Utilization and metabolism of dietary sterols in the honey bee and the yellow fever mosquito

The honey bee,Apis mellifera, does not convert C₂₈ and C₂₉ phytosterols to cholesterol as found in most previous studies of phytophagous or omnivorous insects, but instead the workers and queens selectively transfer 24-methylenecholesterol, sitosterol and isofucosterol from their endogenous sterol pools to the brood larvae regardless of the sterol in the worker diet. Administering radiolabeled sterols by feeding and injection has made it possible to trace this selective transfer through a second generation of the honey bee. In further comparative sterol metabolism studies, the yellow fever mosquito,Aedes aegypti, was shown to be capable of dealkylating and converting a radiolabeled C₂₉ dietary sterol ([¹⁴C] sitosterol) to cholesterol. Metabolic studies with several radiolabeled dietary sterols and an inhibitor of steroid metabolism in the yellow fever mosquito further verified this capability.     

Comparative studies of metabolism of 4-desmethyl, 4-monomethyl and 4,4-dimethyl sterols inManduca sexta

To investigate the metabolism and possible deleterious effects of 4-methyl and 4,4-dimethyl steroids inManduca sexta, the 4,4-dimethyl sterols lanosterol and cycloartenol, the 4-methyl sterol obtusifoliol and the 4,4-dimethyl pentacyclic triterpenoid α-amyrin were fed in an artificial agar-based diet at various concentrations. Utilization and metabolism of these four compounds were compared with sitosterol, stigmasterol, brassicasterol, ergosterol and 24-methylenecholesterol, 24-alkyl sterols that are readily dealkylated and converted to cholesterol inManduca and in most phytophagous insects. None of the 4-methylated compounds significantly inhibited development except at very high dietary concentrations. The Δ²⁴-bonds of lanosterol and cycloartenol were effectively reduced by theManduca Δ²⁴-sterol reductase enzyme, as is the Δ²⁴-bond of desmosterol which, in most phytophagous insects, is an intermediate in the conversion of sitosterol, stigmasterol and other C₂₈ and C₂₉ phytosterols to cholesterol. On the other hand, the 24-methylene substituent of obtusifoliol was not dealkylated. Each of the 4-desmethyl C₂₈ and C₂₉ sterols was readily converted to cholesterol, and a significant amount of 7-dehydro-cholesterol was derived from ergosterol metabolism. The reason for the differences in substrate specificity of these sterols is not clear, but the information may be useful in the development of new, specific, mechanism-based inhibitors of sterol metabolism.     

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.     

Selective sterol transfer in the honey bee: Its significance and relationship to other hymenoptera

The honey bee,Apis mellifera, is one of only a few species of phytophagous insects known to be unable to convert C-24 alkyl phytosterols to cholesterol. Regardless of the dietary sterols available to worker bees, the major tissue sterol of brood reared by the workers is always 24-methylenecholesterol, followed by sitosterol and isofucosterol. Normally, little or no cholesterol is present in honey bee sterols. The maintenance of high levels of certain sterols is accomplished through a selective transfer of sterols from the endogenous sterol pools of the workers to the developing larvae through the brood food material secreted from the hypopharyngeal and mandibular glands and/or the honey stomach of the workers. The selective uptake and transfer of radiolabeled C₂₇, C₂₈ and C₂₉ sterols have been studied to correlate these aspects of sterol utilization with the discovery of an unusual molting hormone (ecdysteroid) in honey bee pupae as the major ecdysteroid of this stage of development. The phylogenetic implications of this selective transfer phenomenon in the honey bee and comparison with sterol metabolism in certain other hymenopteran species emphasize the diversity of steroid biochemistry in insects.     

Neutral sterols of sawflies (symphyta): Their relationship to other hymenoptera

To investigate sterol utilization in sawflies, the neutral sterols of four species of sawflies were determined by gas-liquid chromatography and mass spectrometry, and compared to the respective dietary plant material. Cholesterol was the predominant (55–76%) sterol in all species and stages of sawflies examined. Host plants, however, contained primarily sitosterol (50–88%), along with other 24-alkylsterols and only 0.5–5.9% cholesterol, indicating that the sawflies examined are capable of dealkylating the C₂₈ and C₂₉ phytosterols in their diet to cholesterol. Comparative sterol metabolism in Hymenoptera is discussed.     

