bluuebunny/crossref_metadata_2025 huggingface dataset:
Biosynthesis of makisterone A and 20‐hydroxyecdysone from labeled sterols by the honey bee, Apis mellifera
Authors: Feldlaufer M. F. , Herbert E. W. , Svoboda J. A. , Thompson M. J.
Published: February 2005

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.
The neutral sterols of Megalotomus quinquespinosus (say) (hemiptera: Alydidae) and identification of makisterone a as the major free ecdysteroid
Authors: Feldlaufer M. F. , Svoboda J. A. , Aldrich J. R. , Lusby W. R.
Published: February 2005

Last‐stage nymphs of the broad‐headed bug, Megalotomus quinquespinosus contain the C28 ecdysteroid makisterone A as their major ecdysteroid. No ecdysone or 20‐hydroxyecdysone was detected in whole body extracts analyzed by high performance liquid chromatography and radioimmune assay. Analyses of the neutral sterols of this phytophagous hemipteran revealed that the sterol composition of the nymphs was highly reflective of their dietary sterols. The most abundant nymphal sterols were sitosterol (46.6%), Δ7‐stigmastenol (13.8%) and spinasterol (13.4%). Cholesterol accounted for only 0.2% of the total sterols and indicates that this species is incapable of converting 24‐alkyl sterols to cholesterol.
Maturation of locust corpora allata during the reproductive cycle: Effect of reserpine on allatotropic activity, juvenile hormone‐III biosynthesis, and oocyte development
Authors: Gadot Michal , Faktor Ouriel , Applebaum Shalom W.
Published: February 2005

Reserpine, at doses of 20–175 μg per g body weight, severely retards oogenesis in newly emerged adult female migratory locusts (Locusta migratoria migratorioides) but does not increase mortality during the first 9 days and only slightly delays somatic growth. Total protein, and hemolymph vitellogenin content particularly, are significantly reduced in reserpine‐treated locusts. The synthesis of juvenile hormone III (JH‐III) following adult emergence, essential for induction of vitellogenesis and subsequent oogenesis, is dependent on the maturation and activation of the corpora allata (CA). CA of 7‐ to 8‐day‐old female locusts, treated with reserpine at day 1 after adult emergence, are only marginally active in vitro and are only slightly stimulated by an allatotropic factor. The basal activity and response of CA from the reserpine‐treated locusts resembles that of newly emerged locusts, suggesting that reserpine specifically retards the initial maturation of the locust CA. Recovery of basal CA activity is evident on days 12–13 in reserpine‐treated locusts, but responsiveness to the allatotropic factor is not recovered. Starvation of newly emerged females for 3 days and subsequent feeding did not effect ooctye development or CA activity. Cerebral content of the allatotropic factor, assayed on days 7–8, is not reduced by the reserpine treatment.
25‐Azasteroid inhibition of development and conversion of C28 and C29 phytosterols to cholesterol in Spodoptera litura (F.)
Authors: Kuthiala A. , Agarwal H. C. , Thompson Malcolm J. , Svoboda James A.
Published: February 2005

Larvae of Spodoptera litura (F.) grown on an artificial diet completed larval development in 19.2 days and attained a maximum weight of 873.2 mg. When fed dietary concentrations of 50 ppm of 25‐azacholesterol or 10 ppm of 25‐azacholestane, the larval developmental period increased to 28.4 and 23.4 days, and the larval weights were 447.5 and 542.3 mg, respectively. Both compounds induced distinct melanization effects and caused production of larval‐pupal intermediates and severe mortality. Treatments with concentrations of 50 ppm or more of either azasteroid caused a decline in pupal period and earlier eclosion and emergence of abnormal adults. Egg laying and hatchability decreased with increasing concentrations of azasteroids in the larval diets.

When 1 ppm or more of 25‐azasteroid is added to the artificial diet, the insect larvae contain identifiable amounts of desmosterol, in addition to cholesterol, campesterol, and sitosterol, which are present in Spodoptera grown on artificial diet alone. Desmosterol accumulation in the insect body is due to an inhibition of the Δ24‐sterol reductase by 25‐azasteroids. An increase in the concentration of these azasteroids in the diet results in an increase in sitosterol concentration and simultaneous reduction in the cholesterol levels due to inhibition of conversion of sitosterol. This inhibition appears to be more pronounced with 25‐azacholestane treatment than with 25‐azacholesterol.
Synergistic action of diethyl maleate on the morphogenetic and antigonadotropic activity of precocenes on the seed bug Oxycarenus Iavaterae (F.)
Authors: Bellés X. , Baldellou M. , Messeguer A.
Published: February 2005

Freshly ecdysed‐third instar larvae and adult females of the seed bug Oxycarenus Iavaterae were treated with diethyl maleate (1 or 10 μg/specimen, topically applied), a commonly used depletor of glutathione levels, prior to the application of model precocenes (P1, P2, and EP2; 1–10 μg/specimen, topically applied). The combined treatment resulted in a significant increase of the characteristic antijuvenile hormone effects elicited by the above allatocidins, namely, induction of precocious metamorphosis in larvae and inhibition of ovarian development in adult females. These results, which indicated that diethyl maleate exerted a definite synergistic action, constitute the first example of a synergism strategy to the improvement of precocene activity.
Effects of starvation, chilling, and injury on endocrine gland function in Galleria mellonella
Authors: Malá Jaroslava , Sehnal Frantisek , Kumaran A. Krishna , Granger Noelle A.
Published: February 2005

Starvation, chilling, and injury of last instar Galleria mellonella larvae typically elicit extra larval molts or a delay in pupation. The primary sites of action and the nature of the signals by which these treatments affect development are not known. However, since the connections of the brain to the nerve cord are crucial for the effects of starvation and chilling, these signals apparently affect the brain‐centered program of developmental regulation via the nerve cord. Chilling, and occasionally starvation, cause extra larval molts in last instar larvae treated prior to the nervous inhibition of their corpora allata; release of a cerebral allatotropin, which stimulates the production of juvenile hormone, appears to be involved in this effect. After this time, a delay in pupation is the principal effect of starvation and chilling, and is apparently due to a temporal inhibition of the release of the prothoracicotropic hormone. Chilling also appears to inhibit unstimulated ecdysteroid production by the prothoracic glands.

