Sterols of the oyster,Crassostrea virginica

A commericial sample of the oyster,Crassostrea virginica, obtained from Maryland waters of the Chesapeake Bay, contained 31 desmethylesterols and at least eight 4-monomethylsterols. The combined gas liquid chromatography-mass spectra of the minor components showed the presence of 6 unusual sterols, 24-ethylcholest-22-en-3β ol, 4α-methyl-24-ethylcholestan-3β-ol, occelasterol, (24E)-24-propylidene-cholest-5-en-3β-ol, (24ZO-24-propylidene-cholest-5-en-3β-ol, and 24-methylene-cholestanol. The C-24 configuration of 24-ethylcholest-5-enol, 24-methyl-cholesta-5,22-dienol, and 24-ethylcholesta-5,22 dienol were elucidated by 220 MHz nuclear magnetic resonance spectrometry.     

Phosphono-lipid content of the oyster,Crassostrea virginica,in three physiological conditions

The distribution of lipid-bound phosphorus between the phosphodiester and phosphonate forms was determined for the oyster in three physiological conditions. The adductor muscle had the highest level of phosphonate phosphorus, 92 μg/g wet weight tissue, or 40.4% of all muscle phospholipids. The total phospholipid level (μg/g wet weight tissue) increased in all tissues of the oyster during starvation, as did the percentage of phosphonolipids in all tissues except the adductor muscle, in which it remained constant. These data suggest that during starvation the phosphonate bonds are conserved at the expense of phosphodiester bonds in oyster lipids.     

Polyunsaturated fatty acids and neutral lipids in developing larvae of the oyster,Crassostrea virginica

The fatty acid composition of oyster larvae at various stages, as well as of the algal diet, were determined by gas liquid chromatography (GC). Saturated fatty acids are the major fatty acid components in all larval stages and account for 34–62%, 30–35% and 35–81% of the neutral, polar and total lipids of algal-fed larvae respectively. Weight percentage of saturated fatty acid in “starved” larvae was consistently higher (63–81%) during the whole period. The total polyunsaturated fatty acids were higher in the polar lipids than in the neutral lipids. The concentration of the ω3 fatty acids also was comparatively higher in the polar lipids than in the neutral lipids. In the total and neutral lipid fractions, the weight percentage of polyunsaturated and ω3 fatty acids was higher in the eyed than in the pre-eyed (pediveliger) larvae. Eicosapentaenoic acid (20∶5ω3) and 22∶6ω3 were not detected in lipids of “starved” and young larvae. There was an accumulation of 20∶5ω3, 22∶6ω3, and total ω3 fatty acids in the older larvae. Lipid classes were separated by thin layer chromatography (TLC). There was no qualitative change in lipid composition during larval development, but a marked increased of triacylglycerol in larvae up to the stage of maturation in algae-fed larvae. Contribution number 1195 of the Virginia Institute of Marine Science, Gloucester Point, VA 23062     

The fatty acid composition of three unicellular algal species used as food sources for larvae of the American oyster (Crassostrea virginica)

The total lipid and fatty acid content of 3 algal species,Pyramimonas virginica, Pseudoisochrysis paradoxa andChlorella sp., which have been successful as food sources for rearing larvae of the American oyster,Crassostrea virginica, was determined. Of the fatty acids of ω6 and ω3 families which have been shown to be essential fatty acids for normal growth in many animals, only the ω6 fatty acids were found to be higher in these 3 species of algae than in the traditional oyster larvae diet which consists of the algaeMonochrysis lutheri andIsochrysis galbana. The major fatty acid constituents of the total lipids of the 3 species were the C12, C14, C16 and C18 saturated fatty acids and the C16 and C18 mono- and polyunsaturated acids. These components constituted 70–93% of the total lipid in cultures of all ages. There were modest amounts of C20 and C22 polyunsaturated acids; some of these existed only in trace amounts. InP. virginica andChlorella sp., hexadecanoic acid was dominant (23–39%). The presence of large quantities of tetradecanoic acid (22–26%) and oleic acid (17–21%) was characteristic ofP. paradoxa. Chlorella sp. had the highest proportion of octadecatrienoic acid (18∶3ω3) which accounted for up to 17% of the total lipids. γ-Linolenic acid (18∶3ω6) was found only inChlorella sp., but in the 5th-day culture only. The lowest proportion of total polyethylenic acid was inP. paradoxa; however, lipid analyses showed this alga had the most lipid/individual cell. Some variations were observed in the fatty acid composition with age of the culture. Contribution No. 883 of the Virginia Institute of Marine Science, Gloucester Point, VA 23062.     

