bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of the brain and corpus cardiacum on egg production in Rhodnius prolixus
Authors: Davey K. G.
Published: February 2005

Using total egg production corrected for size of blood meal as an index of the activity of the corpus allatum (CA), the effects of various surgical manipulations of the neuroendocrine system have been examined. Isolation of the CA from its nervous connections increases egg production well beyond that of a normal insect, thus confirming that the CA is at least partly controlled by inhibitory nerves from the brain. Removal of the corpora cardiaca (CC) reduces the level of this increased egg production, and decapitation anterior to the CC results in a level of egg production that is greater than that found in females decapitated between the CA and CC. Implanting a CC together with a CA into a decapitated female results in a higher egg production than implanting a CA alone. These results demonstrate that an allatotropic influence is exerted by the CC. Experiments designed to examine the role of the brain were inconclusive and did not eliminate the possibility that the allatotropin from the CC originated in the brain.
Biochemical and immunological studies on an α‐glycerophosphate dehydrogenase from the tephritid fly, Anastrepha suspensa
Authors: Sarma Ranjit , Kitto G. Barrie , Berlocher Stewart , Bush Guy L.
Published: February 2005

A rapid and efficient procedure has been developed for the purification of α‐glycerophosphate dehydrogenase from the tephritid fly Anastrepha suspensa. This procedure is applicable to the isolation of the enzyme from other tephritids. The A. suspensa α‐glycerophosphate dehydrogenase is dimeric with a molecular weight of 70,000 and a subunit molecular weight of 35,000. The pH optimum of the enzyme is 7.0. The amino acid composition is compared with that of other α‐glycerophosphate dehydrogenases. By means of the quantitative microcomplement fixation procedure the A. suspensa α‐glycerophosphate dehydrogenase is compared immunologically to a variety of other tephritid and dipteran α‐glycerophosphate dehydrogenases.
Variation in thickness and protein content of the cuticle of the female of Glossina austeni
Authors: Solowiej S. , Davey K. G.
Published: February 2005

The thickness and total protein content of the ventral abdominal cuticle of the female tsetse, Glossina austeni, increase during the early part of each pregnancy cycle, reaching a maximum at approximately 2 days after ovulation. They decrease thereafter, and reach a minimum value just before larviposition. Virgin females do not exhibit a cycle of protein content or thickness in the cuticle. Preliminary data on the incorporation of [3H]tyrosine or [3H]leucine into the water‐soluble proteins of the ventral abdominal cuticle at the time of the second larviposition suggest that there is rapid turnover of protein in the cuticle at this time. These observations are consistent with the net storage of protein in the cuticle during the early part of pregnancy cycle followed by a net depletion of that store as the nutritional demands of the rapidly growing larva in utero exceed the capacity of the ingested blood meals to supply them.
Isolation and characterization of vitellin from the fruitfly, Dacus oleae
Authors: Levedakou Eleni N. , Sekeris Constantine E.
Published: February 2005

Vitellin was isolated from mature eggs of Dacus oleae. A combination of anion‐exchange chromatography and gel filtration was used for purification of the protein. The molecular weight of isolated vitellin, as determined by Sephacryl S‐300 chromatography, was approximately 300,000. Electrophoresis on SDS‐polyacrylamide gels demonstrated the presence of vitellin subunits with molecular weight of 47,000 and 49,000. Isoelectric focusing on polyacrylamide gels revealed a series of polypeptides with isoelectric points covering an acidic pH region of 5.7 to 6.2. Immunodiffusion, immunoelectrophoresis, and immunoblotting were used for further characterization of vitellin.
Ecdysteroid production by a continuous insect cell line
Authors: Ward G. B. , Kelly T. J. , Woods C. W. , Marks E. P.
Published: February 2005

The spent medium from ten established cell lines was extracted and tested for ecdysteroids by radioimmunoassay. Of the seven lepidopteran lines tested, only IAL‐TNDI and MRRL‐CH showed evidence of ecdysteroid production. However, the results were erratic and difficult to evaluate and these lines were dropped from further consideration. However, of the three cockroach cell lines tested, one, UMBGE 4, produces ecdysteroid and consistently releases virtually all of it into the medium. The main ecdysteroid was identified as ecdysone and the increase was logarithmic during the first 11 days of the subculture, with a decrease from day 11 to day 14.