The effect of injury is not mediated by the nerve cord, but appears to involve an inhibitory humoral factor that affects either the brain or the prothoracic glands themselves. Injury also stimulates juvenile hormone production, an effect which is enhanced when the brain is separated from the nerve cord and which is evidenced by a delay of ecdysis and the occasional retention of some larval features in the ecdysed insects.

None of the effects of these various treatments on the brain and the endocrine glands persist when the brains or glands are implanted into untreated hosts.
Disruption of antennal morphogenesis in Bracon hebetor by exposure to triethylamine
Authors: Abbott Barbara D.
Published: February 2005

Exposing third instar larvae of the wasp Bracon hebetor to triethylamine (TEA) results in malformed antennae in eclosing adults. The purpose of this research was to examine the effects of TEA at the cellular level. Rate of growth of treated antennal buds (56–76 h) and mean size (76–96 h) were significantly reduced. Internal pH was elevated for 5 h after TEA exposure. Cell size remained constant except in treated buds from 76 to 96 h, when diameter was reduced. The mean mitotic index (MI) was reduced and a normal peak at 72–76 h was eliminated. Cell death (DI) increased. Increased DI and decreased MI from 0 to 5 hours after exposure are attributed to increased pH. TEA can form reactive alkylating intermediates, and loss of the MI peak 12 h after exposure accompanied by increased DI may be due to alkylation. It is concluded that the reduced number of segments is due to an overall reduction in total number of cells. Abnormal segmentation may involve alkylation induced somatic mutations.
Effects of intestinal insulin‐like peptide on glucose catabolism in male adult Locusta migratoria
Authors: Khay A. Ben , Gourdoux L. , Moreau R. , Dutrieu J.
Published: February 2005

An insulin‐like peptide (ILP) extracted from midgut of 25‐day‐old male adult Locusta migratoria can modify the relative activity of the two main pathways of glucose catabolism. The effect of ILP on the activity of the pentose cycle and the glycolytic‐citric acid cycle in Locusta migratoria was evaluated by a radiorespirometric method by means of [1‐14C] glucose and [6‐14C]glucose as substrates. The time course of the ILP effect was determined. The insulin‐like peptide increases the relative activity of the pentose cycle. This effect is rapid and of short duration. Injected mammalian insulin has a similar effect.
The role of alcohols in pheromone biosynthesis by two noctuid moths that use acetate pheromone components
Authors: Teal P. E. A. , Tumlinson J. H.
Published: February 2005

Primary alcohols varying in chain length from C13 to C16, and in number, position, and geometric configuration of double bonds, were applied in dimethyl sulfoxide to the surface of the female sex pheromone glands of Heliothis subflexa (Gn.) and Hydraecia micacea (Esper). Capillary gas chromatographic analysis of extracts of the treated glands indicated that the alcohols were converted to the corresponding aldehydes by H. subflexa females and to the acetates by H. micacea females. Conversions of the alcohols showed no preferences for molecular weight, number, position, or geometry of the double bonds in either species. Application of the acetates of the primary alcohols to the gland surface of H. subflexa females resulted in the production of both the corresponding alcohols and aldehydes, while neither alcohols nor aldheydes were produced when acetates were applied to the glands of H. micacea. In addition, application of the acetates to the gland surface of Heliothis virescens (F.) resulted in the production of both the corresponding alcohols and aldehydes. However, no evidence was found to indicate that acetates are ever produced by the pheromone gland of females of H. virescens.
Protein cross‐linking by peroxidase: Possible mechanism for sclerotization of insect cuticle
Authors: Hasson Chad , Sugumaran Manickam
Published: February 2005

Incubation of test proteins with horseradish peroxidase in the presence of hydrogen peroxide and a catechol resulted in polymerization and precipitation of test proteins. SDS‐PAGE readily revealed the generation of dimers, trimers, and higher oligomers in the reaction mixture. With the exception of 3,4‐dihydroxyphenylalanine, dopamine, and norepinephrine, most other catechols tested participated in protein polymerization. The inability of these three catechols to accomplish polymerization is attributed to their high rate of intramolecular cyclization, which results in melanin formation. Radioactive studies with [3H]N‐acetyldopamine clearly reveal both intermolecular and intramolecular cross‐linking of test proteins by peroxidase. Based on these studies a possible mechanism for sclerotization and the biological significance of peroxidase in cuticle is discussed.
Isolation and identification of 20‐hydroxyecdysone from a lepidopteran continuous cell line
Authors: Lynn D. E. , Feldlaufer M. F. , Lusby W. R.
Published: February 2005