Sterol composition and biosynthesis in the sponge Spheciospongia vesparia

The sterol composition of Spheciospongia vesparia from the Caribbean Sea is reported. Three groups of sterols, Δ⁵, Δ⁰, Δ^5,7, were found. The isolated 5α,8α-epidioxy sterols appeared to be predominantly, if not exclusively, artifacts. The auto-oxidation of native Δ^5,7-sterols to 5α,8α-epidioxy sterols has been investigated. A transformation of dietary Δ⁵-sterols to Δ^5,7-sterols has been demonstrated in Spheciospongia vesparia. © 1998 SCI     

Sterol biosynthesis by the arbuscular mycorrhizal fungus Glomus intraradices

Ri-T-DNA-transformed carrot roots were used for investigating sterol metabolism by the arbuscular mycorrhizal (AM) fungus Glomus intraradices under three distinct experimental conditions: (i) a symbiotic stage (fungus still attached to the host roots); (ii) a detached stage (fungus physically separated from the roots); and (iii) a germinating stage (germinating spores). In all three stages, G. intraradices was found to contain a mixture of 24-alkylated sterols, with 24-methyl and 24-ethyl cholesterol as the main compounds, but no ergosterol, the predominant sterol in most fungi. Feeding experiments with [1-¹⁴C]sodium acetate were performed to check the ability of the fungus to synthesize sterols. Whatever the experimental conditions, G. intraradices was able to actively take up exogenous acetate and to incorporate it into sterols and their precursors. Our data provide first evidence for de novo sterol synthesis by an AM fungus.     

Absence of sterol biosynthesis in oyster tissue culture

An oyster heart tissue culture was used to investigate the ability of the American oyster (Crassostrea virginica) to synthesize sterols from [¹⁴C]acetate. Lipids were extracted from cultured oyster heart cells and no label was found in the purified fatty acid fraction, 90% of which was located in the saturated and monounsaturated fractions. Although these results cannot rule out the possibility of sterol synthesis in other oyster tissues, this and previous evidence are in support of the hypothesis that the American oyster is unable to synthesize sterols.     

Sterol composition and biosynthesis in sorghum: Importance to developmental regulation

Sterol composition and biosynthesis have been examined in seeds, germinating seeds and blades from fally matured leaves ofSorghum bicolor in various stages of development’from seedlings (seven-day plants) to flowering (66-day) plants. The profile of the dominant free sterols of seeds was similar to that of leaf blades; both contained cholesterol, 24α-methylcholesterol (campesterol), 24β-methylcholesterol (dihydrobrassicasterol), 24α-ethylcholesterol (sitosterol) and 24α-ethylcholesta-5,22-dienol (stigmasterol). Sufficient sterol intermediates were identified in the plant to indicate separate post-cycloartenol pathways to sterolic end products. The total free sterol content of the seed (μg/seed) increased somewhat during the 20 hr germination period. However, as the plant developed (seven to 48 days), there was a logarithmic increase in the leaf blade sterol content (μg/leaf blade) which plateaued at the onset of floral differentiation (ca. day 41). Over the next 18 days (48 to 66 days—period of inflorescense development), the sterol content rapidly decreased. In the early stages of plant development, the leaf blade pentacyclic triterpenoid (PT) content was negligible. With the onset of floral differentiation, PT content increased logarithmically, reaching a plateau level that surpassed the sterol content as flowering progressed. These results imply that a critical mass of sterol is associated with sorghum for floral induction. Sterol loss from the leaves of the flowering plants presumably was compensated for by the diversion of 2,3-oxidosqualene (SO) from sterol synthesis to PT production. Additional feeding and trapping experiments with [2-¹⁴C]mevalonic acid, [2-³H]cycloartenol, [24-³H]lanosterol [4-¹⁴C]sitosterol and [4-¹⁴C]cholesterol fed to germinating seeds and leaves from flowering plants demonstrated that sorghum possessed a cycloartenolbased pathway; germinating seeds synthesized 24-alkylsterols but not cholesterol, although cholesterol was identified in both dry and germinating seeds by gas chromatography-mass spectroscopy (GC-MS); and mature leaves synthesized cholesterol and 24α-alkylsterols but not 24β-methylcholesterol.     

