Christopher C. Parrish

Determination of Total Lipid, Lipid Classes, and Fatty Acids in Aquatic Samples

The hydrophobic nature of lipids provides a convenient means of separating them from other compounds in an aqueous sample matrix. Extraction in nonpolar solvents is universally employed and is the basis of the operational definition of lipids. This approach is used routinely in algal biosynthetic studies in which the fate of a radiolabeled precursor is followed into the lipid pool. By adding 14C bicarbonate to a sample from the field (Wainman and Lean, 1992) or a culture (Rai, 1995) and then later extracting the sample with a water-immiscible organic solvent, the “lipid fraction of carbon fixation” (LFCF) can be determined (Wainman and Lean, 1992). By performing a chromatographic separation before counting, this procedure can be further refined to determine the subclasses in which the 4C ends up (Smith and D’ Souza, 1993). Subfractionation is important when a differentiation between allocation to lipid storage and membrane synthesis is required. The radiolabeling approach is convenient, sensitive, and not prone to contamination. However, many ecological studies are not amenable to this approach, and so chemical analysis of the constituents of lipid extracts has to be performed.

Effects of docosahexaenoic, eicosapentaenoic, and arachidonic acids on the early growth, survival, lipid composition and pigmentation of yellowtail flounder (Limanda ferruginea): a live food enrichment experiment

The role of dietary ratios of docosahexaenoic acid (DHA, 22:6n−3), eicosapentaenoic acid (EPA, 20:5n−3) and arachidonic acid (AA, 20:4n−6) on early growth, survival, lipid composition, and pigmentation of yellowtail flounder was studied. Rotifers were enriched with lipid emulsions containing high DHA (43.3% of total fatty acids), DHA+EPA (37.4% and 14.2%, respectively), DHA+AA (36.0% and 8.9%), or a control emulsion containing only olive oil (no DHA, EPA, or AA). Larvae were fed differently enriched rotifers for 4 weeks post-hatch. At week 4, yellowtail larvae fed the high DHA diet were significantly larger (9.7±0.2 mm, P<0.05) and had higher survival (22.1±0.4%), while larvae fed the control diet were significantly smaller (7.3±0.2 mm, P<0.05) and showed lower survival (5.2±1.9%). Larval lipid class and fatty acid profiles differed significantly among treatments with larvae fed high polyunsaturated fatty acid (PUFA) diets having higher relative amounts of triacylglycerols (18–21% of total lipid) than larvae in the control diet (11%). Larval fatty acids reflected dietary levels of DHA, EPA and AA while larvae fed the control diet had reduced amounts of monounsaturated fatty acids (MUFA) and increased levels of PUFA relative to dietary levels. A strong relationship was observed between the DHA/EPA ratio in the diet and larval size (r2=0.75, P=0.005) and survival (r2=0.86, P=0.001). Following metamorphosis, the incidence of malpigmentation was higher in the DHA+AA diet (92%) than in all other treatments (∼50%). Results suggest that yellowtail larvae require a high level of dietary DHA for maximal growth and survival while diets containing elevated AA exert negative effects on larval pigmentation.

Lipid biogeochemistry of plankton, settling matter and sediments in Trinity Bay, Newfoundland. II. Fatty acids

The fatty acid biogeochemistry of a fjord-like bay in eastern Newfoundland, Canada, was investigated to determine sources and fates of organic material. Samples of plankton, settling particu- late matter and sediments were examined and fatty acids were used to identify their sources. Centric diatoms, as well as the dinoflagellate Ceratium tripos, were the dominant phytoplankton, producing, through primary production, large fluxes of settling particulate matter. Mean fatty acid fluxes of 8.827.4 mg m ˇ2 d ˇ1 were produced in the 1994, 1995 and 1997 seasons. Fatty acid distributions suggested that the acids were almost totally marine in origin with very few terrestrial plant indicators present and that bacterial biomass, as indicated by odd/branched chain fatty acids, was minimal. Short- term sediment trap studies with mercury chloride poisoning did not reveal a significant diAerence in fatty acid composition in traps with and without poison; however, dissolution of fatty acids from par- ticles can introduce large errors in flux and concentration measurements. Fatty acid concentrations in sediments were approximately 1% of the concentration found in settling particulate matter. Fatty acids of bacterial and terrestrial origin were present at higher concentrations in the sediments and it appears that the fatty acids of marine origin are eAectively recycled by diagenetic processes at the sediment- water interface. This implies that, in terms of fatty acid nutrients, the lower food web in this area is very eAcient as there is a minimal loss of the high flux of fatty acids through burial in sediments. # 1998 Elsevier Science Ltd. All rights reserved