Extracts of three continuous cell lines from the cabbage looper, Trichoplusia ni, were assayed for the presence of ecdysteroids. While no evidence of ecdysteroids was present in the extracts of the ovarian (TN‐368) or embryonic (IPLB‐TN‐R2) cell lines, radioimmunoassays on extracts of media and extracts of cell pellets from imaginal disc cell cultures (IAL‐TND1) were positive. The immunoreactive material from both cells and media co‐migrated with a 20‐hydroxyecdysone standard on reversed‐phase high‐performance liquid chromatography (HPLC). The immunoreactive fractions from the cell extract were chromatographed on silica HPLC and subjected to mass spectral analysis. Both of these analyses indicated that the unknown compound was 20‐hydroxyecdysone. Radioimmunoassay indicated up to 28 ng of ecdysone equivalents in cells (3.75 x 107 cells) from 50 ml of IAL‐TND1 cultures, which is equivalent to 120 ng of 20‐hydroxyecdysone based on relative reactivity of the antiserum used in this study. This report presents the first evidence of 20‐hydroxyecdysone production by a continuous insect cell line and also the first to show that cells from imaginal discs are capable of ecdysteroid synthesis.
Ecdysteroid action on moth epithelial tissues and cell lines
Authors: Oberlander H. , Leach C. E. , Lanka S. , Willis J. H.
Published: February 2005

The spontaneous formation of multicellular, fluid‐filled vesicles in primary cultures of pupal wings of Hyalophora cecropia is inhibited by 20‐hydroxyecdysone (20‐OH‐ecdysone). Physiological levels of ecdysteroids inhibit the ability of larval hemolymph to induce such vesicles in an established cell line (IAL‐TND1) derived from imaginal discs of Trichoplusia ni (Hübner). This cell line grew as vesicles for its first year in culture even in the absence of hemolymph, but then converted to growing as multicellular aggregates. Aggregate‐conditioned media but not vesicle‐conditioned or control media displayed ecdysteroid activity in an imaginal disc bioassay. Larval hemolymph had no influence on the imaginal discs' response to ecdysteroids. Hence, its action in promoting vesicle formation is not to prevent the action of ecdysteroids. We postulate that the change in morphology of TND1 cell cultures from vesicles to aggregates was accompanied by their attaining (or enhancing) the capacity to produce ecdysteroids.
Long‐term cholesterol labeling as a convenient means for measuring ecdysteroid production and catabolism in vivo: Application to the last larval instar of Pieris brassicae
Authors: Beydon Philippe , Lafont René
Published: February 2005

In vivo biosynthesis of ecdysteroids during the last larval instar of Pieris brassicae was investigated by administering [3H] cholesterol followed byhigh‐performance liquid chromatography analysis of the resulting [3H] ecdysteroids. The demonstration that the specific activity of the ecdysteroids synthesized at a given time is always identical with that of cholesterol indicates that the cholesterol pool is uniformly labeled, and this allows us to easily calculate the amounts of ecdysteroids produced by animals. The total amount of ecdysone produced throughout the last larval instar was measured as 1.17 nmol/insect. This quantity is more than three‐fold the maximal level of molting hormones (ecdysone +20‐hydroxyecdysone) reached during the instar (0.37 nmol/animal) because a high catabolic activity occurs at the beginning of the hormone production period. Larvae thus differ from pupae, where catabolism is minimal when ecdysone synthesis takes place, resulting in a more “economical” system.
Succinate metabolism related to lipid synthesis in the housefly Musca domestica L
Authors: Halarnkar Premjit P. , Heisler Charles R. , Blomquist Gary J.
Published: February 2005

The metabolism of succinate was examined in the housefly Musca domestica L. The labeled carbons from [2,3‐14C]succinate were readily incorporated into cuticular hydrocarbon and internal lipid, whereas radioactivity from [1,4‐14C]succinate was not incorporated into either fraction. Examination of the incorporation of [2,3‐14C]succinate, [1‐14C]acetate, and [U‐14C]proline into hydrocarbon by radio‐gas‐liquid chromatography showed that each substrate gave a similar labeling pattern, which suggested that succinate and proline were converted to acetyl‐CoA prior to incorporation into hydrocarbons. Carbon‐13 nuclear magnetic resonance showed that the labeled carbons from [2,3‐13C]succinate enriched carbons 1, 2, and 3 of hydrocarbons with carbon‐carbon coupling showing that carbons 2 and 3 of succinate were incorporated as an intact unit. Radio‐high‐performance liquid chromatographic analysis of [2,3‐14C]succinate metabolism by mitochondrial preparations showed that in addition to labeling fumarate, malate, and citrate, considerable radioactivity was also present in the acetate fraction. The data show that succinate was not converted to methylmalonate and did not label hydrocarbon via a methylmalonyl derivative. Malic enzyme was assayed in sonicated mitochondria prepared from the abdomens and thoraces of 1‐ and 4‐day‐old insects; higher activity was obtained with NAD+ in mitochondria prepared from thoraces, whereas NADP+ gave higher activity with abdomen preparations. These data document the metabolism of succinate to acetyl‐CoA and not to a methylmalonyl unit prior to incorporation into lipid in the housefly and establish the role of the malic enzyme in this process.
Diflubenzuron‐Induced alterations during in vitro development of Tenebrio molitor pupal integument
Authors: Soltani N. , Quennedey A. , Delbecque J. P. , Delachambre J.
Published: February 2005

The effects of diflubenzuron (DFB) in Tenebrio molitor pupae were first investigated on cuticle secretion induced by 20‐hydroxyecdysone in vitro. The sternal integuments were treated by DFB either 3 days before culture or during culture. DFB, when applied before culture, did not prevent the molting hormone from inducing a new cuticle deposition by integument explants in vitro. However, this cuticle showed several architectural alterations and a thickness reduction. When applied during the culture in the presence of 20‐hydroxyecdysone, DFB at high dose (≥ 20 μg/ml) was able to inhibit cuticle secretion, but lower doses (⩽ 10 μg/ml) resulted in epicuticle deposition. These observations confirm in vivo studies showing antagonistic effects of DFB and ecdysteroids at the level of epidermal cells.