UMBGE 4 is a vesicle cell line which also tested positive for chitin synthesis. When the pH of the medium was lowered from pH 7.4 to pH 6.3, both the chitin synthesis and the ecdysone synthesis dropped by roughly 50%.
Isolation of two arginine vasopressin‐like factors from ganglia of Locusta migratoria
Authors: Schooley David A. , Miller Christine A. , Proux Jacques P.
Published: February 2005

Tissues of Locusta migratoria are known to contain a material which crossreacts with an antibody against arginine vasopressin (AVP), and this factor has been correlated with the diuretic hormone of this species. In this paper, we report the isolation of two AVP‐like factors from suboesophageal ganglia and thoracic ganglia of Locusta migratoria. The less abundant, more hydrophobic of these AVP‐like factors shows diuretic activity in an assay where excretion of amaranth dye from Locusta migratoria hemolymph is used as the scoring criterion. After extracting a total of ∼ 51,000 ganglia with an acidic solvent, the crude extract was prepurified by batch adsorption/elution from disposable reversed‐phase cartridges. The prepurified extract was then sequentially purified by reversed‐phase liquid chromatography using solvent programs of substantially differing selectivity. The more abundant factor was isolated to apparent homogeneity in three steps, while the less abundant factor required four or five steps. Use of a C4 reversed‐phase column minimized losses of the minor, more hydrophobic factor.
Some aspects of activity regulation of Galleria mellonella PTTH cells
Authors: Muszyńska‐Pytel Malgorzata
Published: February 2005

Using the Galleria prothoracicotropic bioassay, five small neurosecretory cells occurring in each dorsolateral part of protocerebrum of Galleria mellonella brain were identified as prothoracicotropic hormone (PTTH) cells. It was found that the critical period for the release of PTTH from a brain implanted in neck‐ligated larva lasts up to the third day after implantation. The content of paraldehyde‐fuchsin positive neurosecretory material (NSM) in PTTH cells was determined during the penultimate and last larval instar, during pupal instar, and in starved or poststarvation fed or space‐deprived last instar larvae. Two peaks of NSM in PTTH cells were found in the penultimate instar (in freshly molted, and 76‐h‐old larvae), four peaks in the last instar larvae (in freshly molted, and in 67‐, 132‐, and 174‐h‐old larvae), and one peak in the pupal instar (in 56‐76‐h‐old pupae). It was also observed that upon starvation NSM accumulated in PTTH cells, while after 3 h of poststarvation feeding it was released. In permanent space‐deprived last instar larvae no NSM occurred in PTTH cells. In all investigated larval instars a rapid release of NSM from PTTH cells was found a few hours after molt associated with the beginning of the feeding period. The significance of the NSM content in PTTH cells is discussed in relation to ecdysteroid titer.
Sodium inward currents through calcium channels in mealworm muscle fibers
Authors: Yamamoto Daisuke
Published: February 2005

The contribution of Na+ ions to the nonsynaptic electrogenesis was studied in the larval muscle fibers of mealworm, Tenebrio molitor, using currentclamp and voltage‐clamp techniques. Na‐dependent graded responses were generated by depolarizing current stimuli in Ca2+‐free solutions. These responses were insensitive to tetrodotoxin and were blocked by Co2+. Large inward‐going currents were elicited by step depolarizations in Ca2+‐free solutions under voltage‐clamp conditions. The inward currents were totally eliminated by removal of Na+ from the bathing solution. These results indicate that the calcium channel of mealworm muscle is permeable to Na+.
Purification and characterization of three major hemolymph proteins of an insect, Calpodes ethlius (lepidoptera, hesperiidae)
Authors: Palli Subba Reddy , Locke Michael
Published: February 2005

