Dietary Sterol Preference in the Silkworm,Bombyx mori

Since insects are unable to biosynthesize sterols de novo, sterols must be obtained from dietary sources. Although it has been reported that β-sitosterol is crucial for larval growth in the silkworm, Bombyx mori, little has been investigated concerning the dietary selection of sterols by Bombyx larvae. Here, we demonstrate that Bombyx larvae have the following sterol preference: β-sitosterol >> ergosterol > cholesterol = stigmasterol. Interestingly, Bombyx larvae preferred ergosterol, an inhibitory sterol on larval growth, indicating that sterol selection following first contact of the diet with the mouthpart might be different from the sterol recognition mechanism present in sterol metabolism.     

Breaking Caenorhabditis elegans the easy way using the Balch homogenizer: an old tool for a new application

The biochemical quantification of sterols in insects has been difficult because only small amounts of tissues can be obtained from insect bodies and because sterol metabolites are structurally related. We have developed a highly specific and sensitive quantitative method for determining of the concentrations of seven sterols—7-dehydrocholesterol, desmosterol, cholesterol, ergosterol, campesterol, stigmasterol, and β-sitosterol—using a high performance liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry (HPLC/APCI-MS/MS). The sterols were extracted from silkworm larval tissues using the Bligh and Dyer method and were analyzed using HPLC/APCI-MS/MS with selected reaction monitoring, using cholesterol-3,4-¹³C₂ as an internal standard. The detection limits of the method were between 12.1 and 259 fmol. The major sterol in most silkworm larval tissues was cholesterol, whereas only small quantities of the dietary sterols were detected. Thus, a simple, sensitive, and specific method was successfully developed for the quantification of the sterol concentrations in each tissue of an individual silkworm larva. This method will be a useful tool for investigating to molecular basis of sterol physiology in insects, facilitating the quantification of femtomole quantities of sterols in biological samples.     

STEROLS OF 14 SPECIES OF MARINE DIATOMS (BACILLARIOPHYTA)1

The sterol compositions of 14 species of marine diatoms were determined by gas chromatography and gas chromatography-mass spectrometry. A variety of sterol profiles were found. The sterols 24-methylcholesta-5,22E-dien-3β-ol, cholest-5-en-3β-ol, and 24-methylcholesta-5,24(28)-dien-3β-ol, previously described as the most common sterols found in diatoms, were major sterols in only a few of the species. In light of this and other recent data, it is clear that these three sterols are not typical constituents of many diatom species. Most of the centric species examined had 24-methylcholesta-5,24(28)-dien-3β-ol and 24-methylcholest-5-en-3β-ol as two of their major sterols. The exception was Rhizosolenia setigera, which possessed cholesta-5,24-dien-3β-ol as its single major sterol. In contrast to the centric species, the pennate diatoms examined did not have any particular sterols common to most species. Minor levels ofΔ⁷-sterols, rarely found in large amounts in diatoms, were found in four species. C₂₉sterols were found in many species; seven contained 24-ethylcholest-5-en-3β-ol and three contained 24-ethylcholesta-5,22E-dien-3β-ol, reinforcing previous suggestions that C₂₉ sterols are not restricted to higher plants and macroalgae. 24-Ethylcholesta-5,22E-dien-3β-ol may prove to be useful for taxonomy of the genus Amphora and the order Thalassiophysales. A major sterol of Fragilaria pinnata was the uncommon algal sterol 23,24-dimethylcholesta-5,22E-dien-3β-ol. Cholesta-5,24-dien-3β-ol was the only sterol found in the culture of Nitzschia closterium. This differed from previous reports of 24-methylcholesta-5,22E-dien-3β-ol as the single major sterol in N. closterium. Two C₂₈ sterols possessing an unusual side chain were found in Thalassi-onema nitzschioides, a C_28:2 sterol (16%) and a C_28:1 sterol in lower abundance (2.5%), which may be 23-methylcholesta-5,22E-dien-3β-ol and 23-methyl-5α-cholest-22E-en-3β-ol, respectively. The species Cylindrotheca fusiformis, T. nitzschioides, and Skeletonema sp. may be useful as direct sources of cholesterol in mariculture feeds due to their moderate to high content of this sterol.     