In another series of experiments, the DFB effects were analyzed without addition of exogenous molting hormone in vitro. Because it had been observed in previous studies that pupal epidermal explants of Tenebrio secrete low but significant amounts of ecdysteroids in the culture medium, this in vitro secretion was measured by radioimmunoassay after DFB treatment. It was observed that DFB, when applied either before or during culture, significantly reduced the hormonal secretion in vitro. This reduction, observed at the level of epidermal cells, could be homologous with the diminution of the endogenous ecdysteroid peak previously described after in vivo DFB treatment in Tenebrio pupae.
Molecular characteristics of lipophorin, the juvenile hormone‐binding protein in the hemolymph of the Colorado potato beetle
Authors: de Kort C. A. D. , Koopmanschap A. B.
Published: February 2005

Lipophorin, the protein that specifically binds juvenile hormone in the hemolymph of the Colorado potato beetle, Leptinotarsa decemlineata, is a high‐density lipoprotein of Mr ∼ 574,000. Lipophorin contains 43% lipid and is composed of two apoproteins: apolipophorin I (Mr ∼ 251,000) and apolipophorin II (Mr ∼ 78,000). Both apoproteins contain mannose residues. Carotenoids make up a substantial part of the lipid fraction. Lipophorin constitutes about 25% of the total hemolymph proteins. Its concentration in the hemolymph (26 μM in 4‐day‐old long‐day and 40 μM in 4‐day‐old short‐day beetles) changes with different physiological conditions concomitant with changes in total protein content. Lipophorin specifically binds 10R‐juvenile hormone III with high affinity. The dissociation constant for 10R‐juvenile hormone III is 12 ± 2 nM. One lipophorin molecule contains one specific juvenile hormone‐binding site. The concentration of binding sites therefore equals that of lipophorin in hemolymph.
Major plasma proteins of larvae of the southwestern corn borer, Diatraea grandiosella
Authors: Lenz C. J. , Venkatesh K. , Chippendale G. M.
Published: February 2005

The major protein fraction of the larval hemolymph of the southwestern corn borer, Diatraea grandiosella, was shown to be composed of approximately six native proteins, and accounted for up to 55% of the plasma proteins. The apparent molecular weights of these proteins ranged from 350,000 to 500,000, as determined by nondenaturing electrophoresis at a neutral pH. Lower apparent molecular weights were obtained when the major protein fraction was subjected to electrophoresis at a high pH under nondenaturing conditions, indicating that these proteins dissociated under alkaline conditions. The isoelectric points of the major hemolymph proteins fell between 5.6 and 5.9. Denaturing electrophoresis and two‐dimensional electrophoresis showed that the native proteins were composed of subunits having apparent molecular weights of 78,000, 84,000, and 87,000. These subunits were the major labeled polypeptides found in the hemolymph of feeding last instar larvae 24 h after they had been injected with [3H]leucine. They were also the major polypeptides synthesized when the fat body of last instar prediapausing larvae was incubated in vitro for 4 h in Grace's medium containing [3H]leucine. Immunoelectrophoresis confirmed that the major hemolymph proteins were present in the larval fat body. In addition, these proteins were the major proteins present in the hemolymph of diapasuing larvae.
Effects of precocene II on female‐specific hemolymph polypeptides in Oncopeltus fasciatus
Authors: Martínez T. , Garcerá M. D.
Published: February 2005

Using polyacrylamide gel electrophoresis (PAGE) under denaturing conditions, two major polypeptides of 200,000 and 170,000 daltons were detected in the hemolymph of mature female Oncopeltus fasciatus, but they were not found in the hemolymph of males or newly emerged females. Those polypeptides constituted the two major bands of early vitellogenic oocytes; however, they were absent from the yolk of mature eggs. The slower‐migrating band (200,000 daltons) appears to correspond to a vitellogenic protein already identified in O. fasciatus, whose synthesis has been suggested to be independent of juvenile hormone (JH). Treatment of newly emerged adult females with the corpus allatum cytotoxin precocene II prevented the appearance of the female‐specific bands and induced an important accumulation of other proteins in the hemolymph. Yolk deposition was also inhibited in those animals. Topical application of JH to precocene‐treated females restored the appearance of the 200,000 and 170,000 dalton polypeptides in the hemolymph. These results suggest that JH is required for the synthesis of female‐specific polypeptides in O. fasciatus.
Regulation of long‐term oviposition in the house cricket, Acheta domesticus: Roles of prostaglandin and factors associated with sperm
Authors: Murtaugh Michael P. , Denlinger David L.
Published: February 2005

The transfer of spermatophore contents derived from testes during mating greatly stimulates ovipositional activity for long periods of time in the house cricket, Acheta domesticus (L.). Since prostaglandins appear to play a role in reproduction in several insect species, and since prostaglandin synthesis enzymes occur in cricket testes and spermatophores, we investigated the role of prostaglandins in the regulation of long‐term oviposition. Inactivation of prostaglandin synthesis enzymes in males or females using specific inhibitors failed to block mating‐induced increases in egg laying. However, males lacking sperm because of X‐irradiation were unable to induce oviposition even though they mated, transferred spermatophores, and had high levels of prostaglandins in both testes and spermatophores. X‐irradiation was also used to generate males with nonfunctional sperm. Females mated to these animals readily laid eggs, which failed to develop. It appeared that sperm or a factor associated with sperm induced long‐term oviposition in female house crickets. Prostaglandin synthesis enzymes transferred from the male to females may have other roles in the female, for example, in sperm maintenance in the spermatheca. Previous observations strongly suggest that prostaglandins induce egg laying behavior and activity; they may be synthesized by female enzymes that are regulated by male‐derived factors.
Monoclonal antibodies specific for apoproteins of lipophorins from the migratory locust
Authors: Schulz Thomas K. F. , Van Der Horst Dick J. , Amesz Hans , Voorma Harry O. , Beenakkers Ad M. Th.
Published: February 2005