Like many other Lepidoptera, fifth‐stage Calpodes larvae have three major hemolymph proteins. Their molecular weights were estimated by 3‐15% nondenaturing polyacrylamide gel electrophoresis (N‐PAGE) as 470,000 (arylphorin; Ar), 580,000 (storage protein 2; SP2) and 720,000 (storage protein 1; SP1). Carbohydrate is associated with all three, but only Ar has lipid. The three proteins have been purified by preparative N‐PAGE and sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis. On 3‐15% SDS gels, Ar dissociated into 82,000 Mr subunits, SP2 into 86,000 Mr subunits, and SP1 into both 86,000 and 90,000 Mr subunits. The 470,000 Mr protein is identified as Ar because it is rich in aromatic amino acids. The 580,000 and 720,000 Mr proteins are rich in glycine and are called storage proteins. Electron microscopy of negatively stained preparations shows that each polymer has a different geometrical arrangement of subunits. SP1 is a cube made from eight subunits. SP2 is a hexamer in the form of a pentahedral prism. Ar is probably an octahedron made from six subunits. All three geometrical arrangements could permit the presence of a central carrying space.
Mode of action of proctolin on locust visceral muscle
Authors: Lange Angela B. , Orchard Ian , Lam William
Published: February 2005

Proctolin increases the frequency and amplitude of myogenic contractions and results in a sustained contraction of the oviducts of Locusta migratoria. The possible mode of action of proctolin receptors on this visceral muscle has been investigated. Calcium‐free saline, containing either 20 mM magnesium ions or 100 μM EGTA, inhibited myogenic contractions, lowered basal tension, and abolished all the effects of proctolin following a 20 min incubation. These effects were reversible upon washing with normal saline. Similar results were obtained with normal saline containing 10 mM cobalt ions. Nifedipine at 50 μM lowered basal tension, abolished myogenic contractions, and reduced the proctolin‐induced sustained contraction by 42‐62% at 0.5 nM proctolin and by 33‐37% at 5 nM proctolin. Similar results were obtained with 100 μM verapamil. Proctolin was still capable of eliciting considerable contractions (25‐67% of controls) in preparations depolarized with 100 mM potassium saline. The removal of calcium from the high‐potassium saline reversibly abolished the potassium‐induced contraction and reversibly blocked the action of proctolin. Nifedipine was ineffective in blocking the action of proctolin in high‐potassium saline. Neither cyclic AMP levels nor cyclic GMP levels of the lateral oviducts were elevated by proctolin in the presence of a phosphodiesterase inhibitor. The results indicate that proctolin mediates its effects via an influx of external calcium ions. This calcium appears to enter through two channels, a voltage‐dependent channel and a receptor‐operated channel. Cyclic nucleotides do not appear to be involved in the action of proctolin in this visceral muscle.
Effects of steroid metabolism inhibitors and ecdysteroid analogs on honey bee sterol metabolism and development
Authors: Svoboda James A. , Herbert Elton W. , Thompson Malcolm J.
Published: February 2005

The effects of two azasteroids, an alkyl amine, and two ecdysteroid analogs on honey bee (Apis mellifera L.) sterol metabolism and development were compared by feeding each compound in a chemically defined diet supplemented with 24‐methylenecholesterol. Although each of the inhibitors, 25‐azacoprostane, 25‐azacholestane, or N,N‐dimethyldodecanamine, has been shown to severely inhibit steroid metabolism and larval development in a number of species of omnivorous and phytophagous insects, none of them affected honey bee brood development to the adult stage. Compared to the controls, there was a strikingly higher relative percentage of 24‐methylenecholesterol in the sterols from prepupae reared by workers fed diets containing each of these inhibitors, but the level of this sterol was reduced considerably in the adults reared from the brood in these test colonies. Each of the ecdysteroid analogs, 22,25‐dideoxyecdysone or 2,22,25‐trideoxy‐5β‐hydroxyecdysone, severely suppressed larval development when fed in the chemically defined diet. This effect apparently results from the presence of the dietary ecdysteroid or its metabolite in the brood food placed in the larval cells or the inability of the worker bees fed the ecdysteroid to include some essential factor in the brood food. The importance of this information relative to the possible application of certain steroid inhibitors to control pathogens of honey bees is discussed.
Properties of copper‐dependent o‐diphenol oxidase activity in the potato aphid Macrosiphum euphorbiae (thomas)
Authors: Skiba Paul J. , Mullin Christopher A.
Published: February 2005

