Sterol utilization in honey bees fed a synthetic diet: Analysis of prepupal sterols

The sterols of prepupal honey bees, Apis mellifera L., from brood reared by workers fed chemically-defined synthetic diets containing cholesterol, campesterol, sitosterol, stigmasterol, 24-methylenecholesterol, or no sterol over a 12-week period were isolated, identified, and quantified. The major sterol present in each prepupal sample was 24-methylenecholesterol, but significant levels of sitosterol and isofucosterol were also present in every case, as was a very small percentage of desmosterol (usually < 1%). This is the first report of isofucosterol being identified in the sterols of the honey bee. A considerably larger percentage of each dietary sterol was found in prepupae reared by workers fed that particular sterol in the diet. This was most dramatic in the case of the cholesterol diet in which case cholesterol content increased to as much as 17.2% of the prepupal sterols, whereas cholesterol had not exceeded 2.2% in samples from other diet regimens. However, stigmasterol comprised no more than 6.3% of the total sterols in any sample from prepupae fed the stigmasterol diet. The preponderance of 24-methylenecholesterol in all prepupae, regardless of the dietary sterol provided to the workers, as well as the lesser quantities of sitosterol and isofucosterol present in all samples, suggest a unique system of utilization and metabolism of these dietary sterols by the worker bees. Apparently they make available to the brood varying amounts of unchanged dietary sterol plus considerable and fairly constant portions of 24-methylenecholesterol, sitosterol, and isofucosterol drawn from their own sterol pools.     

Biosynthesis of makisterone A and 20-hydroxyecdysone from labeled sterols by the honey bee,Apis mellifera

In an effort to determine the sterol precursor(s) of the 28-carbon ecdysteroid, makisterone A, honey bee pupae (13 days post-oviposition) were injected with radiolabeled sterols and subsequently examined for labeled ecdysteroids. High performance liquid chromatography of the pupal extracts revealed that [³H]campesterol was converted to a compound that behaved chromatographically identical to authentic makisterone A, and [¹⁴C]cholesterol was incorporated into a compound chromatographically like 20-hydroxyecdysone. No incorporation of either 24-[³H]methylenecholesterol or [¹⁴C]sitosterol into an ecdysteroid was observed. The neutral sterols of uninjected honey bee pupae contained 49.8% 24-methylenecholesterol on a relative percent basis and, with three other C²⁸ and C²⁹ sterols, accounted for over 99% of the total sterols present.     

Effects of dietary sterols on development and reproduction of southwestern corn borer Diatraea grandiosella Dyar

Southwestern corn borer larvae, Diatraea grandiosella Dyar, were reared on artificial diets containing individual sterols (cholesterol, sitosterol, or stigmasterol) in concentrations ranging from 0.05 to 0.2%. Female larvae developed to pupae more rapidly as sitosterol and stigmasterol were increased in the diets. Increased cholesterol concentrations did not affect the larval period significantly, and development was not as rapid as with the phytosterols. Female larvae developed at significantly slower rates in all diets than did males, except at the highest concentrations of sitosterol and stigmasterol. Female pupae and adults were significantly heavier than the males, and pupal and adult weight increased as sterol concentrations increased. Number of eggs laid per fertilized female and egg hatchability were significantly increased as concentrations of the three sterols were increased in the larval diets. Sitosterol-reared females produced more eggs than did females reared on other sterols but egg hatchability was not significantly different among sterols.     

Makisterone C: A 29-carbon ecdysteroid from developing embryos of the cotton stainer bug,Dysdercus fasciatus

An ecdysteroid RIA was used to determine the ecdysteroid titer in developing embryos of Dysdercus fasciatus and revealed that peak titer occurred approximately 120 h post-oviposition. Analysis of neutral sterols at this time indicated sitosterol to be the predominant neutral sterol with lesser amounts of campesterol. Embryonic sterols were highly reflective of the sterols found in the cotton seed diet upon which previous generations of the bugs had fed. Analysis of the embryonic extract for ecdysteroids indicated the presence of both makisterone A and the 29-carbon ecdysteroid makisterone C. Isolation of these compounds was accomplished by reversed-phase and silica HPLC in conjunction with RIA, and the identification of both compounds was confirmed by mass spectrometry. No ecdysone or 20-hydroxyecdysone was detected in the embryonic sample.     

