish species

The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using MiSeq paired‐end (PE; 2 × 150 bp) technology. To critically evaluate the method′s performance with degraded/digested DNA, the diets of two zooplanktivorous fish species from the Bay of Biscay, European sardine (Sardina pilchardus) and European sprat (Sprattus sprattus), were analysed. The taxonomic resolution and quantitative potential of the 18S V9 metabarcoding was first assessed both in silico and with mock and field plankton samples. Our method was capable of discriminating species within the reference database in a reliable way providing there was at least one variable position in the 18S V9 region. Furthermore, it successfully discriminated diet between both fish species, including habitat and diel differences among sardines, overcoming some of the limitations of traditional visual‐based diet analysis methods. The high sensitivity and semi‐quantitative nature of the 18S V9 metabarcoding approach was supported by both visual microscopy and qPCR‐based results. This molecular approach provides an alternative cost and time effective tool for food‐web analysis.     

Growth of North Alboran Sea sardine larvae estimated by otolith microstructure, nucleic acids and protein content

Wet mass and DNA, RNA and protein content increased significantly with standard length ( L _S) of sardine Sardina pilchardus larvae, collected in January 1995, in the Bay of Málaga, North Alboran Sea. L _S, wet mass and DNA, RNA and protein content were closely related allometrically to otolith radius ( R ). Larval daily length increments decreased but DNA, protein and wet mass daily increments increased with larval age. Daily length increments showed a negative and poor relationship with long-term otolith growth. In contrast, DNA, protein and wet mass daily increments were positively correlated. Differences between observed and back-calculated otolith radius-at-age indicated that larvae with slow otolith growth were under represented in older age groups, suggesting the existence of growth-selective mortality. Recent otolith growth, estimated from the mean widths of the last six increments, increased with age and R . Individual RNA: DNA and protein: DNA ratios were correlated significantly, although weakly, with L _S and larval growth.     

In vitro inhibition of microbial flora of fish by nisin and lactoperoxidase system

AIMS: To test the antimicrobial effects of nisin and lactoperoxidase system (LP system) against sardines flora. This study is part of a programme designed to investigate the preservability of fish using these inhibitors as potential biopreservatives. METHODS AND RESULTS: Antimicrobial effects of nisin and LP system alone or in combination were tested by the agar diffusion method against bacterial strains isolated from sardines (Sardina pilchardus). Nisin inhibited only Gram-positive bacteria, whereas LP system inhibited all strains studied. The combination of nisin (100 IU ml-1) and LP system (10 level) was significantly more effective than LP system or nisin alone against all strains, excepting Aeromonas salmonicida subsp. salmonicida and Vibrio alginolyticus. CONCLUSION: These results clearly demonstrated the efficiency of LP System-nisin combination for inhibiting spoilage flora of fish. SIGNIFICANCE AND IMPACT OF THE STUDY: Because LP system has a broad activity spectrum, it may be an interesting additional hurdle to improve the safety of food preservation by nisin. Combination of nisin and LP system could be of great interest as biopreservatives for fish and fish products.