Metabolism of 26-[14C]hydroxyecdysone 26-phosphate in the tobacco hornworm,Manduca sexta L., to a new ecdysteroid conjugate: 26-[14C]hydroxyecdysone 22-glucoside

Following injection into female Manduca sexta pupae, [¹⁴C]cholesterol is converted to a radiolabeled C₂₁ nonecdysteroid conjugate as well as ecdysteroid conjugates, which in ovaries and newly-laid eggs consist mainly of labeled 26-hydroxyecdysone 26-phosphate. During embryogenesis, as the level of 26-hydroxyecdysone 26-phosphate decreases there is a concurrent increase in the amount of a new, labeled ecdysteroid conjugate. This conjugate, which is the major ecdysteroid conjugate (9.4 μg/g) in 0- to 1-hour-old larvae was identified as 26-hydroxyecdysone 22-glucoside by nuclear magnetic resonance and chemical ionization mass spectrometry. This is the first ecdysteroid glucoside to be identified from an insect. The disappearance of 26-hydroxyecdysone 26-phosphate in 0- to 1-hour-old larvae indicates that the 26-hydroxyecdysone 22-glucoside is derived from 26-hydroxyecdysone 26-phosphate. 3-Epi-26-hydroxyecdysone was the major free ecdysteroid isolated from these larvae and 3-epi-20,26-dihydroxyecdysone was the next most abundant ecdysteroid isolated. Interestingly, the 0- to 1-hour-old larvae contained the highest levels of 3α-ecdysteroids per gram of insect tissue (8.7 μg/g) to be isolated from an insect, yet there was a complete absence of the corresponding free 3β-epimers. The ecdysteroid conjugate profiles of ovaries and 0- to 1-hour-old larvae are discussed. Methodology is presented that permits the efficient separation of free and conjugated ecdysteroids and nonecdysteroid conjugates (C₂₁-steroid conjugates).     

Midgut and fatbody mitochondrial transhydrogenase activities during larval-pupal development of the tobacco hornworm, Manduca sexta

Midgut and fatbody mitochondria from fifth larval instar Manduca sexta display a membrane-associated transhydrogenase that catalyzes a reversible hydride ion transfer between NADP(H) and NAD(H). The NADPH-forming activity occurs as a nonenergy- or energy-linked activity with energy for the latter derived from either electron transport-dependent NADH or succinate utilization, or ATP hydrolysis by Mg⁺⁺-dependent ATPase. During the ten-day developmental period preceding the larval-pupal molt (fifth larval instar), significant peaks in the mitochondrial transhydrogenase activities of midgut and fatbody tissues were noted and these peaks were coincident with the onset of wandering behavior and with the fifty-fold increase in ecdysone 20-monooxygenase (E20-M) activity previously reported for M. sexta midgut. Since E20-M preferentially uses NADPH in catalyzing ecdysone conversion to the physiologically active molting hormone, 20-hydroxyecdysone, the physiological and developmental significance of the mitochondrial, NADPH-forming energy-linked transhydrogenations were made apparent. Moreover, that the increases in all transhydrogenase activities resulted from de novo enzyme synthesis were indicated by the cycloheximide-dependent reductions in these activities.     

DNA transfection in the ecdysteroid-responsive GV1 cell line from the tobacco hornworm, Manduca sexta

Summary The embryonic cell line, GV1, from Manduca sexta was transiently transfected with DNA constructs of the Drosophila hsp70 promoter fused to either a β-galactosidase (pXH70ZT) or a chloramphenicol acetyl transferase (HSP-CAT-1) reporter gene using lipofectin. Optimal cell density, DNA:lipofectin ratio, and time of incubation were varied to determine the optimal conditions: 2 × 10⁵ cells/ml, 1:3, and 5 h. Under these conditions, the transfection efficiency was about 40%. Heat inducibility of two hsp70 constructs was compared. The HSP-CAT-1, containing 1127 bp of upstream sequence, was more sensitive to heat shock than that of pXH70ZT, containing only 194 bp of upstream sequence. Thus, the 1127 bp hsp70 promoter appears to be a better inducible promoter in these cells. A 2 kb fragment of the proximal promoter region of the MHR3 gene containing a putative ecdysone response element was shown to be responsive to 20-hydroxyecdysone after its transfection into these cells.     