The fate of radiolabelled C28 and C29 phytosterols in the honey bee

The fate of radiolabelled campesterol, sitosterol and 24-methylenecholesterol fed in chemically-defined diets to honey bee (Apis mellifera L.) workers was determined. At various intervals, sterols of prepupae, newly emerged adults and queens were analyzed qualitatively, quantitatively and radiochemically and it was determined that there was not sufficient radioactivity associated with cholesterol and/or desmosterol in any of the samples to verify that any of the three C₂₈ and C₂₉ sterols was dealkylated and converted to cholesterol. Similarly, there was no evidence for the conversion of campesterol or sitosterol to 24-methylenecholesterol. It was concluded that the major portion of the sterols incorporated into the tissues of the brood larvae originated from the worker bees used to establish the colony. There is good evidence supporting the premise that the workers can make available sterols from their endogenous pools to the nutrient in the hive and that they can replenish these sterols with those from the artificial diet. The queen is also able to replenish sterols utilized in egg production from those obtained by the workers from the artificial diet, and at the end of nine weeks queens contained more than four times as much sterol, on a ‘μg sterol per g fresh weight’ basis, than was found in fertile queens at the beginning of the test period.     

A reappraisal of sterol biosynthesis and metabolism in aphids

Aphids of Schizaphis graminum (Rondani) (biotype C) reared on its host-plant, Sorghum bicolor (L.) Moench, sequestered campesterol, stigmasterol and sitosterol. Aphids reared for 72 hr on holidic diets supplemented with [4-¹⁴C]-sitosterol contained both [¹⁴C]-sitosterol and [¹⁴C]-cholesterol, indicating that these aphids are capable of dealkylation at C-24. When aphids were reared on artificial diets containing [2-¹⁴C]-mevalonic acid, no detectable amounts of radioactively labelled desmethyl sterols, nor metabolic intermediates in sterol synthesis (i.e. squalene, 2,3-oxidosqualene, 4,4-dimethyl and 4-monomethyl sterols) were found to accumulate in their tissues. The relevance of these findings to previous research suggesting the ability of aphids, via their symbiotes, to synthesize sterols is discussed.     

Sterols of roots and nitrogen-fixing root nodules of some non-leguminous species

Sterols of both roots and nodules of three species of Alnus were found to consist only of sitosterol, whereas Casuarina cunninghamiana contained substantial amounts of campesterol, stigmasterol and sitosterol. In all four cases more sterol was extracted from nodules than from roots.     

The effects of the structure of sterols on the development of Heliothis zea

Heliothis zea was reared on artificial diets which lacked supplementation with plant materials but were supplemented with different sterols in order to determine how certain structural features of a sterol molecule affect the development of this insect. We found that sitosterol and cholesterol supported a more rapid rate of growth than did campesterol. Larvae did not moult when they ingested 5-pregnen-3β-ol. Larvae utilized spinasterol more efficiently than lathosterol. Such a pronounced effect was not observed in the Δ⁵-series. The substitution of a Δ⁷-bond (spinasterol, dihydrospinasterol, lathosterol) for the Δ⁵-bond (stigmasterol, sitosterol, cholesterol) in the 24-ethyl- and desalkylsterols reduced the rate of growth of the larvae. Although larvae developed normally on cholesterol, the addition of a Δ⁷-bond to give the Δ^5,7-diene system apparently altered the functionality of the molecule because 7-dehydrocholesterol did not support larval development. The growth of larvae was also inhibited, although not prevented, when they consumed diets which contained ergosterol. In contrast, the larvae completed their development more rapidly on brassicasterol which lacked the Δ⁷-bond. Cholestanol supported the complete development of the insect. H. zea is unusual among investigated insects because it develops both on cholestanol and lathosterol but does not utilize ergosterol efficiently and fails to grow on 7-dehydrocholesterol.     

Sterol/steroid metabolism and absorption in a generalist and specialist caterpillar: Effects of dietary sterol/steroid structure,mixture and ratio

Insects cannot synthesize sterols de novo, so they typically require a dietary source. Cholesterol is the dominant sterol in most insects, but because plants contain only small amounts of cholesterol, plant-feeding insects generate most of their cholesterol by metabolizing plant sterols. Plants almost always contain mixtures of different sterols, but some are not readily metabolized to cholesterol. Here we explore, in two separate experiments, how dietary phytosterols and phytosteroids, in different mixtures, ratios, and amounts, affect insect herbivore sterol/steroid metabolism and absorption; we use two caterpillars species – one a generalist (Heliothis virescens), the other a specialist (Manduca sexta). In our first experiment caterpillars were reared on two tobacco lines – one expressing a typical phystosterol profile, the other expressing high amounts/ratios of stanols and 3-ketosteroids. Caterpillars reared on the control tobacco contained mostly cholesterol, but those reared on the modified tobacco had reduced amounts of cholesterol, and lower total sterol/steroid body profiles. In our second experiment, caterpillars were reared on artificial diets containing known amounts of cholesterol, stigmasterol, cholestanol and/or cholestanone, either singly or in various combinations and ratios. Cholesterol and stigmasterol-reared moths were mostly cholesterol, while cholestanol-reared moths were mostly cholestanol. Moth tissue cholesterol concentration tended to decrease as the ratio of dietary cholestanol and/or cholestanone increased. In both moths cholestanone was metabolized into cholestanol and epicholestanol. Interestingly, M. sexta generated much more cholestanol than epicholestanol, while H. virescens did the opposite. Finally, total tissue steroid levels were significantly reduced in moths reared on diets containing very high levels of cholestanol. We discuss how dietary sterol/steroid structural differences are important with respect to sterol/steroid metabolism and uptake, including species-specific differences.     