Developmental Changes in the Sterol Composition and the Glycerol Content of Cuticular and Internal Lipids of Three Species of Flies

The glycerol concentration and the composition of cuticular and internal sterols in three medically and forensically important fly species, viz., Musca domestica, Sarcophaga carnaria, and Calliphora vicina, were analyzed. The cuticular and internal lipid extracts were separated by HPLC‐LLSD, after which the sterol fraction was characterized by GC/MS in total ion current (TIC) mode. The cuticular lipids of M. domestica larvae contained seven sterols, while in pupae and females, six sterols were identified. Five sterols were found in the cuticular lipids of M. domestica males. The internal lipids of M. domestica larvae and pupae contained six and seven sterols, respectively, while those of male and female flies contained only five sterols. Sitosterol, cholesterol, and campesterol were the dominant sterols in M. domestica, while campestanol, stigmasterol, sitostanol, and fucosterol were identified in low concentrations or in traces. In contrast, cuticular and internal lipids of S. carnaria and C. vicina contained only cholesterol. Glycerol was identified in all stages of M. domestica, S. carnaria, and C. vicina. For all the three examined fly species, the present study clearly showed species‐specific developmental changes in the composition of cuticular and internal sterols as well as in the glycerol concentration.     

Sterols of the heterotrophic dinoflagellate,pfiesteria piscicida (dinophyceae): is there a lipid biomarker?1

Within U.S. waters, blooms of the dinoflagellate, Pfiesteria piscicida, have been recorded on an almost regular basis in the Chesapeake Bay and surrounding mid‐Atlantic regions for the last two decades. Despite the apparent significance of such blooms to the environment and human health and the attendant economic consequences, little work has addressed the physiology and biochemistry, particularly that of sterol composition, of P. piscicida. GC‐MS characterization of trimethylsilyl ether derivatives of sterols from free sterol and sterol ester fractions was performed in an effort to determine whether P. piscicida produces unique sterols that may serve as potential biomarkers. This characterization revealed that like most dinoflagellates, the majority of sterols was present as free sterols. Furthermore, the profile of free sterols was found to resemble those of photosynthetic dinoflagellates, with the dominant compound being the previously reported dinoflagellate sterol, dinosterol. A number of other 4α‐methyl‐substituted sterols and steroidal ketones common to other dinoflagellates were also identified. No strong candidate(s) for a unique sterol biomarker was present.     

Sterols in Germinating Seeds and Developing seedlings of Longleaf Pine, Pinus palustris

Sterols in germinating embryos and young seedlings of longleaf pine (Pinus palustris Mill.) were identified and quantities determined for different periods after germination. Sterol analyses were performed by gas-liquid chromatography (GLC) and verified by combination of GLC-mass spectrometry. Campesterol and β-sitosterol were two major sterols which accounted for most of the sterol composition while stigmasterol was present in very small amounts. No cholesterol was revealed by GLC-mass spectrometry although there was a minor peak appearing on the sterol gas-liquid chromatograms with a retention time close to that of authentic cholesterol. By fractionation, three different forms of sterols were obtained: steryl esters, steryl glycosides, and free sterols. The sterols were mainly found in the esterified fraction, while steryl glycosides and free sterols only made up a small portion of the total sterol value. The total sterol content in general increased during seedling development, and this increase reflected mainly a change in steryl esters. The low levels of both free and glycosidic sterols remained nearly unchanged throughout the experimental germination period.     

Sterol Composition of the Arthrospores and Mycelium of the Fungus Mucor hiemalis

Sterol composition of the arthrospores and mycelium of the fungus Mucor hiemalis 1156 was studied by the method of chromatography–mass spectrometry. Along with ergosterol, the major sterol of the culture studied, ten minor sterols were identified, which were either precursors or products of ergosterol degradation. The content of individual sterols differed substantially in arthrospores and mycelium, which represent different stages of ontogenetic development of the fungus. In arthrospores, the content of ergosterol was lower than in mycelium (55.9 and 78.0%, respectively). Among the precursors of ergosterol, methylated sterols predominated in arthrospores (24.1% versus 11.6% in mycelium). Eburicol and 4,4-dimethylfecosterol were the major methylated sterols of arthrospores (10.6 and 8.1%, respectively). In addition, two uncommon and extremely rare sterols, 1-dihydro-dehydroneoergosterol and dehydroneoergosterol, were identified (for the first time in M. hiemalis). These substances, containing a complex system of conjugated double bonds in their A and B rings, are the products of ergosterol degradation. The data on sterol composition are discussed in terms of their morphogenetic implication.     