A new ecdysteroid, 2-deoxy-5beta,20-dihydroxyecdysone from the fruits of Diploclisia glaucescens

Chemical investigation of ethyl acetate extract of the fruits of Diploclisia glaucescens of the family Menispermaceae furnished a new ecdysteroid 2-deoxy-5beta,20-dihydroxyecdysone, together with 20-hydroxyecdysone, 3-deoxy-1beta,20-dihydroxyecdysone, 2-deoxy-20-hydroxyecdysone, 24-ethyl-20-hydroxyecdysone (makisterone C). Latter two ecdysteroids are reported first time from the family Menispermaceae.     

Changes in fat body urate synthesizing capacity during the larval-pupal transformation of the tobacco hornworm, Manduca sexta

Large quantities of uric acid or urates are deposited in the fat body of tobacco hornworms, Manduca sexta, between the larval and pupal stages in development. The cause of this increased deposition was investigated by measuring fat body urate synthesizing capacity (USC) during the larval-pupal transformation (LPT). An 85% loss in USC occurs between the late-feeding larval and newly-ecdysed pupal stages. Urate synthesizing capacity, per se, is not responsible for the increase in fat body urate deposition, as evidenced by comparable rates of urate deposition in insects whose USCs differ by a factor of three. Rather, the increased deposition is caused by an increase in substrate availability. The loss in USC is programmed in two steps. The first programmed loss occurs by the end of the feeding fifth larval instar, since hornworms ligated at the pink stripe (PS) stage and measured at the time of the larval-pupal ecdysis (LPE) exhibit an increased retention of USC relative to controls. The second programmed loss in USC occurs between PS + one and PS + two day stages in development. A single administration of 20-hydroxyecdysone to hornworms ligated at the PS stage causes a restoration of this loss in USC by PS + two days, which is further sustained until the LPE. Unexpectedly, when measured immediately after the LPE, the second programmed loss in USC can be delayed until PS + 3 days if non-ligated hornworms are daily administered 20-hydroxyecdysone. The possibility is raised that 20-hydroxyecdysone does not act alone in causing the loss in fat body USC.     

The ecdysteroids from young embryonated eggs of the tobacco hornworm

26-Hydroxyecdysone, which is the major free recoverable ecdysteroid of older age groups of embryonated eggs of the tobacco hornworm was also the major component in 4- to 18-hour-old embryonated eggs. The other 3β-ecdysteroids, ecdysone, 20-hydroxyecdysone, and 20,26-dihydroxy-ecdysone, were also present and accounted for an the molting hormone activity; 26-hydroxyecdysone was devoid of molting hormone activity in the house fly assay. 20-Hydroxyecdysone was a minor component, which confirms the earlier observations that the main metabolic route for ecdysteroids during embryonic development is that leading to 26-hydroxy-ecdysone, whereas formation of 20-hydroxyecdysone is a minor pathway. A new 3α-ecdysteroid, 3-epi-26-hydroxyecdysone, also devoid of molting hormone activity, was the second major ecdysteroid isolated from the eggs. 3-Epi-20,26-dihydroxyecdysone was detected in very minute amounts. In additon to the six 3β-and 3α-ecdysteroids there were at least an equivalent number of unknown ecdysteroids an of which lacked molting hormone activity. Their physical properties including chromatographic behavior are discussed.     