High levels of plant sterols and cholesterol precursors in cerebrotendinous xanthomatosis

We measured the cholestanol, cholesterol precursor (lathosterol), and plant sterol (campesterol and sitosterol) concentrations of serum and bile in 11 patients with cerebrotendinous xanthomatosis. The mean values of serum cholestanol, lathosterol, campesterol, and sitosterol were, respectively, 8.4-, 2.5-, 2.7-, and 1.4-times higher in the patients than in normal control subjects (n = 26). Cholestanol (6.7-fold) and campesterol (3.7-fold) levels in bile (n = 4) were also elevated in the patients. There was no significant difference of serum sterol levels between patients with coronary artery disease and those without it. Chenodeoxycholic acid treatment for periods ranging from 6 months to 3 years and 4 months lowered serum lathosterol (57.7% reduction) and campesterol (57.8%) levels in parallel with cholestanol (70.8%) level, but the sitosterol level (19.7%) decreased less. Thus, increased levels of cholesterol precursor (lathosterol), plant sterols (campesterol and sitosterol), and cholestanol were found in the serum and bile in cerebrotendinous xanthomatosis. Chenodeoxycholic acid treatment effectively reduced the levels of these sterols, except for sitosterol.     

Die quantitative verteilung der sterine und sterinderivate in organen von Solanum dulcamara

The following sterols were found in the roots, stems, leaves, unripe and ripe fruits of Solanum dulcamara: cholesterol, sitosterol, stigmasterol, campesterol, brassicasterol, isofucosterol and 24-methylenecholesterol. The most abundant components are cholesterol, sitosterol and stigmasterol (77–84%). In all parts of the plant the sterols are present in the free form and as esters, glycosides and acylated glycosides. The total sterol content and the content of combined forms were determined photometrically. In the leaves 58% of the sterols were found in the form of glycoside (26%), acylated glycoside (29%) and ester (2%). In the roots only 25% of the sterol were found in combined form. In the other organs the ratio of free and combined sterols was intermediate. In all cases, the ester fraction was the least.     

Absorbability of plant sterols and their distribution in rabbit tissues

Rabbits were fed a low cholesterol diet containing 2% plant sterols for 10 weeks to determine the absorbability of these sterols and their deposition in the tissues. We found campesterol and beta-sitosterol in the blood and tissues. The plasma campesterol levels were 4.34--13.3 mg/100 ml, whereas, beta-sitosterol levels were 0.41--1 mg/100 ml. Stigmasterol was not detected. The total plasma plant sterol concentration was about 10% of the total plasma sterol. The mean terminal plasma cholesterol concentration averaged 60% higher (55 vs. 88 mg/100 ml, P less than 0.001) than the mean initial value. Campesterol was the preponderant sterol in all tissues studied, including the aorta. Sitosterol was found in small amounts in the tissues of the abdominal organs. Stigmasterol was not detected in any tissue studied. Esterified campesterol and sitosterol were detected in trace amounts in most tissues. Campesterol and sitosterol, particularly the former, accumulated in the tissues including the aorta.     

Influence of Temperature on Sterol Biosynthesis in Triticum aestivum

Sitosterol, campesterol, stigmasterol, and cholesterol were isolated from green wheat (Triticium aestivum var. Monon) seedlings. Sitosterol was the predominant sterol extracted from the shoot, root, and crown tissue. Cholesterol accounted for less that 1% of sterol in shoot tissue with only trace amounts in the root. A temperature change from 10 to 1 C resulted in a general decrease in sitosterol, stigmasterol, and campesterol in the shoot tissue. The cholesterol level was not altered significantly by the temperature change. The sterols in the root responded in a manner very different from those in the shoots. With the reduction in temperature, sterols first decreased and then recovered over a period of 7 to 14 days to levels that were equal to or exceeded the original levels. From these experiments, it would appear that root tissue can acclimate to the lower temperatures and continue sterol synthesis at the normal rate. The level and response of sterols in the crown tissue were intermediate between the root and shoot tissue. At 10 C the crown response was similar to that of root tissue, whereas, at 1 C the response more closely resembled that of the shoot.     