Sterol biosynthesis inhibitors: Their current status and modes of action

The mechanism of each of the reactions in the post-squalene segment of the fungal and higher plant sterol biosynthetic pathway is outlined. The inhibitors of the enzymes catalyzing the reactions are described and how inhibition is brought about is explained in the areas where it is known. Based on a paper presented at the Symposium on Plant and Fungal Sterols: Biosynthesis, Metabolism and Function, held at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

Lipids ofCrassostrea virginica. I. Preliminary investigations of aldehyde and phosphorous containing lipids in oyster tissue

Oyster tissue contained 2.4% lipid, 0.14 μmole aldehyde per milligram lipid and at least 10 μg phosphorous per milligram lipid. The neutral lipid represented 58%, the glycolipid 6%, and the polar lipid 36% of the total lipid recovered after silicic acid column chromatography. Aldehydes were found in all fractions, but the presence of plasmalogen was verified in only the neutral and polar lipid fractions. At least 68% of the plasmalogen in oyster lipid was found in the polar lipid fraction. At least 13% of the phosphorous in oyster lipids was present as phosphonolipid. The distribution of phosphate and phosphonate lipids was: diacyl phospholipid 38.1%, plasmalogen phospholipid 21.8%, sphingophosphonolipid 13.5%, glyceryl ether phospholipid 8.3%, sphingophospholipid 6.9%, plasmalogen phosphonolipid 6.4%, diacyl phosphonolipid 2.6%, and glyceryl ether phosphonolipid 2.4%. When the per cent of phosphorous as phosphonolipid within the plasmalogen and glyceryl ether classes was calculated, similar values were obtained. These results support the hypothesis that there is a product precursor relationship between these two classes of lipids. Some of the data taken from a thesis to be submitted to the Graduate School, University of Maryland, by Leslye Johnson in partial fulfillment of the requirements for the M.S. degree in biochemistry.     

Sterol metabolism in the nematodeCaenorhabditis elegans

The metabolism of various dietary sterols and the effects of an azasteroid on sitosterol metabolism in the free-living nematodeCaenorhabditis elegans was investigated. The major unesterified sterols ofC. elegans in media supplemented with sitosterol, cholesterol or desmosterol included 7-dehydrocholesterol (66.5%, 40.5%, 31.2%, respectively), cholesterol (6.7%, 52.3%, 26.9%), lathosterol (4.4%, 3.6%, 1.7%) and 4α-methylcholest-8(14)-en-3β-ol (4.2%, 2.1%, 3.8%). Esterified sterols, representing less than 20% of the total sterols, were somewhat similar except for a significantly higher relative content of 4α-methylcholest-8(14)-en-3β-ol (23.3%, 23.4%, 10.6%). ThusC. elegans not only removes the substituent at C24 of dietary sitosterol but possesses the unusual ability to produce significant quantities of 4α-methylsterols. WhenC. elegans was propagated in medium supplemented with sitosterol plus 5 μg/ml of 25-azacoprostane hydrochloride, the azasteroid strongly interfered with reproduction and motility ofC. elegans and strongly inhibited the Δ24-sterol reductase enzyme system; excluding sitosterol, the major free sterols of azacoprostane-treatedC. elegans were cholesta-5, 7, 24-trien-3β-ol (47.9%), desmosterol (9.4%), fucosterol (2.1%) and cholesta-7,24-dien-3β-ol (2.0%). These 4 sterols are likely intermediates in the metabolism of sitosterol inC. elegans.     