Sterol composition and phytosterol utilization and metabolism in the milkweed bug

Analysis of the sterols of the milkweed bug,Oncopeltus fasciatus (Dallas) and dietary sunflowerseeds revealed that there is little, if any, conversion of dietary C₂₈ or C₂₉ phytosterols to cholesterol in this phytophagous insect. The dietary sterols are apparently utilized with little alteration both during development to the adult stage and egg production, and cholesterol comprises <1% of the sterols in either adult males and females or in the eggs. The significance of these findings are discussed in light of the recent discovery that the C₂₈-ecdysone, makisterone A, is the predominant molting hormone in the enbryonated egg of the milkweed bug.     

Impact of 10 Dietary Sterols on Growth and Reproduction of Daphnia galeata

In crustaceans, cholesterol is anEssential nutrient, which they must directly obtain from their food or by bioconversion from other dietary sterols. Eukaryotic phytoplankton contain a great variety of sterols that differ from cholesterol in having additional substituents or different positions and/or number of double bonds in the side chain or in the sterol nucleus. In this study, we investigated to what extent these structural features affect the growth and reproduction of Daphnia galeata in standardized growth experiments with the cyanobacterium Synechococcus elongatus supplemented with single sterols as food source. The results indicated that ⁵ (sitosterol, stigmasterol,Desmosterol) and ^5,7 (7-dehydrocholesterol, ergosterol) sterols meet the nutritional requirements of the daphnids, while the ⁷ sterol lathosterol supports somatic growth and reproduction to a significantly lower extent than cholesterol. Dihydrocholesterol (⁰) and lanosterol (⁸) did not improve the growth of D. galeata, and growth was adversely affected by the ⁴ sterol allocholesterol. Sterols seem to differ in their allocation to somatic growth and reproduction. Thus, structural differences of dietary sterols have pronounced effects on life-history traits of D. galeata.     

Growth support and metabolism of phytosterols inParamecium tetraurelia

The basis of the growth requirement ofParamecium for one of several structurally similar phytosterols is not known. Previous research has indicated that selective esterification of only growth-promoting sterols may be a key. In this study, it was found that under certain conditions sterols that fail to support growth (e.g., cholesterol) can be esterified in large amounts inParamecium. We found no compelling evidence to support the hypothesis that steryl esters serve a specialized role in the fatty acid metabolism of the cell. Octadecenoic acid, essential for cell growth, was the major fatty acid in both steryl esters and triglycerides. It was also shown thatP. tetraurelia can dehydrogenate Δ⁰ and Δ⁷, as well as Δ⁵-3β-hydroxy sterols, to yield the conjugated 5,7-diene derivative. These results indicate the presence of a Δ⁵, in addition to a Δ⁷, desaturase of the sterol nucleus in this ciliate. Two C₂₄ α-ethyl sterols, Δ²²-stigmasterol (Δ²²) and stigmastanol (Δ⁰), were shown for the first time to promote growth. Finally, we found that non-growth-promoting sterols may compose a high percentage of the free sterols of the surface membrane without adversely affecting cell growth or viability. These data support the conclusion that the growth requirement for select phytosterols inParamecium does not involve the structural or functional role of “bulk” sterols in cell membranes.     

Neutral sterol metabolism in insects

Since they are unable to biosynthesize sterols, many phytophagous and omnivorous insects satisfy their cholesterol requirement by side chain dealkylation of the C-24 alkyl group of dietary C₂₈ and C₂₉ phytosterols. However, not all insects that can dealkylate the phytosterol side chain produce cholesterol. In addition, certain insects,e.g., some Hymenoptera, Hemiptera, and Diptera, are unable to dealkylate the sterol side chain. Although C₂₇ ecdysteroids (molting hormones), which are biosynthesized from cholesterol, are the major ecdysteroids in most insects, many of those species that are unable to dealkylate phytosterols utilize campesterol as a precursor for the C₂₈ ecdysteroid makisterone A. The considerable diversity of steroid utilization between certain insect species makes it difficult to generalize about insect steroid biochemistry. The ability to disrupt certain unique aspects of steroid utilization and metabolism in insects might be exploited for developing new insect control technology. 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.     