Dietary sterol preference in the silkworm, Bombyx mori

Since insects are unable to biosynthesize sterols de novo, sterols must be obtained from dietary sources. Although it has been reported that beta-sitosterol is crucial for larval growth in the silkworm, Bombyx mori, little has been investigated concerning the dietary selection of sterols by Bombyx larvae. Here, we demonstrate that Bombyx larvae have the following sterol preference: beta-sitosterol > ergosterol > cholesterol = stigmasterol. Interestingly, Bombyx larvae preferred ergosterol, an inhibitory sterol on larval growth, indicating that sterol selection following first contact of the diet with the mouth part might be different from the sterol recognition mechanism present in sterol metabolism.     

The role of ABC proteins Aus1p and Pdr11p in the uptake of external sterols in yeast: dehydroergosterol fluorescence study

Uptake of external sterols in the yeast Saccharomyces cerevisiae is a multistep process limited to anaerobiosis or heme deficiency. It includes crossing the cell wall, insertion of sterol molecules into plasma membrane and their internalization and integration into intracellular membranes. We applied the fluorescent ergosterol analog dehydroergosterol (DHE) to monitor the initial steps of sterol uptake by three independent approaches: fluorescence spectroscopy, fluorescence microscopy and sterol quantification by HPLC. Using specific fluorescence characteristics of DHE we showed that the entry of sterol molecules into plasma membrane is not spontaneous but requires assistance of two ABC (ATP-binding cassette) pumps – Aus1p or Pdr11p. DHE taken up by uptake-competent hem1ΔAUS1PDR11 cells could be directly visualized by UV-sensitive wide field fluorescence microscopy. HPLC analysis of sterols revealed significant amounts of exogenous ergosterol and DHE (but not cholesterol) associated with uptake-deficient hem1Δaus1Δpdr11Δ cells. Fluorescent sterol associated with these cells did not show the characteristic emission spectrum of membrane-integrated DHE. The amount of cell-associated DHE was significantly reduced after enzymatic removal of the cell wall. Our results demonstrate that the yeast cell wall is actively involved in binding and uptake of ergosterol-like sterols.     

Sterols of Amphidinium species in the Operculatum Clade: Predominance of cholesterol instead of Δ8(14) sterols previously considered Amphidinium-specific biomarkers

The dinoflagellates Amphidinium carterae and Amphidinium corpulentum have been previously characterized as having Δ⁸⁽¹⁴⁾-nuclear unsaturated 4α-methyl-5α-cholest-8(14)-en-3β-ol (C_28:1) and 4α-methyl-5α-ergosta-8(14),24(28)-dien-3β-ol (amphisterol; C_29:2) as predominant sterols, where they comprise approximately 80% of the total sterol composition. These two sterols have hence been considered as possible major sterol biomarkers for the genus. Here, we have examined the sterols of four recently identified species of Amphidinium (Amphidinium fijiense, Amphidinium magnum, Amphidinium theodori, and Amphidinium tomasii) that are closely related to Amphidinium operculatum as part of what is termed the Operculatum Clade to show that each species has its sterol composition dominated by the common dinoflagellate sterol cholesterol (cholest-5-en-3β-ol; C_27:1), which is found in many other dinoflagellate genera, rather than Δ⁸⁽¹⁴⁾ sterols. While the Δ⁸⁽¹⁴⁾ sterols 4α-methyl-5α-cholest-8(14)-en-3β-ol and 4α,23,24-trimethyl-5α-cholest-8(14),22E-dien-3β-ol (C_30:2) were present as minor sterols along with another common dinoflagellate sterol, 4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol (dinosterol; C_30:1), in some of these four species, amphisterol was not conclusively observed. From a chemotaxonomic perspective, while this does reinforce the genus Amphidinium's ability to produce Δ⁸⁽¹⁴⁾ sterols, albeit here as minor sterols, these results demonstrate that caution should be used when considering Δ⁸⁽¹⁴⁾ sterols, especially amphisterol, as Amphidinium-specific biomarkers within these species where cholesterol is the predominant sterol.     

STEROLS OF PORPHYRIDIUM(RHODOPHYTA)1,2

Species of the unicellular Porphyridium have been examined for their sterol content. Clones of 4 species maintained in axenic, chemically-defined culture were analyzed—these included P. sordidum Geitler, P. purpureum (Bory) Ross, P. aerugineum Geitler and P. violaceum Kormnann(P. griseum Geitler was not available to use for examination). The major sterol was 22-dehydrocholesterol in all except P. aerugineum in which there was a mixture of this sterol, cholesterol and higher sterols. Traces of C₂₈ and C₂₉ sterols were detected in most instances as well.     