Hormonally-regulated differential expression of the two duplicated insecticyanin genes,ins-a and ins-b,during development of the tobacco hornworm,Manduca sexta

The effects of juvenile hormone (JH) and 20-hydroxyecdysone (20E) on the developmental expression of the two insecticyanin genes, ins-a and ins-b, were investigated with two gene-specific probes. Removal of the corpora allata (-CA, source of JH) clearly delayed and down-regulated the epidermal expression of these genes but enhanced their expression in the fat body during the early development of the fifth instar. Application of JH I to the -CA larvae at the time of head capsule slippage completely restored the normal epidermal expression pattern of the two genes in the early fifth instar, then INS-a mRNA declined prematurely whereas INS-b mRNA remained similar to that in the intact larvae. By contrast, in the fat body of -CA larvae, the exogenous JH had little effect on the levels of INS-a mRNA, but enhanced expression of INS-b mRNA relative to intact larvae. Culture of epidermis from day 1 fifth instar larvae with 40 ng/ml 20E for up to 24 h accelerated the loss of INS-a mRNA without affecting the levels of INS-b mRNA. Both mRNAs declined in isolated larval abdomens over a 24 h period, and this decline was slowed by 1 g methoprene (a JH analog). Together these results indicate that JH controls the levels of the two mRNAs in both the epidermis and fat body, with additional factors involved in regulating these genes in the fat body during the molt and in the epidermis during the growth phase.     

Studies on the black box: Incorporation of 3-oxo-7-dehydrocholesterol into ecdysteroids by Drosophila melanogaster and Manduca sexta

It has long been hypothesized that the oxidation of 7-dehydrocholesterol (7dC), made from dietary cholesterol (C), to 3-oxo-7dC (3-oxo-Δ^5,7C) immediately precedes the unknown “Black Box” oxidations that lead to the formation of the first up-stream intermediate exhibiting the highly characteristic ecdysteroid structure of the steroid molting hormone of insects, crustaceans and some other arthropods. Perhaps rate-limiting and under the control of the prothoracicotropic hormone (PTTH), the biosynthesis of 3-oxo-7dC and its subsequent oxidative modifications have been difficult to study because of their apparent instability, i.e. no intermediates between 7dC and the diketol (3-oxo-25,22,2-trideoxyecdysone) have ever been observed or identified in insect prothoracic gland incubations with radiolabelled precursors. However, we show that 3-oxo-7dC can be converted into lipophilic, photosensitive, ketone-blocked (PSKB) ketal derivatives which will release 3-oxo-7dC when and where desired following brief irradiation with innocuous long-wave (365 nm) UV-light both in vivo and in vitro. In this manner, 3-oxo-7dC is quickly and efficiently incorporated into ecdysteroids by adult male and female Drosophila raised on a diet containing the PSKB ketals and in prothoracic glands of Manduca sexta incubated with the ketals emulsified into media. The instability of 3-oxo-7dC and its spontaneous transformation into extensively electron-delocalized intermediates will be discussed in relation to a possible mechanism of the Black Box oxidations eventually leading to the production of the active molting hormone 20-hydroxyecdysone (20E).     

Temporal control of urate oxidase activity during development of the third-instar larva of Drosophila: The role of 20-hydroxyecdysone

The tissue-specific enzyme urate oxidase is confined exclusively to the Malpighian tubules of Drosophila melanogaster and expressed only in the third-instar larva and the adult. Shortly before pupariation urate oxidase activity declines precipitously and is not detectable 24 hours later. That 20-hydroxyecdysone is the factor that triggers the disappearance of urate oxidase activity in late third-instar larvae is demonstrated using the temperature sensitive mutant ecd¹ which at the nonpermissive temperature of 29°C fails to accumulate a sufficient concentration of 20-hydroxyecdysone necessary for puparium formation and thus remains a third-instar larva for 1 to 2 weeks before death. Both the life cycle and the temporal profile of urate oxidase activity in ecd¹ larvae at 19°C is identical to that of the wild type. However, at 29°C ecd¹ third-instar larvae retain high urate oxidase activity. A precipitous decline in urate oxidase activity is observed when ecd¹ larvae at 29°C are fed 20-hydroxyecdysone. These data implicate 20-hydroxyecdysone in the process that controls the rapid decline of urate oxidase activity at the time of puparium formation. In whole homogenates of Malpighian tubules, the urate oxidase polypeptide was identified in SDS-polyacrylamide gels by its Rf with respect to homogeneously pure Drosophila urate oxidase and also by immunoprecipitation with rabbit anti-Drosophila urate oxidase IgG. Throughout development the amount of the urate oxidase polypeptide is correlated with the magnitude of urate oxidase activity.     