The relationship between dietary phytosterols and the sterols of wild and cultivated oysters

Wild oysters (Crassostrea virginica) contained cholesterol, 24-methyl-cholesta-5, 22-dienol, 24-methylenecholesterol, 22-dehydrocholesterol, 24-methylcholesterol, 24-ethylcholesterol, 24-norcholesta-5, 22-dienol, 24-ethylcholesta-5, 22-dienol and fucosterol. The same species was cultivated on a defined diet ofThalassiosira pseudonana andIsochrysis sp. The dietary algae were cultured and their sterol compositions were analyzed by gas chromatography and mass spectroscopy.T. pseudonana andIsochrysis sp. had 24-methylenecholesterol and 24-methyl-cholesta-5, 22-dienol as their major sterols. The sterol composition of the cultivated oysters revealed the predominance of cholesterol (19%), 24-methyl-cholesta-5, 22-dienol (21%) and 24-methylenecholesterol (46%). Therefore, oysters must be able to bioconvert phytosterols to cholesterol, concentrate dietary cholesterol, or synthesize cholesterol de novo.     

Sterols of the phylum Zygomycota: Phylogenetic implications

The sterol composition of 42 fungal species representing six of the eight orders of the Zygomycota was determined using gas-liquid chromatography-mass spectrometry to assess whether the distribution of major sterols in this phylum has taxonomic or phylogenetic relevance. Ergosterol, 22-dihydroergosterol, 24-methyl cholesterol, cholesterol, and desmosterol were detected as the major sterols among the species studied. Ergosterol was the major sterol of the Dimargaritales, Zoopagales, and 13 of the 14 Mucorales families included in this study. Desmosterol appeared to be the characteristic sterol of the Mortierellaceae (Mucorales). 24-Methyl cholesterol was the major sterol of the Entomophthorales genera Entomophthora, Conidiobolus and Basidiobolus, but cholesterol was the sole sterol detected in Delacroixia coronatus. The Kickxellales species analyzed in this study were characterized by 22-dihydroergosterol as the major sterol. These results suggest that certain orders of the Zygomycota may be distinguished on the basis of major sterol. Also, if sterol structure has phylogenetic implications, then orders might be arranged in the order Kickxellales (C₂₈Δ^5,7) → Dimargaritales, Zoopagales and Mucorales (C₂₈Δ^5,7,22) on the basis of evolution of the predominant and presumably most competent sterol, ergosterol. Although the Entomophthorales would be expected to be more primitive than the above orders based on the predominance of C₂₈Δ^5,, it is not apparent from these data that members of the Zygomycota with ergosterol or its precursors as major sterols evolved from this taxon or the Chytridiomycota.     

Effect of Plant Sterols and Tannins on Phytophthora ramorum Growth and Sporulation

Elicitin-mediated acquisition of plant sterols is required for growth and sporulation of Phytophthora spp. This study examined the interactions between elicitins, sterols, and tannins. Ground leaf tissue, sterols, and tannin-enriched extracts were obtained from three different plant species (California bay laurel, California black oak, and Oregon white oak) in order to evaluate the effect of differing sterol/tannin contents on Phytophthora ramorum growth. For all three species, high levels of foliage inhibited P. ramorum growth and sporulation, with a steeper concentration dependence for the two oak samples. Phytophthora ramorum growth and sporulation were inhibited by either phytosterols or tannin-enriched extracts. High levels of sterols diminished elicitin gene expression in P. ramorum; whereas the tannin-enriched extract decreased the amount of ‘functional’ or ELISA-detectable elicitin, but not gene expression. Across all treatment combinations, P. ramorum growth and sporulation correlated strongly with the amount of ELISA-detectable elicitin (R ²?=?0.791 and 0.961, respectively).     

Inhibition of hepatic sterol and squalene biosynthesis in rats fed di-2-ethylhexyl phthalate

Di-2-ethylhexyl phthalate (DEHP), a commonly used plasticizer, was found to be an inhibitor of the biosynthesis of hepatic nonsaponifiable lipids in the rat. The addition of DEHP at levels of 0.5% or 1.0% to a stock diet of rats resulted in a decreased conversion of acetate-1-¹⁴C and mevalonate-5-³H into squalene, C₃₀ sterols, and C₂₇ sterols by liver minces or slices, in vitro. In studies conducted with 0.5% DEHP feeding from 2 to 11 days, the degree of inhibition was found to increase with the duration of DEHP feeding; the inhibition of³H-mevalonate conversion to squalene and sterols developed more slowly, being reduced to ca. 70% of control values in 11 days, whereas¹⁴C-acetate conversion was reduced to ca. 35% of control values during the same period.³H-mevalonate conversion to sterols and squalene was, however, found to be suppressable to the same extent as¹⁴C-acetate conversion when diets containing 1.0% DEHP were fed for 18 days. The inhibitory effect of dietary DEHP on sterol and squalene biosynthesis from¹⁴C-acetate and³H-mevalonate by rat liver preparations is unlikely to be accounted for by the negative feedback of cholesterol secondary to hepatic sterol accumulation since, in these studies, hepatic total lipid and hepatic total sterol levels were simialr in control and DEHP-fed rats.     