Fine measurement of ergosterol requirements for growth of Saccharomyces cerevisiae during alcoholic fermentation

Yeasts can incorporate a wide variety of exogenous sterols under strict anaerobiosis. Yeasts normally require oxygen for growth when exogenous sterols are limiting, as this favours the synthesis of lipids (sterols and unsaturated fatty acids). Although much is known about the oxygen requirements of yeasts during anaerobic growth, little is known about their exact sterol requirements in such conditions. We developed a method to determine the amount of ergosterol required for the growth of several yeast strains. We found that pre-cultured yeast strains all contained similar amounts of stored sterols, but exhibited different ergosterol assimilation efficiencies in enological conditions [as measured by the ergosterol concentration required to sustain half the number of generations attributed to ergosterol assimilation (P₅₀)]. P₅₀ was correlated with the intensity of sterol synthesis. Active dry yeasts (ADYs) contained less stored sterols than their pre-cultured counterparts and displayed very different ergosterol assimilation efficiencies. We showed that five different batches of the same industrial Saccharomyces cerevisiae ADY exhibited significantly different ergosterol requirements for growth. These differences were mainly attributed to differences in initial sterol reserves. The method described here can therefore be used to quantify indirectly the sterol synthesis abilities of yeast strains and to estimate the size of sterol reserves.     

Sterol Composition and Ecdysteroid Content of Eggs of the Root-knot Nematodes Meloidogyne incognita and M. arenaria

Free and esterified sterols of eggs of the root-knot nematodes Meloidogyne incognita races 2 and 3 and M. arenaria race 1 were isolated and identified by gas-liquid chromatography-mass spectrometry. The major sterols of eggs of each race were 24-ethylcholesterol (33.4-38.8% of total sterol), 24-ethylcholestanol (18.3-25.3%), 24-methylcholesterol (8.6-11.7%), 24-methylcholestanol (7.7-12.5%), and cholesterol (4.6-11.6%). Consequently, the major metabolic transformation performed by Meloidogyne females or eggs upon host sterols appeared to be saturation of the sterol nucleus. The free and esterified sterols of the same race did not differ appreciably, except for a slight enrichment of the steryl esters in cholesterol. Although the sterol composition of Meloidogyne eggs differed from that of other life stages of other genera of plant-parasitic nematodes, the three Meloidogyne races could not be distinguished from each other by their egg sterols. Ecdysteroids, compounds with hormonal function in insects, were not detected by radioimmunoassay in the Meloidogyne eggs either as free ecdysteroids or as polar conjugates.     

Sterol Accumulation and Composition in Developing Zea mays L. Kernels

Kernels were collected from three maize (Zea mays L.) inbreds from 10 days after pollination until kernel maturity. Sitosterol, campesterol, and stigmasterol were the major sterols at all stages of kernel development. Cholesterol was less than 1% of the dry weight. The three major sterols accumulated during kernel development, but at a rate slower than dry weight. The ratio of the sterols did not vary greatly among the inbreds. At maturity, the three inbreds, Wf9, Oh43, and Ky226, had sterol levels of 325, 228, and 173 micrograms per kernel, respectively. Sitosterol accounted for 75 to 85% of the sterol. The relative amount of stigmasterol decreased during the linear phase of development, while sitosterol increased in the free fraction and campesterol increased in the steryl ester fraction.     

A dynamic role for sterols in embryogenesis of Pisum sativum

Molecular roles of sterols in plant development remain to be elucidated. To investigate sterol composition during embryogenesis, the occurrence of 25 steroid compounds in stages of developing seeds and pods of Pisum sativum was examined by GC-MS analysis. Immature seeds containing very young embryos exhibited the greatest concentrations of sterols. Regression models indicated that the natural log of seed or pod fr. wt was a consistent predictor of declining sterol content during embryonic development. Although total sterol levels were reduced in mature embryos, the composition of major sterols sitosterol and campesterol remained relatively constant in all 12 seed stages examined. In mature seeds, a significant decrease in isofucosterol was observed, as well as minor changes such as increases in cycloartenol branch sterols and campesterol derivatives. In comparison to seeds and pods, striking differences in composition were observed in sterol profiles of stems, shoots, leaves, flowers and flower buds, as well as cotyledons versus radicles. The highest levels of isofucosterol, a precursor to sitosterol, occurred in young seeds and flower buds, tissues that contain rapidly dividing cells and cells undergoing differentiation. Conversely, the highest levels of stigmasterol, a derivative of sitosterol, were found in fully-differentiated leaves while all seed stages exhibited low levels of stigmasterol. The observed differences in sterol content were correlated to mRNA expression data for sterol biosynthesis genes from Arabidopsis. These findings implicate the coordinated expression of sterol biosynthesis enzymes in gene regulatory networks underlying the embryonic development of flowering plants.     