Sterol metabolism in the nematodePanagrellus redivivus

Panagrellus redivivus was propagated in media containing three structurally different sterols: 7-dehydrocholesterol, campesterol or stigmastanol. Nematodes propagated with 7-dehydrocholesterol contained mostly lathosterol and 7-dehydrocholesterol. Nematodes propagated with campesterol contained mostly cholesterol and cholestanol. Nematodes propagated with stigmastanol contained mostly cholestanol. The sterol ester fraction was enriched with 4α-methylsterols and contained the same sterols as the free sterol fraction except for nematodes propagated with 7-dehydrocholesterol, where no dietary sterol was found in the ester fraction.P. redivivus is capable of reducing the Δ⁵-bond, C−24 dealkylation and methylating the sterol nucleus at C−4.     

Techniques for delivery of arachidonic acid to pacific oyster, <Emphasis Type="Italic">Crassostrea gigas</Emphasis>, spat

The present study tested two techniques for dietary supplementation of Crassostrea gigas spat with PUFA, such as arachidonic acid (AA). The first technique consisted of a preliminary enrichment and growth of an algal concentrate (T-ISO, Isochrysis sp.) with AA dissolved in an ethanol solution, the whole culture then being fed to the spat. This enrichment increased the AA weight percentage in T-ISO neutral and polar lipids from 0.6 to 22.4% and from 0.4 to 6.8%, respectively. The second delivery technique was direct addition separately of free AA dissolved in ethanol solution and algal concentrate (T-ISO+AA) to the spat-rearing tank. To test the efficiency of these delivery techniques, oyster spat were supplemented with AA-enriched T-ISO, T-ISO+AA, and T-ISO alone. The possible biological impacts of these dietary treatments were assessed by measuring growth, condition index, and TAG content of oyster spat. Dry weight and condition index of spat fed AA-enriched T-ISO decreased by 24 and 49%, respectively, after 26 d of feeding; basically, TAG content declined 88% after 34 d of conditioning. When AA was added directly to seawater, spat growth and condition index were comparable with those of oysters fed T-ISO alone. AA incorporation in oyster tissues was assessed by analysis of the FA compositions in both neutral and polar lipid fractions. After 34 d, AA content in neutral lipids reached 7 and 11.7% in the spat fed, respectively, AA-enriched T-ISO and T-ISO+AA, as compared with 1.1% in spat fed only T-ISO. AA incorporation was greater in polar lipids than in neutral lipids, reaching 7.8 and 12.5% in spat fed AA-enriched T-ISO and T-ISO+AA, respectively. A direct addition of PUFA along with the food supply represents an effective and promising means to supplement PUFA to oyster spat.     

Use of low-field-NMR and MRI to characterize water mobility and distribution in pacific oyster (Crassostrea gigas) during drying process

Low-field nuclear magnetic resonance (LF-NMR) and magnetic resonance imaging (MRI) were used to monitor the water mobility and distribution of Pacific oyster during drying process. The results show the mobilities of bulk, immobilized, and free water were reduced, and the immobilized water was removed dramatically. T₂-weighted images displayed the water decrease from the external surface to inner center of oyster during drying. In addition, excellent correlations between the total moisture content and T₂₂ and A₂₂ were observed with coefficients 0.9777 and 0.9832, respectively. Principal component analysis showed the drying degree of oyster could be monitored based on raw relaxation data. Thus, the result revealed that LF-NMR and MRI have great potential in assessing water mobility and distribution in oyster during drying process.     

Sterol metabolism in the tobacco hornworm,Manduca sexta—A review

A number of intermediates involved in the dealkylation and conversion of the major C₂₈ and C₂₉ phytosterols to cholesterol in insects were first isolated and identified in studies with the tobacco hornworm,Manduca sexta, carried out in our laboratory. We also investigated the effects of a variety of known sterol metabolism inhibitors inManduca, particularly those affecting the Δ²⁴-sterol reductase enzyme, and synthesized and tested a number of new inhibitors as well. In-depth studies of ecdysteroids inManduca during embryogenesis and during pupal-adult development provided new information on molting hormone content, biosynthesis, and metabolism. In addition, this insect has been utilized in the study of three specific enzyme systems of ecdysteroid metabolism, namely 20-monooxygenase, 3-epimerase, and phosphotransferase, which are critical to activation and deactivation of molting hormones in insects. Based on a paper presented at the symposium on the “Regulation of Biosynthesis and Function of Isopentenoids,” Atlanta, Georgia, May 1994.     