The molting hormones from the embryonated egg of the tobacco hornworm, Manduca sexta (L.)

26-Hydroxyecdysone is the predominant molting hormone in 24- to 44-hour-old embryonated tobacco hornworm eggs, accounting for approximately 80% of the ecdysones present at this stage of development. This molting hormone was previously shown to be the major ecdysone present in 48- to 64-hour-old embryonated eggs of this insect. During both of these periods of embryonic development in the hornworm 20-hydroxyecdysone is a minor component, in contrast to its presence as the major ecdysone in the hornworm during certain stages of post-embryonic development.     

The effect of Achyranthes japonica extract on larval survival and development and oviposition behavior of Plutella xylostella L. (Lepidoptera: Plutellidae)

The extract of Achyranthes japonica was tested for effects on larval survival and development and the oviposition behavior of the diamondback moth, Plutella xylostella L. Chinese cabbage dipped in A. japonica extract solution showed 51–80% antifeedant activity for 5 days against P. xylostella larvae, and more larvae were also on untreated cabbage leaves 24 h after release. The mortality of P. xylostella larvae increased proportionally to the duration of dipping time in the extract, and both pupation and emergence rates of larvae feeding only on treated cabbage were lower than those for larvae raised on untreated or with a choice of cabbage. The 20-hydroxyecdysone (20E) concentration in leaves was approximately 549, 1232, 1275, and 1426 μg/g at 6, 12, 24, and 48 h after dipping treatment, respectively. Notably, naive females laid more eggs on untreated cabbage than on treated cabbage, and females from larvae raised on treated Chinese cabbage also preferred the non-treated leaves. Our results are in contrast to those from earlier studies using various insect models that confirmed most females prefer to lay eggs on the host type that was eaten in the larval stage (Hopkins host selection principle). Cabbage dipped in the A. japonica solution for 24 h caused 59% larval mortality and inhibited both pupation and emergence rates of the larvae when exposed to plants 15 and 22 days after planting in the field, with the 20E concentration in the treated cabbage leaves at 1600.9 ± 122.36 and 1386.8 ± 24.69 μg/g, respectively. Therefore, the biological effectiveness could be attributed to the 20E in the treated cabbage leaves.     

The effects of juvenile hormone, 20-hydroxyecdysone and precocene II on activity of corpora allata and the mode of negative-feedback regulation of these glands in the adult Colorado potato beetle

When the titre of juvenile hormone III in female Leptinotarsa decemlineata was elevated by the implantation of supernumerary corpora allata or by the injection of the hormone, the rate of endogenous hormone production by the host glands was significantly restrained, as determined by the short-term in vitro radiochemical assay. From denervation studies, it is suggested that during phases of elevated juvenile hormone titre, the corpus allatum activity is regulated via humoral as well as neural factors requiring intact nerve connections. Restrainment of gland activity appears to be mainly via the neural pathway. Isolated corpora allata were not influenced by 10^?5 M juvenile hormone III added to the incubation medium in vitro.Studies with farnesenic acid revealed that the final two enzymatic steps in the biosynthetic pathway of juvenile hormone are also diminished during prolonged neural inhibition of the corpora allata.20-Hydroxyecdysone and precocene II had no apparent effect on the corpus allatum activity of Leptinotarsa decemlineata.     