Lipid and Phenolic Biomarkers in Marine Ecosystems: Analysis and Applications

Biomarkers are compounds or groups of compounds that can be used as signatures of individual organisms or groups of organisms, or of certain environmental processes. Lipid and phenolic biomarkers can be used to assess the health of an ecosystem and the degree to which it has been influenced by terrestrial and anthropogenic inputs. Lipid classes and fatty acids can be used to determine production of marine biogenic material of dietary value to pelagic and benthic organisms. Polycyclic aromatic hydrocarbons and 5β-stanols such as coprostanol can be used to determine pollutant loading from oil spillage or sewage and the phenanthrene/methylphenanthrene ratio can be used specifically as an indicator of wood burning. N-alkanes and thermochemolysis products in cores can show the sensitivity of sediments to changes in land use patterns near the land margin. The relationship between marine and terrestrially derived products in sediment cores can be used to indicate the degree to which land use changes have impacted the pattern of marine biogenic productivity in the area. Stable isotope and multivariate analyses are particularly useful for biomarker validation.

Dissolved and particulate marine lipid classes: a review

There are similar lipid compounds present in the dissolved and particulate fractions of seawater, but the processes governing their concentrations in each fraction appear to be different. Although they are composed primarily of atoms of carbon and hydrogen, marine lipid compounds can be subdivided into as many as 16 individual classes according to their chemical structure. These classes are present in the dissolved and particulate fractions of seawater at the μgl−1 level. The compounds in most of these classes have known functions in various types of cells, or known anthropogenic sources. It is possible to distinguish energy storage clases, membrane classes, pollutants and indicators of lipid degradation. Individual classes or groups of certain classes may be used to indicate the presence of certain types of organisms as well as their physiological state and activity. The presence of other classes at the μgl −1 level can provide a signal of deteriorating water quality.

Dietary fish oils limit adipose tissue hypertrophy in rats.

Total body mass, fat pad mass, and fat cell size were examined after feeding rats diets containing 20% triglycerides from fish oil or lard. Although food consumption, weight gain, and fat balance on the two diets were similar, lard-fed rats had 77% more fat in perirenal fat pads and 51% more fat in epididymal fat pads compared with fish oil-fed rats. There was no difference between the two groups in fat cell number in each region; however, adipocytes were significantly smaller in fish oil-fed rats. Thus dietary fish oil appears to limit triglyceride accumulation in adipose tissue and thereby limit fat cell trophic growth.

Essential fatty acids in aquatic food webs

Aquatic ecosystems occupy the largest part of the biosphere, and lipids in those systems provide the densest form of energy. Total lipid energy can be used to predict features of animal population dynamics such as egg production by fish stocks. Difficulties in determining the relationship between spawner biomass and the number of offspring produced (recruitment) have led researchers to look at lipids (Marshall et al. 1999). A positive association between recruitment and liver weights in cod prompted an investigation of total lipid energy as a proxy for total egg production by fish stocks. Marshall et al. (1999) found a highly significant linear relationship between total egg production and total lipid energy, and they suggested this approach should be used at other trophic levels too. Total lipid content of fish has also been connected to climate-induced community changes (Litzow et al. 2006). It is hypothesized that this relates to the dietary availability of just two fatty acids which were positively correlated with total lipid content.

Eicosapentaenoic Acid Regulates Scallop (Placopecten magellanicus) Membrane Fluidity in Response to Cold