Spleen lymphocytes from mice immunized with locust native low‐density lipophorin A+ (LDLp) were fused with nonproducing myeloma cells, strain Sp 2/0. Hybridomas that were isolated from the fused cells produced antibodies specific for LDLp and the high‐density lipophorin Ayellow (HDLp).

Monoclonal strains were generated through cloning by limiting dilution from those hybridomas synthesizing antibodies specific for apolipophorins (apoLp)‐I, ‐II, and ‐III of LDLp. Additionally, a hybridoma strain that was obtained after fusion of lymphocytes from mice immunized with apoLp‐III produced antibodies that bind to apoLp‐III and native LDLp.

Some features of LDLp and HDLp were studied using these antibodies. It could be demonstrated that apoLp‐I and apoLp‐II are not immunochemically identical and are exposed in the native particle of both LDLp and HDLp. It was also shown that in both lipophorins apoLp‐II is less exposed than apoLp‐I, whereas in LDLp apoLp‐III is mainly exposed; some apoLp‐III could also be detected in HDLp.

Tween‐20, a nonionic detergent, appears to affect the binding of anti‐apoLp‐I, ‐II, and ‐III to both LDLp and HDLp. The monoclonal antibodies specific for locust apolipophorins do not bind to the respective apoproteins of lipophorins from other insects.
Metabolism of polyunsaturated fatty acids by larvae of the waxmoth, Galleria mellonella
Authors: Stanley‐Samuelson David W. , Jurenka Russell A. , Loher Werner , Blomquist Gary J.
Published: February 2005

The metabolic fates of linoleic (18:2n6) and linolenic (18:3n3) acids injected into the hemocoel of fifth instar larvae of the waxmoth, Galleria mellonella, were examined by radio‐high‐pressure liquid chromatography and radio‐gas‐liquid chromatography. In addition to undergoing β‐oxidation and incorporation into neutral and phospholipid fractions, a portion of both of these C18 fatty acids was elongated and desaturated to longer chain and more unsaturated polyenoics. Radioactivity from linoleic acid was recovered in components that coeluted with 18:3, 18:4, 20:3, and 20:4. Radioactivity from linolenic acid was recovered in an unidentified component and in components that coeluted with 18:4, 20:3, and 20:5. Labeled arachidonic and eicosapentaenoic acids injected into waxmoth larvae were converted to prostaglandins, suggesting that one aspect of the biological significance of the elongation/desaturation reactions is to generate precursors for prostaglandin biosynthesis.
Single‐channel recordings of chloride currents in primary cultured Drosophila neurons
Authors: Yamamoto Daisuke , Suzuki Nobuyuki
Published: February 2005

Single‐chloride‐channel currents were recorded from primary cultured Drosophila neurons by means of the gigaohm‐seal patch‐clamp technique. Small inward‐going current channels were observed in excised inside‐out patches with the external face of the membrane exposed to bathing solutions devoid of K+, Na+, and Ca2+. The inward current was affected by changing the anions but not the cations bathing the cytoplasmic face of the patch. Complete replacement of CI− by glutamate eliminated the current. The current was maintained with intracellular solutions containing NO3− in place of CI−. The single‐channel conductance was estimated to be 7 ps with CI−, and 11 ps with NO3− at 10°C. Possible functions of this anion‐selective channel have been discussed.
Properties of an ATP‐dependent Ca2+ pump and (Ca2+ + Mg2+)‐ATPase in brain synaptosome membrane vesicles from the bertha armyworm, Mamestra configurata Wlk
Authors: Luo Ma , Bodnaryk Robert P.
Published: February 2005

Biochemical and kinetic properties under identical substrate and reaction conditions were obtained for an ATP‐dependent Ca2+ pump and (Ca2+ + Mg2+)‐ATPase in synaptosome membrane vesicles prepared from the brain of the moth, Mamestra configurata. Both the ATP‐dependent Ca2+ pump and (Ca2+ + Mg2+)‐ATPase had single, high‐affinity binding sites for ATP (Km = 14 and 116 μM, respectively), Ca2+free (Km = 0.13 nM and 0.072 nM, respectively), and Mg2+ (Km = 1.1 mM and 0.07 mM, respectively). Both systems were relatively little affected by K+ and were insensitive to ouabain, an inhibitor of (Na+ + K+)‐ATPase. The results indicate that the ATP‐dependent Ca2+ pump and (Ca2+ + Mg2+)‐ATPase are functionally coupled in synaptic membranes and constitute a mechanism for Ca2+ transport in the brain of M. configurata.

