Orlando J. Luis

Age-related changes in antioxidant enzyme activities, fatty acid composition and lipid peroxidation in whole body gammarus locusta (crustacea: amphipoda)

The main aim of this work was to provide baseline data on aspects of pro-oxidant and antioxidant processes in different life-stages of the marine amphipod Gammarus locusta. The activities of antioxidant enzymes and levels of lipid peroxidation were determined in whole body juveniles, subadults, and male and female adults of a laboratory population of G. locusta. Fatty acid composition of individuals at these different stages of development was also characterised in order to examine the contribution that polyunsaturated fatty acids (PUFA) might make to the peroxidation status of animals. The antioxidant enzymes, measured in whole body 100,000 supernatants, comprised catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD; EC 1.15.1.1) and glutathione peroxidase (GPX; EC 1.11.1.9). Fatty acids were analysed as fatty acid methyl esters (FAME). Lipid peroxidation was examined in terms of the levels of lipid peroxides determined as thiobarbituric acid-reactive malondialdehyde equivalents. Age-related changes were seen in antioxidant enzyme status: levels of SOD (p<0.01) and GPX (p<0.001) activities decreased progressively during development from juveniles to adults. Sex-related changes in GPX activity were also seen, the levels being higher in adult males than females (p<0.001). The amount of FAME present in whole body amphipod also changed over the life span. Among PUFA, the eicosapentaenoic (C20:5n-3), arachidonic (C20:4n-6) and docosahexaenoic (C22:6n-3) were the most abundant acids in this species, and both their individual concentrations and total PUFA increased progressively with age (up to 3.3-fold; p<0.001). The latter changes may contribute to the explanation of the observed differences in peroxidation status of the animals with age; thus, levels of lipid peroxides increased up to 40% in adult males compared to other age-classes (p<0.01). Overall, the decline in antioxidant enzyme activities, coupled with increased levels of PUFA, as the individual grows older, may render the older animals more susceptible to lipid peroxidation and oxidative stress.

Seasonal changes in lipid content and composition of the polychaete Nereis (Hediste) diversicolor

The lipid content and composition of Nereis (Hediste) diversicolor O. F. Muller (Annelida, Polychaeta, Nereidae) a mud-dwelling, intertidal errant polychaete in the Tagus estuary (Portugal), were examined on the monthly basis by lipid extraction, TLC and capillary GC. In this estuary, N. diversicolor is by far the dominant species among polychaeta and the main food item in the natural diet of several flatfishes. The biochemical elucidation of its lipid structure and distribution throughout the year, described in this study, provides information not only about the physiological role of lipids in the animal under consideration but also about dietary fatty acid requirements of some flatfishes in the wild and under laboratory conditions. The total lipid content varied between a maximum of 19.3% lyophilized dry weight in February (4.4% fresh weight) and a minimum of 6.6% in August (1.9% fresh weight). The major lipid classes were triacylglycerol, phospholipid, free sterol, free fatty acid, sterol ester/wax ester and alkyldiacylglycerol. The fatty acid composition was rather unsaturated with a 1:2 mean ratio of n-3: n-6. The major fatty acids were C160:0, C18:1n-9, C18:2n-6, and C20:5n-3; there were smaller amounts of C180:0, C18:1n-11, C18:1n-7, C18:3n-3, C20:1, C20:2n-6, C20:4n-6, C22:2, C22:5n-3, and many other fatty acids were detected at trace levels. The unsaturation ranged from 36.9 mg/g dry weight in summer to 107.4 mg/g in winter. An accumulation of fatty acids from plant origin was evident, in particular linoleic acid (C18:2n-6), which was quantitatively one of the major fatty acids throughout the year.

Broodstock diet effect on sea urchin Paracentrotus lividus (Lamarck, 1816) endotrophic larvae development: potential for their year-round use in environmental toxicology assessment.

The effect of captive broodstock diet on fertilization and endotrophic larvae development of the sea urchin Paracentrotus lividus was assessed. Maize grain and five inert pelleted diets were tested, during a three-month experimental period. Maize flour, wheat flour, soybean flour, maize/wheat flour (MWF) and maize/soybean flour mixes were used as vegetal sources for inert feed. Gonad index, percent egg fertilization and larvae malformation occurrence were compared with the results obtained from wild sea urchins (W). Whole egg total amino acid composition was concomitantly analyzed as a tool to explain eventual endotrophic larvae malformations caused by lack of specific nutrients. For all treatment groups (wild and captive), percent egg fertilization values above 96% were always observed, fulfilling the requisites (70-90%) necessary to conduct environmental monitoring bioassays, according to USEPA (2002). Similar values for normal percent larval development were only obtained from P. lividus broodstock subjected to an inert feeding diet based on a maize/wheat flour mix (85.0±1.45%), in comparison to wild P. lividus (82.5±1.75%). Likewise, no statistical differences on resultant whole egg total amino acid composition were observed between P. lividus fed MWF and wild treatments. Moreover, statistical differences between MWF and all the other captive feeding treatments were found for six out of the seventeen amino acids analyzed. This study demonstrates the possibility to obtain high values for P. lividus endotrophic larvae percent normal development based on broodstock held in captivity as long as an appropriate inert diet is provided.

Stocking Density and Captive Sea Urchin Paracentrotus lividus (Lamarck, 1816) Gamete Production and Fertilization

ABSTRACT Given that sea urchins eggs and larvae have been proposed as putative prey in marine larviculture, broodstock rearing conditions must be optimized for this purpose. In the current study, adult sea urchins Paracentrotus lividus were reared in 400-L tanks for 5 mo under 4 stocking densities (100, 200, 300, and 400 sea urchins/m2 of tank base area) to determine their effect on spawning performance and fertilization rate, and on broodstock month relative growth. For all stocking densities, more than 60% of sea urchins induced to spawn released a large number of gametes (more than 200 × 106 spermatozoa or more than 500 × 103 eggs per spawning). The percentage of males with large emissions was higher at a density of 300 sea urchins/m2. Irrespective of broodstock stocking density, fertilization rate was always higher than 90%. Lower relative growth was recorded with densities of 300 sea urchins/m2 and 400 sea urchins/m2 compared with 100 sea urchins/m2. We conclude that under the rearing conditions adopted in this study, captive P. lividus broodstock can be at least reared up to 400 sea urchins/m2 without impairing reproductive performance.