The lipid core of a biological membrane requires a certain degree of structural rigidity, but it must also be sufficiently fluid to permit lateral movement of the constituent lipids and embedded proteins. Ectotherms can counteract the ordering effects of reduced temperature by changing the structure of their membranes, a process known as homeoviscous adaptation (1). Although the content of unsaturated fatty acids in the membranes of ectothermic animals is generally known to increase in response to cold (2), no clear and direct relationship between unsaturated fatty acids and membrane fluidity has been established in marine organisms. For example, phospholipid molecular species containing docosahexaenoic acid (22:6 3) are believed to be important in controlling finfish membrane fluidity (3–6), but a direct correlation between 22:6 3 and membrane fluidity has not been found (4, 5, 7, 8). In contrast, we show here a simple but very strong relationship between fluidity and a single polyunsaturated fatty acid, eicosapentaenoic acid (20:5 3), in gill membranes from a marine bivalve mollusc, the sea scallop Placopecten magellanicus. Phospholipids are the main structural elements of biological membranes, and their physical characteristics are key determinants of membrane structure and function. Many vital cell activities that depend on the optimal functioning of membranes are therefore sensitive to the chemistry of the membrane lipids (9) and to environmental conditions, such as temperature and pressure, that perturb the phase behavior and dynamics of lipids in membranes (10). Under extreme or variable conditions, organisms can exploit the tremendous chemical diversity among membrane lipids to defend the physical properties of the membrane (10). Thus in ectotherms, where changes in temperature cause important membrane perturbations, the usual adaptive response includes a modification of lipid composition (11). Sessile animals living in Newfoundland waters must maintain membrane structure and function in the face of extreme cold in deep waters (as low as 1.4°C) or seasonally highly variable conditions in surface waters (as much as 22°C in 6 months) (12). In the present study, we exposed sea scallops to a 10°C decrease in temperature for up to 3 weeks and then examined the relationship between the fatty acid composition of branchial phospholipids and membrane fluidity. Vesicles were prepared from the gills of scallops acclimated to temperatures of 15 and 5°C. After three weeks of thermal acclimation, the structural order of the phospholipids was measured by electron spin resonance (ESR) spectroscopy at five temperatures (0–20°C) that span the physiological range of Placopecten magellanicus (Fig. 1). The vesicles prepared from gills of 5°C-acclimated scallops were significantly (ANCOVA, P 0.03) less ordered than vesicles from 15°C-acclimated scallops. Temperature acclimation had shifted the order parameter curve 1–2°C toward lower assay temperatures, giving a homeoviscous efficacy (13) of 14%. Such a partial adjustment towards an ideal or complete homeoviscous response has also been found in crabs (14) and crayfish (15). In these invertebrates, the costs of perfect compensation may be too high, or the benefits too low. On the other hand, the ESR measurements in this study were made with the spin probe 5-doxyl stearic acid, reflecting the homeoviscous response in the outer region of the purified lipid bilayer. It is possible that the response deeper in the bilayer, in the actual region of alkenyl chain unsaturation, would have been greater (16). Received 30 November 2001; accepted 22 March 2002. 1 Present address: Department of Physiology and Experimental Medicine, The George Washington University Medical Center, Ross Hall Room 402, 2300 Eye Street, NW, Washington, DC 20037. 2 To whom correspondence should be addressed. E-mail: cparrish@ mun.ca Reference: Biol. Bull. 202: 201–203. (June 2002)

Feeding frequency affects food consumption, feeding pattern and growth of juvenile yellowtail flounder (Limanda ferruginea).

Feeding experiments were carried out on juvenile yellowtail flounder (Limanda ferruginea) (6.8±0.2 g), a candidate for cold-water aquaculture in eastern North America. At about 7 °C, feeding frequency was shown to have a significant (P<0.05) effect on food consumption and growth, with fish fed to apparent satiation in two or four daily meals consuming more food and growing better than fish fed less often (once daily and two meals every other day). Fish fed two meals every other day ate significantly more (P<0.05) in the morning (8.4±0.4 mg) than in the afternoon (6.0±0.2 mg), whereas there were no differences in meal sizes between feedings for other groups. Behavioural observations revealed that fish fed fewer meals per day ingested more pellets per feeding (1.1±0.1 vs. 0.7±0.1 pellet/min), showed more activity and foraged more often throughout the day than fish fed four times daily or twice daily (3.5±0.3 vs. 2.1±0.2 behaviour/min). Low incidences of aggression indicated that yellowtail flounder probably do not form feeding hierarchies; however fish fed twice daily showed a significant increase in the coefficient of variation (CV) for body weight over the course of the experiment (P<0.05). Fish fed twice daily gained significantly more weight (P<0.05) and had the lowest FCR (0.89), thus it is recommended that fish at this stage of grow-out be fed twice per day.

Cholestane as a digestibility marker in the absorption of polyunsaturated fatty acid ethyl esters in Atlantic salmon

Salmonid fish require long-chain n−3 fatty acids in their diet. The digestibility of different chemical forms of fish oil fatty acids, fed as triacylglycerols, free fatty acids or ethyl esters, was examined in 300 g farmed Atlantic salmon (Salmo salar) using cholestane as an indicator of fat absorptionin lieu of the chromium oxide (Cr2O3) which is commonly used as a marker in digestibility studies. It was established that the two digestibility markers gave similar results. Conveniently, cholestane does not require a separate analysis if fatty acids are to be determined by appropriate gas-liquid chromatography. The long-chain polyunsaturated fatty acids were particularly well absorbed, the apparent digestibility being 90–98% when feeding triacylglycerols or free fatty acids. However, the digestibility of monounsaturated fatty acids (75–94%) was lower, and lower still for saturated fatty acids (50–80%). Ethyl esters of fatty acids were significantly less well absorbed (P<0.05) than were the corresponding fatty acids in free acid or triacylglycerol form. Irrespective of dietary fat type, only free fatty acids were identified in feces, indicating total hydrolysis of triacylglycerols and ethyl esters.