Although moth brain (Ca2+ + Mg2+)‐ATPase is maximally active at nanomolar concentrations of free calcium ion, the enzyme retains at least one‐half of its maximal activity at micromolar calcium concentrations, indicating either that the enzyme has two binding sites for calcium (a high‐affinity site at nanomolar Ca2+free and a low‐affinity site at micromolar Ca2+free), or that there are two enzymes with high and low affinity for calcium, respectively. Calcium extrusion from brain neurones of M. configurata may operate in a two‐stage, concentration‐dependent process in which a first stage, low‐affinity pump reduces intraneuronal calcium to a concentration at which a second stage, high‐affinity pump becomes activated.
Enhancement by farnesol and farnesoic acid of juvenile‐hormone biosynthesis in induced low‐activity locust corpora allata
Authors: Couillaud Franck , Mauchamp Bernard , Girardie Adrien , de Kort Stan
Published: February 2005

Exogenous farnesol or farnesoic acid (FA) stimulates juvenile hormone III (JH III) biosynthesis by isolated corpora allata from Locusta migratoria in a dose‐dependent manner. Farnesol and FA also stimulate a dose‐dependent accumulation of substantial amounts of methyl farnesoate (MF), identified by gas chromatography‐mass spectroscopy (GCMS) analysis, in the corpora allata. Lower quantities of MF were found in the incubation medium. Corpora allata, denervated 2 days prior to assay, showed low spontaneous rates of JH biosynthesis which were stimulated by farnesol and FA. The dose‐response curves for control and denervated corpora allata were similar. During oocyte maturation the rate of farnesol and FA stimulation of JH biosynthesis increased gradually. However, after transection of nervus corporis allati 1 (NCA‐1), the rate of stimulated JH synthesis was maintained at preoperative levels. Although the spontaneous rate of JH biosynthesis decreased rapidly after NCA‐1 transection, denervated glands could still be stimulated by farnesol or FA to produce large amounts of JH. These results suggest that the low spontaneous rate of JH biosynthesis in denervated corpora allata is not caused by inhibition of the final steps of JH biosynthesis.
Ecdysteroid regulation of amino sugar uptake in a lepidopteran cell line derived from imaginal discs
Authors: Porcheron P. , Oberlander H. , Leach C. E.
Published: February 2005

The hormonal regulation of uptake of amino sugars was examined in a cell line derived from imaginal wing discs of the Indianmeal moth, Plodia interpunctella (Hübner). The cell line, IAL‐PID2, responded to treatment with 20‐hydroxyecdysone with increased uptake of N‐acetyl‐D‐glucosamine, N‐acetyl‐galactosamine, and D‐glucosamine. Uptake was stimulated with 3‐O‐methyl D‐glucose, and to a lesser extent with 2‐deoxy‐D‐glucose, but not with D‐glucose or D‐mannose. The requirements for optimum hormonal stimulation of uptake of N‐acetyl‐D‐glucosamine were similar to those for uptake of this chitin precursor by intact wing discs. The PID2 cell line is responsive to treatment with 20‐hydroxyecdysone and offers promise of being amenable to a detailed analysis of the mode of action of ecdysteroids on the uptake of amino sugars.
Profile of free and conjugated ecdysteroids and ecdysteroid acids during embryonic development of Manduca sexta (L.) following maternal incorporation of [14C]cholesterol
Authors: Thompson Malcolm J. , Svoboda James A. , Lozano Ruben , Wilzer Kenneth R.
Published: February 2005

The levels of both free and conjugated ecdysteroids, maternally labeled from [14C]cholesterol, of six different age groups of Manduca sexta eggs were quantitatively determined. Eggs 0–1‐h old contain about 2.5 and 35 μ/g of the 2‐ and 26‐phosphates of 26‐hydroxyecdysone, respectively, and 1 μg/g of 26‐hydroxyecdysone. During embryogenesis of 26‐hydroxyedcdysone 26‐phosphate is hydrolyzed to 26‐hydroxyecdysone, which reaches a peak titer in 1–18‐h‐old eggs; the level of 26‐hydroxyecdysone 2‐phosphate remains rather constant. Additionally, other metabolic modifications such as hydroxylation, conjugation, epimerization, and oxidation are occurring; and as the level of the 26‐hydroxyecdysone 26‐phosphate decreases there is a progression of other ecdysteroids. C‐20 hydroxylation first appears in 24–40‐h‐old eggs and reaches peak activity in 48–64‐h‐old eggs, where 20‐hydroxyecdysone and 20, 26‐dihydroxyecdysone are both present at peak titer but the latter is the major free ecdysteroid. Ecdysone is observed at measurable levels only in the three age groups of eggs between 1 and 64 h‐old. C‐3 epimerase activity also appears at 24–40 h and continually increases throughout embryogenesis to the point that 3‐epi‐26‐hydroxyecdysone and 3‐epi‐20, 26‐dihydroxyecdysone are the major free ecdysteroids in 96‐h‐old eggs. A new ecdysteroid conjugate, 26‐hydroxyecdysone 22‐glucoside, first appears at 24–40h and becomes the major conjugate in 72–80‐h‐old eggs; it represents an apparent end‐product as its peak titer is reached and maintained throughout the final embryonic stages. 20‐Hydroxyecdysonoic acid occurs in 48–64‐h‐old eggs, and along with 3‐epi‐20‐hydroxyecdysonoic and ecdysonoic acids in 72–88‐h‐old eggs. 20‐Hydroxyecdysonoic acid peaks during the latter time interval, and as its titer subsequently falls, there is a concurrent increase in the level of 3‐epi‐20‐hydroxyecdysonoic which was identified as the second major component of the ecdysteroid conjugate fraction of 0–1‐h‐old larvae. Our results indicate that there is little or no biosynthesis of ecdysteroids during embryogenesis; that the materal ecdysteroid conjugate 26‐hydroxyecdysone 26‐phosphate serves as source for 26‐hydroxyecdysone and the numerous metabolites; that 26‐hydroxyecdysone and 20,26‐dihydroxyecdysone may be the active hormones during embryonic development; and that glucosylation, epimerization, and formation of acids cosntitute inactivation processes. A scheme of the proposed pathways involved in the metabolism of 26–hydroxyecdysone 26‐phosphate in the developing eggs of m. sexta is presented.
Ecdysteroids in Carausius eggs during embryonic development
Authors: Fournier Bernard , Radallah Driss
Published: February 2005