Effects of cholestyramine and squalene feeding on hepatic and serum plant sterols in the rat

Hepatic and serum phytosterol concentrations were compared in the rat under basal conditions and during activated cholesterol and bile acid production due to squalene and cholestyramine feeding. Both treatments consistently decreased hepatic and serum levels of sitosterol and campesterol and, unlike esterified cholesterol, esterified plant sterols were not increased in liver during squalene feeding. Serum levels of phytosterols were decreased quite proportionately to those in the liver. The hepatic levels of sitosterol and campesterol closely correlated with each other, but not with cholesterol levels. The percentage esterification of both phytosterols was lower than that of cholesterol. The results indicate that activation of hepatic sterol production leads to depletion of hepatic plant sterols. It is suggested that poor esterification of plant sterols may contribute to this decrease.     

Same Host-Plant,Different Sterols: Variation in Sterol Metabolism in an Insect Herbivore Community

Insects lack the ability to synthesize sterols de novo, which are required as cell membrane inserts and as precursors for steroid hormones. Herbivorous insects typically utilize cholesterol as their primary sterol. However, plants rarely contain cholesterol, and herbivorous insects must, therefore, produce cholesterol by metabolizing plant sterols. Previous studies have shown that insects generally display diversity in phytosterol metabolism. Despite the biological importance of sterols, there has been no investigation of their metabolism in a naturally occurring herbivorous insect community. Therefore, we determined the neutral sterol profile of Solidago altissima L., six taxonomically and ecologically diverse herbivorous insect associates, and the fungal symbiont of one herbivore. Our results demonstrated that S. altissima contained Δ⁷-sterols (spinasterol, 22-dihydrospinasterol, avenasterol, and 24-epifungisterol), and that 85% of the sterol pool existed in a conjugated form. Despite feeding on a shared host plant, we observed significant variation among herbivores in terms of their qualitative tissue sterol profiles and significant variation in the cholesterol content. Cholesterol was absent in two dipteran gall-formers and present at extremely low levels in a beetle. Cholesterol content was highly variable in three hemipteran phloem feeders; even species of the same genus showed substantial differences in their cholesterol contents. The fungal ectosymbiont of a dipteran gall former contained primarily ergosterol and two ergosterol precursors. The larvae and pupae of the symbiotic gall-former lacked phytosterols, phytosterol metabolites, or cholesterol, instead containing an ergosterol metabolite in addition to unmetabolized ergosterol and erogsterol precursors, thus demonstrating the crucial role that a fungal symbiont plays in their nutritional ecology. These data are discussed in the context of sterol physiology and metabolism in insects, and the potential ecological and evolutionary implications.     

Inhibition of sterologenesis in brain and liver of fetal and suckling rats from dams fed di-2-ethylhexyl phthalate plasticizer