Relationship of the neutral sterols and ecdysteroids of the parasitic mite,Varroa jacobsoni to those of the honey bee,Apis mellifera

The neutral sterols of the parasitic mite Varroa jacobsoni were compared with Apis mellifera carnica drone pupae. Analysis by GLC-mass spectrometry indicated mite sterols were reflective of the sterol composition of the drones; 24-methylenecholesterol was the major sterol in both species, with lesser amounts of sitosterol and isofucosterol. Cholesterol accounted for less than 1% of the total sterols. Ecdysteroid analyses indicated drones contained primarily makisterone A. In addition to makisterone A, mites contained ecdysone and 20-hydroxyecdysone, which accounted for over 66% of the ecdysteroid detected. These results indicate that while V. jacobsoni are apparently unable to convert dietary sterols to cholesterol, they are able to produce significant amount of C(27) ecdysteroids in a low cholesterol environment.     

Characterization of Soybean Plasma Membrane during Development: FREE STEROL COMPOSITION AND CONCANAVALIN A BINDING STUDIES

Plasma membrane preparations from soybean root and hypocotyl contained the following free sterols: cholesterol, campesterol, stigmasterol, and sitosterol. The cholesterol level was relatively low in root plasma membrane (less than 0.5%) but was 1.4 to 2.4% in hypocotyl membrane. The relative levels of the three other sterols fluctuated with cellular development and tissue source. Campesterol level decreased with the development of both root and hypocotyl membrane. With development, stigmasterol increased greatly in root membrane but remained constant in hypocotyl membrane, and sitosterol, the major free sterol component of all membrane preparations, decreased in root membrane but increased slightly in hypocotyl membrane.     

α-Onocerin and sterol content of twelve species of Ononis

The sterols and triterpenoids of 12 species of the genus Ononis were analysed by GLC. α-Onocerin was found in all but one of these species, although in some others its concentration was low. In all species examined, sitosterol was the major sterol; stigmasterol, campesterol, cholesterol and the triterpenoids cycloartenol and 24-methylene cycloartanol also occurred. The patterns of α-onocerin and sterols found seem to be consistent with the accepted classification of species within the genus.     

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.     

Sterol distribution in intracellular organelles isolated from tobacco leaves

All membrane-containing fractions isolated from tobacco leaves contained free sterols, sterol glycosides, and sterol esters. The three sterol forms increased, on a dry weight basis, with a decrease in particle size. The supernatant fraction contained only trace amounts of sterol. The major sterols in all cellular fractions, in the order of decreasing amounts, were: stigmasterol, β-sitosterol, campesterol, and cholesterol. The 500g pellet contained the largest percentage of free sterol, while the 46,000g pellet contained the largest percentage of esterified sterol. The individual sterol composition of the free sterol and sterol glycoside fraction was very similar; however, the composition of the sterol ester fraction varied widely among intracellular fraction. The intracellular distribution pattern of cholesterol-¹⁴C added to the isolation medium provided evidence that the intracellular sterol distribution pattern is not an artifact. These results support the suggestion that sterols in plant cells may have a physiological function associated with membranes.     

Dietary cholesterol utilization by the housefly,Musca domestica

The amount of cholesterol and the distribution of its metabolites were studied in various larval tissues of the housefly (Musca domestica), reared on a high cholesterol diet containing the radioactive cholesterol. The sterol content was also examined in adults derived from larvae reared on increasing dietary cholesterol concentration in separate batches. The results suggest that when the dietary sterol concentration was raised from 0.002 to 0.02% wet weight (an optimal concentration required for a maximal growth and development of insect), the sterol content of six-day old larvae was increased approx. 4-fold. However, a further 10-fold increase of dietary cholesterol (to 0.22% of wet weight) was associated with only a 1.5-fold increase in larval sterol content, and no increase in the sterol content of adult insects derived from such larvae was observed. This increase of sterol content of whole larvae was found to be confined to the larval cuticle and composite gut fractions and may be attributed to unabsorbed cholesterol in the gut, and to a solubilizing effect of the cuticle. These findings suggest that when the cholesterol requirement for the maximal growth of the insect has been reached, larvae are able to regulate the intake of ingested cholesterol, and no more is taken up through the gut even when a high gut concentration of cholesterol is present.The adults contained a higher percentage of esterified sterols than the larvae, approx. 25% of the sterol in females and 14% of sterol in males were esterified.     

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.