Peaks of ecdysteroids were observed during the different phases of embryonic development of intact Carausius eggs or eggs precociously deprived of their exochorion and cultivated under paraffin oil. Several groups of ecdysteroids were separated and analyzed by thin‐layer chromatography (TLC) and high performance liquid chromatography (HPLC) combined with radioimmunoassay. Ecdysteroids were similar in the two categories of eggs, including high‐polarity products (essentially conjugates hydrolyzable by Helix pomatia digestive juice, or alkaline phosphatase), possible ecdysonoic acids (unhydrolyzable polar substances), free hormones, and nonpolar ecdysteroids. Four ecdysteroids were identified by co‐elution during HPLC with reference compounds of 20,26‐dihydroxyecdysone, 20‐hydroxyecdysone, ecdysone, and 2‐deoxy‐20‐hydroxyecdysone. Concentrations of these substances (free and conjugated forms) were studied during the different stages of embryonic development: 20‐hydroxyecdysone and 2‐deoxy‐20‐hydroxyecdysone were the major free ecdysteroids. They showed parallel variations with large peaks at stages VI8 and VII6 whereas ecdysone titers were consistently low. Injected labelled ecdysone was converted efficiently into 20‐hydroxyecdysone, and both compounds underwent 26‐hydroxylation and/or conjugation to polar or apolar metabolites.
Sex pheromone biosynthesis in the leafroller moth Planotortix excessana by Δ10 desaturation
Authors: Foster S. P. , Roelofs W. L.
Published: February 2005

With the use of deuterium‐labeled saturated fatty acids coupled with gas chromatography‐mass spectrometric analysis, biosynthesis of the sex pheromone component (Z)‐8‐tetradecenyl acetate in the greenheaded leafroller moth Planotortrix excessana was shown to proceed via Δ10 desaturation of palmitate. The resultant (Z)‐10‐hexadecenoate is two carbon chain‐shortened to the precursor (Z)‐8‐tetradecenoate. The minor component (Z)‐10‐tetradecenyl acetate is biosynthesized by Δ10 desaturation of myristate. This is the first confirmation of Δ10 desaturation in an eukaryotic system.
Effects of ionophores on vitellogenin uptake by Hyalophora oocytes
Authors: Stynen Dirk , Woodruff Richard I. , Telfer William H.
Published: February 2005

Oocytes of Hyalophora cecropia that were incubated in vitro with [35S]vitellogenin incorporated label within 10 min into an intermediate‐density compartment identified by sucrose density gradient centrifugation. During a subsequent 20‐min chase this presumptive endosomal label was transferred to a compartment with the higher density of protein yolk spheres. When vitellogenin uptake was inhibited by 10 μM nigericin or monensin, or 50 μM carbonyl cyanide m‐cholorophenylhydrazone, a somewhat larger and more focused peak of label accumulated in the endosome region of the gradient, and the transfer of this label to the yolk spheres was blocked. Valinomycin, at concentrations as high as 100 μM, did not inhibit uptake or processing, even though successful insertion into the oocyte membrane could be demonstrated by the effects of this ionophore on the membrane potential and K+ permeability of the follicle. Inhibition of processing by nigericin and monensin is consistent with a model of endocytosis in which the ionophores prevent acidification of the endosomes by promoting H+‐K+ exchange with the cytoplasm. Several alternative possibilities were ruled out by physiological analyses entailing the measurement of cytoplasmic pH and membrane potentials.
Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta
Authors: Ryan Robert O. , Cole Kenneth D. , Kawooya John K. , Wells Michael A. , Law John H.
Published: February 2005

The identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [35S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of Mr ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ∼8.6). Electrophoresis under denaturing conditions reveals a subunit Mr ≈ 50,000 while the native protein has an apparent Mr ∼ 85,000 by gel permeation chromatography. Anti‐PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.
Experimental modifications of an insect vitellin affect its structure and its uptake by oocytes
Authors: Gochoco Caroline H. , Kunkel Joseph G. , Nordin John H.
Published: February 2005

The 18S and 33S vitellins (Vts) of Blattella germanica were subjected to periodate oxidation and digestions with α‐mannosidase, endo‐β‐Nacetylglucosaminidase H (endo‐H), and trypsin to study their effects on Vt structure and function. Periodate oxidation caused 33S Vt to dissociate to a form that cosedimented with 18S Vt upon glycerol gradient centrifugation but had no effect on the sedimentation of 18S Vt. This result implicates the oligosaccharides in stabilization of the 33S structure. Incubation of 18S and 33S Vts with α‐mannosidase and endo‐H revealed that the oligosaccharides of both Vts are largely shielded from attack by both glycosidases. However, the carbohydrate of 18S Vt was 3 to 5 times more susceptible to both enzymes, suggesting that the 18S to 33S transition results in decreased accessibility of the oligosaccharides to both glycosidases. Short‐term exposure of 18S and 33S Vts to trypsin resulted in limited hydrolysis; the Mr 102,000 subunit of each form was cleaved with an Mr79,000 peptide as a major product. However, the sedimentation properties of the Vts and their relative susceptibilities to α‐mannosidase were unchanged; therefore while the Mr102,000 subunit of the Vt is vulnerable to trypsin, it retains its higher‐order structure after limited digestion. Endocytosis of radiolabelled 18S Vt by oocytes in vivo decreased about 15‐fold after its modification by periodate and sixfold after treatment with α‐mannosidase. Limited trypsin digestion also severely diminished its uptake. All injected radioactivity of unmodified 18S and 33S Vts could be recovered from either the hemolymph or ovaries of recipient females. However, modified Vts were taken up from the hemolymph primarily by cells of the pericardium and the fat body, suggesting that these organs participate in a clearance mechanism that recognizes “damaged” Vt molecules.
Correlation of yolk phosphatase expression with the programmed proteolysis of vitellin in Blattella germanica during embryonic development
Authors: Purcell John P. , Quinn Theresa M. , Kunkel Joseph G. , Nordin John H.
Published: February 2005