Sterol synthesis in the liver intestine,and lung of the guinea pig

The relative rates of sterol synthesis in the liver, ileum, and lung of the guinea pig have been studied by measuring the incorporation by tissue slices of¹⁴C-labeled acetate into digitonin-precipitable sterols. The liver showed maximum incorporation of acetate at pH 6.5, the ileum at pH 7.5, and the lung at pH 6.0. The incorporation of acetate approached the maximum rate at a concentration of 10 mM with the liver and lung and 5 mM with the ileum. Using these conditions of assay, sterol synthesis was measured in the liver, ileum, and lung of four groups of guinea pigs killed at 6-hourly intervals. Depending on the time of day, the rate of sterol synthesis in the ileum was from 6 to 14 times that in the liver, while in the lung the rate was up to 3 times that shown by the liver. Additional studies showed that all regions of the small intestine synthesized sterol at a higher rate than the liver, with the highest rate of synthesis occurring in the ileum. The rates observed in the adrenal, testis, muscle, adipose tissue, and skin indicated that these tissues are not quantitatively important sites of sterol synthesis in the guinea pig.     

Microbial Symbionts Shape the Sterol Profile of the Xylem-Feeding Woodwasp, Sirex noctilio

The symbiotic fungus Amylostereum areolatum is essential for growth and development of larvae of the invasive woodwasp, Sirex noctilio. In the nutrient poor xylem of pine trees, upon which Sirex feeds, it is unknown whether Amylostereum facilitates survival directly through consumption (mycetophagy) and/or indirectly through digestion of recalcitrant plant polymers (external rumen hypothesis). We tested these alternative hypotheses for Amylostereum involvement in Sirex foraging using the innate dependency of all insects on dietary sources of sterol and the unique sterols indicative of fungi and plants. We tested alternative hypotheses by using GC-MS to quantify concentrations of free and bound sterol pools from multiple life-stages of Sirex, food sources, and waste products in red pine (Pinus resinosa). Cholesterol was the primary sterol found in all life-stages of Sirex. However, cholesterol was not found in significant quantities in either plant or fungal resources. Ergosterol was the most prevalent sterol in Amylostereum but was not detectable in either wood or insect tissue (<0.001 μg/g). Phytosterols were ubiquitous in both pine xylem and Sirex. Therefore, dealkylation of phytosterols (sitosterol and campesterol) is the most likely pathway to meet dietary demand for cholesterol in Sirex. Ergosterol concentrations from fungal-infested wood demonstrated low fungal biomass, which suggests mycetophagy is not the primary source of sterol or bulk nutrition for Sirex. Our findings suggest there is a potentially greater importance for fungal enzymes, including the external digestion of recalcitrant plant polymers (e.g., lignin and cellulose), shaping this insect-fungal symbiosis.     

Distribution of neutral lipids in the tissues of the oysterCrassostrea virginica

The content of neutral lipids was determined in the tissues of oysters (Crassostrea virginica Gmelin) collected in June, July and March. The lipid content of starved March oysters was also determined. Oyster tissue from the June harvest contained the highest quantities of triglyceride; starved and July (late spawning) oysters had decreased levels of triglycerides in all tissues except the digestive diverticula/gonad. Free sterol content of all the tissues averaged 1.21 mg equivalent cholesterol/g wet weight tissue, and the steryl ester concentration averaged about 10% of this value. Findings of this investigation indicate that the triglyceride content of oyster tissue fluctuates seasonally and is there-fore keyed to the physiological state of the animal. Furthermore, triglycerides may be an important energy reserve for reproductive tissue.     

Sterol and fatty acid composition of the clam,Codakia orbicularis,with chemoautotrophic symbionts

Codakia orbicularis may obtain nutrients from chemoautolithotrophic bacteria. The chemical composition of theC. orbicularis was investigated because of this unusual source of nutrition, and because it is a human food source in the Caribbean. The lipid fraction of these molluscs is discussed in detail. Polyunsaturated fatty acids account for only 11–15% of the total fatty acids, and non-methylene interrupted dienes are present as high as 9.5%. Cholesterol represents about 45% of the total sterols present. Mention of a commercial company or product does not constitute an endorsement by NOAA, National Marine Fisheries Service.