The eupyrene-apyrene dichotomous spermatogenesis of Lepidoptera. The relationship with postembryonic development and the role of the decline in juvenile hormone titer toward pupation.

The relationships between the stages of postembryonic development and the occurrence of eupyrene and apyrene spermatogenesis, and the effects of the decline of the juvenile hormone (J.H.) titer toward pupation in these processes, were studied in the carob moth, Ectomyelois ceratoniae. The accurate timing of the spermatogenetic events was determined daily from the 2nd instar larva to the imago in squashes and electron microscope preparations of testes. Eupyrene spermatids elongate in two phases. In the first, beginning in late 4th instar larva, only flagella elongate, while in the second, beginning in the mid 5th instar larva, both flagella and nuclei elongate. Apyrene spermatogenesis starts just after the beginning of the nuclear elongation of eupyrene spermatids, in the mid 5th instar larva and not in the pupa, as is commonly believed. Using ligatures, topical applications of a J.H. mimic, and testes transplantation, it was found that the nuclear elongation begins in the 5-day-old eupyrene spermatid and cannot be induced earlier; the elongation is inhibited by high titer of the J.H. mimic. Elongation of the flagella, however, is unaffected by fluctuations of the J.H. titer. The onset of the apyrene spermatogenesis, which occurs in the very early 5th instar larva or before, was found to be unrelated to the decline in the J.H. titer toward pupation.     

The activity of the pyruvate dehydrogenase complex in heart and liver from mice during the development of obesity and insulin resistance.

The amount of pyruvate dehydrogenase in the active form (PDHa) was increased 1.7-fold compared with controls in heart muscle of mice 1 week after induction of obesity with a single injection of gold-thioglucose. At 4 weeks post injection, the amount of PDHa was decreased to 32% of control, a value which was observed in later stages of the obesity syndrome. In contrast, liver PDHa was increased and remained at an increased activity during the development of obesity. Despite normal post-prandial serum insulin contents, liver membrane insulin-receptor numbers were decreased 1 week after gold-thioglucose injection, and there was no change in receptor affinity. The decrease in heart PDHa in the obese animals was reversed by a single dose of 2-tetradecylglycidic acid, but this inhibitor of mitochondrial fatty acid oxidation did not affect liver PDHa in these animals. These early and diverse changes in PDHa argue for a multifactorial aetiology in the development of the whole-body insulin resistance seen in older gold-thioglucose-treated obese animals.     

Effect of thyroid hormone analogues on the displacement of 125I-L-triiodothyronine from hepatic and heart nuclei in vivo: possible relationship to hormonal activity

The capacity of iodotyrosines and iodothyronine analogues to displace tracer[¹²⁵I] L-3,5,3′ triiodothyronine from specific nuclear binding sites in rat liver and heart was related to the displacement capacity of nonradioactive triiodothyronine. Iodotyrosines and L-3,3′,5′ triiodothyronine (“reverse T₃”) were devoid of displacement activity. Analogues with 3,5 substitution in the “inner” ring and single “bulk” substitution in the 3′ position in the phenolic ring exhibited the strongest displacement activity. When the distribution, fractional removal rates and metabolic conversion of the analogues were taken into account, displacement activity appeared to correlate well with the reported thyromimetic activity. These results support the biologic relevance of the nuclear sites.     

The dynamics of oxidase activity during cultivation of basidiomycetes from the genus Trametes Fr.

Twenty strains of the wood-degrading fungi from the genus Trametes Fr., capable of synthesizing laccases, were screened according to the changes in the oxidase activity in a submerged culture. The range of maximal efficiency of various species with respect to extracellular oxidase activity was determined. The absence of correlation between the oxidase activity in a submerged culture and the size of colored zone on agar media (Bavendamm reaction) was demonstrated. The most efficient strains, T. hirsita 56 and T. ochracea 92-78, were used to produce laccases, homogeneous according to SDS electrophoresis data. A number of biochemical parameters characteristic of these enzymes were determined.