Proteolytic processing of the vitellin in Blattella germanica eggs occurs 4 days postovulation and is correlated with both the onset of its utilization and the major portion of the embryo's growth. Yolk phosphatase is also expressed coincident with this event, and some aspects of its activation have been investigated. The enzyme is absent from the ooplasm (yolk) during the first 2 days following ovulation but increases approximately 20‐fold in specific activity between days 3 and 4, when assayed at pH 3.9 or 4.8 and 9‐fold at pH 6.5. No activation is observed for yolk‐bound α‐mannosidase, its specific activity remains elevated through the first 6 days following ovulation. This suggests that expression of the phosphatase is regulated independently of that of α‐mannosidase in the yolk. Yolk with active phosphatase can dephosphorylate native vitellin, delipidated vitellin, and phosphocasein. Sucrose density gradient centrifugation of yolk obtained from eggs 4 days postovulation, revealed that phosphatase activity cosediments with material which reacts with antivitellin antibodies, while α‐mannosidase and β‐N‐acetyl glucosaminidase are found near the top of the gradient. Oothecae derived from crossing certain translocational heterozygote males and wild‐type females contain some eggs with severely depressed levels of yolk phosphatase in which embryos do not grow. Vitellin in these eggs fails to undergo proteolytic processing as late as day 5 postovulation and retains the subunit composition of freshly ovulated vitellin.
Adipokinetic response of a flightless grasshopper (Barytettix psolus): Functional components, defective response
Authors: Ziegler Rolf , Ryan Robert O. , Arbas Edmund A. , Law John H.
Published: February 2005

The mature flightless grasshopper Barytettix psolus shows a very small adipokinetic response when injected with extracts of its own corpora cardiaca, although the fat body contains enough lipid for a strong response. When these extracts were injected into Melanoplus differentialis, a grasshopper capable of flight, or the moth Manduca sexta, much stronger adipokinetic responses were observed. Upon analysis of B. psolus extracts by HPLC, two components with adipokinetic activity were obtained. The major component appears to be identical to locust adipokinetic hormone (AKH) I. Extracts of B. psolus corpora cardiaca also activated fat body glycogen phosphorylase in B. psolus. This activation, however, did not result in an increase in hemolymph sugar, probably because of low levels of glycogen in the fat body.

B. psolus hemolymph contains a high‐density lipophorin (HDLp) consisting of the apolipophorins (apoLp) I and II and lipid. Both apoproteins are glycosylated. The hemolymph also contains apoLp‐III, although this apoprotein apparently does not associate with HDLp to form a low‐density lipophorin (LDLp) following AKH or corpora cardiaca extract injections. When B. psolus lipophorin and AKH were injected into Schistocerca americana, lipophorin took up lipids and combined with apoLp‐III, forming LDLp. ApoLp‐III from B. psolus injected into S. americana can also form LDLp, demonstrating that the components are functional. A lipid transfer particle isolated from M. sexta and injected into B. psolus does not improve the adipokinetic response. Thus, it appears that the adipokinetic response of B. psolus is not deficient because of the lack of AKH or functional lipophorins, but may be caused by the lack of a full response to AKH by fat body or the deficiency in hemolymph of some as yet unknown factor.
Midgut amylase, lysozyme, aminopeptidase, and trehalase from larvae and adults of Musca domestica
Authors: Terra Walter R. , Espinoza‐Fuentes F. P. , Ferreira Clélia
Published: February 2005

Based on polyacrylamide gel electrophoresis, density‐gradient ultracentrifugation and thermal inactivation, there is only one major molecular species of each of the following larval enzymes (soluble in water or solubilized in Triton X‐100): membrane‐bound aminopeptidase (pH optimum 8.5; Km 0.21 mM L‐leucine p‐nitroanilide; Mr 322,000), amylase (pH optimum 6.5; Km 0.14% starch; Mr 66,000), lysozyme (pH optimum 3.5; Km 0.3 mg/ml; Mr 24,000); and membrane‐bound trehalase (pH optimum 5.0; Km 1.09 mM trehalose; Mr 94,000). Except for lysozyme, the properties of adult digestive enzymes are different from those described for larval enzymes. Larval aminopeptidase and trehalase were purified by electrophoresis and larval lysozyme (contaminated with amylase) by density‐gradient ultracentrifugation, and were used to raise antibodies in a rabbit.

Antibodies raised against larval aminopeptidase, trehalase, and amylase did not recognize the imaginal enzymes, whereas those against larval lysozyme recognize imaginal lysozyme. The data suggest that the genes coding for digestive enzymes (except for lysozyme) are different in larvae and imagoes.