ORCHID PHYTOALEXINS. II. ISOLATION AND CHARACTERIZATION OF POSSIBLE STEROL COMPANIONS

Four sterols have been isolated from extracts of Cymbidium pseudobulbs infected with Rhizoctonia repens M 32. One of them, ergosterol peroxide, is most probably an artifact of extraction. The other three, sitosterol, stigmasterol, and campesterol, occur in a 70:25:5 ratio. Appearance of phytoalexin(s) in pseudobulb extracts coincides with increase of sterol production. This raises the question whether Cymbidium phytoalexins are related, biosynthetically or structurally, to sterols. Since the same three sterols occur (free or conjugated) in Cattleya and Arundina, but in different ratios to each other than in Cymbidium, they may be of value in chemotaxonomy.     

Sterols of the Toxic Marine Dinoflagellate,Pyrodinium bahamense

Pyrodinium bahamense is a dinoflagellate of concern in subtropical and tropical coastal environments. To date, there is only a single published study on its fatty acids, but no published data on its sterol composition. Sterols, which are membrane‐reinforcing lipids in eukaryotes, display a great diversity of structures in dinoflagellates, with some serving as chemotaxonomic markers. We have examined the sterol compositions of two isolates of P. bahamense from Indian River Lagoon and Tampa Bay, Florida, and have found both to produce three sterols: cholesterol, dinosterol, and 4α‐methylgorgostanol. All three sterols are found in closely related, armored taxa.     

Permeability and membrane sterol distribution in Saccharomyces uvarum and Kluyveromyces bulgaricus grown in presence of polyoxyalkylene glycol-oleic acid condensates

Summary Antifoam agents, such as polyoxyalkylene glycol-oleic acid condensates, increase cell permeability in Saccharomyces uvarum, but decrease cell permeability in Kluyveromyces bulgaricus.In S. uvarum it was found that the increase in cell permeability is related to a significantly higher level of total sterols. In K. bulgaricus, in which a decrease of permeability was observed, the overall level of sterols is lower.Determination of the sterol derivatives showed that in S. uvarum the content of all sterols identified was increased; in K. bulgaricus only the ergosterol content was increased. The difference in behaviour of the two yeasts grown in the presence of antifoam agents could be attributed to an effect of these agents on sterol biosynthesis.     

A SURVEY OF THE STEROL COMPOSITION OF THE MARINE DINOFLAGELLATES KARENIA BREVIS,KARENIA MIKIMOTOI,AND KARLODINIUM MICRUM: DISTRIBUTION OF STEROLS WITHIN OTHER MEMBERS OF THE CLASS DINOPHYCEAE1

The sterol composition of different marine microalgae has been examined to determine the utility of sterols as biomarkers to distinguish members of various algal classes. For example, members of the class Dinophyceae possess certain 4‐methyl sterols, such as dinosterol, which are rarely found in other classes of algae. The ability to use sterol biomarkers to distinguish certain dinoflagellates such as the toxic species Karenia brevis Hansen and Moestrup, responsible for red tide events in the Gulf of Mexico, from other species within the same class would be of considerable scientific and economic value. Karenia brevis has been shown by others to possess two major sterols, (24S)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol (ED) and its 27‐nor derivative (NED), having novel structures not previously known to be present in other dinoflagellates. This prompted the present study of the sterol signatures of more than 40 dinoflagellates. In this survey, sterols with the properties of ED and NED were found in cultures of K. brevis and shown also to be the principal sterols of Karenia mikimotoi Hansen and Moestrup and Karlodinium micrum Larsen, two dinoflagellates closely related to K. brevis. They are also found as minor components of the more complex sterol profiles of other members of the Gymnodinium/Peridinium/Prorocentrum (GPP) taxonomic group. The distribution of these sterols is consistent with the known close relationship between K. brevis, K. mikimotoi, and K. micrum and serves to limit the use of these sterols as lipid biomarkers to a few related species of dinoflagellates.