The effect of di-2-ethylhexyl phthalate (DEHP) on lipid metabolism was studied in liver and brain from fetal rats taken 3 days before parturition from dams receiving dietary DEHP during gestation. In fetuses from rats receiving 0.5% or 1.0% DEHP in a stock diet, the incorporation of¹⁴C-acetate and labeled mevalonate (³H or¹⁴C) into the C₂₇ sterols, C₃₀ sterols, and squalene fractions of brain tissue incubated in vitro was significantly reduced between the confidence limits P<0.05 to P<0.001. When liver from fetuses was incubated with labeled mevalonate, incorporation of label into the C₂₇ sterol and C₃₀ sterol fractions was significantly reduced as well (P<0.02 to P<0.001), whereas incorporation of labeled mevalonate into the squalene fraction was not significantly altered. The incorporation of¹⁴C-acetate into total hepatic lipids of the fetal rats was also studied, and statistically significant reductions in incorporation were observed in the lanosterol fraction (P<0.001), the combined fraction of sterol esters + squalene (P<0.02), and the combined fraction of cholesterol + diglycerides (P<0.01). No significant changes were observed in the incorporation of¹⁴C-acetate into phospholipids, free fatty acids, or triglycerides. In 8-day old suckling rats delivered from dams fed 0.5% DEHP for the last 16 days of gestation and maintained on the same diet during the nursing period, the incorporation of¹⁴C-mevalonate into hepatic C₂₇ sterols, in vitro, was significantly depressed (P<0.05) whereas in corporation into C₃₀ sterols and squalene was similar to control values. In these same suckling rats, body weights were significantly lower in the control group (21.7 vs. 18.8 g, P<0.01), whereas liver weight as a % of body weight was significantly higher (P<0.01) in rats nursing from the DEHP-fed dams. The results indicate that the inhibitory effect of dietary DEHP on lipid metabolism in the mature rat is transmitted across the placental barrier to the developing fetus and that the abnormal pattern of lipid metabolism in rats delivered from DEHP-fed females is only partially restored to normal during the suckling periods.     

Distribution of sterols in the fungi I. Fungal spores

The freely extractable sterols of spores ofLinderina pennispora, Spicaria elegans, Penicillium claviforme, Aspergillus niger, Ustilago nuda, U. maydis, Puccinia graminis, andP. striiformis were examined using mass spectrometric techniques. Each species contained at least 3–5 detectable sterol components in the 4-desmethyl sterol fraction, and, when present, ergosterol was generally the most abundant sterol produced by an individual species. Smaller relative concentrations of fungisterol (ergost-Δ⁷-enol) di- and tetraunsaturated C₂₈ sterols also were found. In some species, fungisterol was the most abundant sterol. In uredospores of rust fungi, stigmast-Δ⁷-enol (C₂₉) was predominant and was accompanied by lower relative concentrations of a diunsaturated C₂₉ sterol and fungisterol. Cholesterol was found only in the teliospores of the corn smut fungus (U. maydis). Application of glass capillary columns to the separation of yeast sterols by gas liquid chromatography is illustrated. One of eight papers presented in the symposium “Phytosterols,” AOCS Spring Meeting, New Orleans, April 1973.     

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.     

Bioorthogonal Probes for Imaging Sterols in Cells

Cholesterol is a fundamental lipid component of eukaryotic membranes and a precursor of potent signaling molecules, such as oxysterols and steroid hormones. Cholesterol and oxysterols are also essential for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Despite their importance, the use of imaging sterols in cells is currently very limited. We introduce a robust and versatile method for sterol microscopy based on C₁₉ alkyne cholesterol and oxysterol analogues. These sterol analogues are fully functional; they rescue growth of cholesterol auxotrophic cells and faithfully recapitulate the multiple roles that sterols play in Hedgehog signal transduction. Alkyne sterol analogues incorporate efficiently into cellular membranes and can be imaged with high resolution after copper(I)‐catalyzed azide–alkyne cycloaddition reaction with fluorescent azides. We demonstrate the use of alkyne sterol probes for visualizing the subcellular distribution of cholesterol and for two‐color imaging of sterols and choline phospholipids. Our imaging strategy should be broadly applicable to studying the role of sterols in normal physiology and disease.     

Insect steroid metabolism

Insects are unable to biosynthesize the steroid nucleus and generally require an exogenous source of sterols. Two salient areas of insect steroid metabolism are the dealkylation and conversion of dietary C₂₈ and C₂₉ plant sterols to cholesterol and other C₂₇ sterols, and the biosynthesis and metabolism of the steroidal insect molting hormones. Certain azasteroids and nonsteroidal amines block this conversion of 24-alkyl sterols to cholesterol and/or disrupt molting and development in insects. These inhibitors have served in charting metabolic pathways for steroids in insects and are serving as models in developing selective pesticidal chemicals and chemotherapeutic agents for use against insects and other invertebrate pests and parasites. The mode of action of some of these inhibitors on molting and development has been investigated in vivo and in vitro. Certain of these inhibitors represent a new class of insect hormonal compounds with a novel mode of action—the disruption of molting hormone metabolism. Research on sterol metabolism in insects provides important information on the comparative biochemistry and physiological functions of steroids in living systems.