James A. Svoboda

Makisterone A: The major ecdysteroid from the pupa of the honey bee, Apis mellifera

Abstract Makisterone A , a 28-carbon moulting hormone, has been identified as the major free pupal ecdysteroid in the honey bee, Apis mellifera. The pupal ecdysteroid was isolated and identified by normal and reversed-phase high performance liquid chromatography in conjunction with a radioimmune assay. The compound was further characterized physico-chemically by both mass spectrometry and nuclear magnetic resonance spectroscopy. No C27 ecdysteroids (i.e. 20-hydroxyecdysone or ecdysone) were detected at this stage of development. This is the first isolation and identification of a 28-carbon ecdysteroid in an insect species from the order Hymenoptera. Utilization of dietary sterols by honey bees is also discussed.

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.

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.

Nonsteroidal secondary and tertiary amines: Inhibitors of insect development and metamorphosis and Δ24-sterol reductase system of tobacco hornwormreductase system of tobacco hornworm

Several new branched and straight chain secondary and tertiary amines were shown to have inhibitive effects upon development and metamorphosis and the Δ24-sterol reductase system in larvae of the tobacco hornworm similar to those previously observed with a number of azasteroids. Certain of the amines which are related structurally to compounds with juvenile hormone activity in insects also blocked development and metamorphosis in three other species of insects. These compounds are lethal or inhibit development in all larval stages and thus differ in action from compounds with juvenile hormone activity where the principal effect is to block the penultimate or ultimate molt.

Ecdysteroids in developing ovaries and EGGS of the tobacco hornworm

Ecdysteroids of ovaries and newly-laid eggs (0- to 1-hour-old) of the tobacco hornworm are present mainly as conjugates (greater than 95%). Newly-laid eggs contain ecdysteroid conjugates equivalent to 21 micrograms of 26-hydroxyecdysone and 0.73 micrograms of ecdysone per gram of eggs. These levels are similar in ovaries of 93-hour-old adult females. In 1- to 18-hour-old eggs more than 63% of the ecdysteroids exist in the free form and the proportion is similar in 48- to 64-hour-old eggs. The ratio of 26-hydroxyecdysone to ecdysone in the conjugated form remains constant during oocyte maturation and embryogenesis. Though 26-hydroxyecdysone is without molting hormone activity in the house fly assay, the exceptionally high concentration of 26-hydroxyecdysone conjugate(s) in ovaries and newly-laid eggs, together with the fact that it is being released during embryogenesis, indicate some physiological role for 26-hydroxyecdysone.

24-Methylenecholesterol: Isolation and identification as an intermediate in the conversion of campesterol to cholesterol in the tobacco hornworm

Abstract24-Methylenecholesterol was positively identified as an intermediate in the dealkylation and the conversion of campesterol to cholesterol in the tobacco hornworm,Manduca sexta (L.). Biochemical and nutritional studies with3H-labeled campesterol and 24-methyl-enecholesterol indicated that the conversion of campesterol to cholesterol in this insect includes the following metabolic sequence: campesterol, 24-methylene-cholesterol, desmosterol, cholesterol.

Ecdysteroid production in Drosophila melanogaster reared on defined diets.

Larvae of Drosophila melanogaster were reared aseptically on defined diets containing either cholesterol, campesterol or sitosterol as the only dietary sterol. Sterol analyses of pupae revealed that insects reared on campesterol and sitosterol diets contained 3.3 and 8.1% cholesterol, indicative of an ability to accumulate this sterol. Ecdysone and 20-hydroxyecdysone were the predominant ecdysteroids in insects from all diet studies, though makisterone A was detected in pupae reared on campesterol and sitosterol.

Unusual composition of sterols in a phytophagous insect, Mexican bean beetle reared on soybean plants

Three saturated sterols, cholestanol, campestanol, and stigmastanol, constituted 54, 72, and 77% of the total sterols of the egg, prepupa, and adult, respectively, of the Mexican bean beetle,Epilachna varivestis (Mulsant), reared on soybean plants. Lathosterol (7-cholesten-3β-ol), possibly formed from cholestanol in this insect, constituted 12, 16, and 11.8% of the total sterols isolated from egg, prepupa, and adult, respectively. None of these sterols have been isolated and identified previously as components of the sterols of a phytophagous insect reared on a natural host plant. Cholesterol, a major sterol of most plant feeding insects studied thus far, comprised less than 5% of the total sterols in any of the stages examined. The unique composition of the sterols in this insect in relation to the sterol composition of the host plant is compared to dietary sterol utilization and metabolism in other phytophagous insects.

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.

Sterol utilization and ecdysteroid content in the house fly, Musca domestica (L.)

Larvae of the house fly, Musca domestica were reared aseptically on diets which contained either cholesterol, campesterol or sitosterol as the dietary sterol at a concentration of 0.1% dry wt. Analysis of puraria (24 h post-pupariation) reared on campesterol or sitosterol diets revealed they contained from 2.7 to 4.6% cholesterol, indicating an ability to accumulate this sterol even where it is present in only minute quantities. Purparia on all diets produced the 27-carbon molting hormones, ecdysone and 20-hydroxyecdysone. When the concentration of campesterol was increased to 0.2% dry wt, puparia also contained the 28-carbon ecdysteroid, makisterone A, although it accounted for only 20.7% of the total ecdysteroid produced.