Potato aphid Macrosiphum euphorbiae (Thomas) was found to contain high amounts of o‐diphenol oxidase activity. Enzyme activity was largely distributed into the postmitochondrial supernatant from Brij‐35 extracted aphids and occurs in a latent form that was activated up to 45‐fold by pretreatment with isopropanol. The aphid enzyme has a broad pH optimum near 6, and utilized L‐dopa (Km = 1.4 mM, Vmax = 348 nmol/min‐mg protein), dopamine, and 4‐methylcatechol the best out of the twelve substrates tested. In addition, this activity is a typical copper‐dependent oxidase in that it is potently inhibited by phenylthiourea (50% inhibition at 30nM) and other copper chelators, including salicylhydroxamic acid. The above properties are common to most insect tyrosinases. However, the aphid enzyme lacked the o‐hydroxylase and laccase components and the optimal activity at higher temperatures that are typical of cuticular tyrosinases of other insects. The high levels of o‐diphenol oxidase in aphids compared to other insects is surprising, since the major function associated with these enzymes, that of melanization and sclerotization of cuticle, is of much less importance to aphids. The possibility that aphids use this enzyme to metabolize dietary phenolics is discussed.
Metabolism and excretion of acetylchromenes by the migratory grasshopper
Authors: Isman Murray B. , Proksch Peter , Witte Ludger
Published: February 2005

The extent of metabolism and excretion of three acetylchromenes (two toxic, one relatively nontoxic) were examined in adult migratory grasshoppers (Melanoplus sanguinipes) following topical administration. Both the total amount excreted (parent plus metabolites) and the proportion of parent compound in the excreta were inversely correlated with contact toxicity. Both toxic and nontoxic acetylchromenes are rapidly absorbed from the cuticle, with maximum excretion of parent and metabolite chromenes from 4 to 8 h posttreatment in each case. Much of the applied compounds (60–80%) apparently remains within the insect, and cannot be recovered by extraction of the insect. Metabolites formed result from simple oxidative and reductive transformations. For all of the compounds tested (including the allatocidin precocene II), the major mode of metabolism results from aliphatic hydroxylation of one of the geminal methyl groups on the chromene. No conjugated metabolites were found in the excreta.
Identification of juvenile hormone III in the hemolymph of Blattella germanica adult females by gas chromatography‐mass spectrometry
Authors: Camps Francisco , Casas Josefina , Sanchez Francisco‐Jose , Messeguer Angel
Published: February 2005

Juvenile hormone III was identified in purified hemolymph extracts of adult females of the cockroach Blatella germanica by gas chromatography‐mass spectrometry with chemical ionization and selected ion monitoring. Under these conditions, juvenile hormones I and II were not detected within the sensitivity ranges of this analytical method. For quantification purposes a 5,5‐bisdeuterated analog of juvenile hormone III was synthesized and used as an internal standard. In general, juvenile hormone III titers obtained correlated with data on oocyte growth and with hormone titers found from in vitro corpora allata incubations along the first gonotrophic cycle.
Chitin metabolism
Authors: Chen Andrew C.
Published: February 2005

Recent studies on chitin metabolism in insect cuticle are reviewed. Differences in enzymes involved in both synthesis and degradation of chitin are discussed. Emphasis is put on the complexity of chitin degradation involving various enzymes. Evidence for the possible existence of an alternative pathway leading to the formation of chitin is introduced. The involvement of hormones in chitin metabolism is also briefly discussed.
Diphenols in hemolymph and cuticle during development and cuticle tanning of Periplaneta americana (L.) and other cockroach species
Authors: Czapla T. H. , Hopkins T. L. , Kramer K. J. , Morgan T. D.
Published: February 2005

Diphenolic compounds in cockroach hemolymph and cuticle were extracted with 1.2 N HCI, partially purified by alumina adsorption, and analyzed by liquid chromatography. Dopamine (DA) is the major catecholamine in hemolymph of Periplaneta americana, Blatta orientalis, Blattella germanica, Gromphadorhina portentosa, and Blaberus craniifer at adult ecdysis, while N‐acetyldopamine (NADA) predominates in hemolymph of Leucophaea maderae. In P. americana, NADA is the second most abundant catecholamine, while N‐β‐alanyldopamine (NBAD), norepinephrine (NE), 3,4‐dihydroxyphenylalanine, 3,4‐dihydroxyphenylethanol, 3,4‐dihydroxyphenylacetic acid, and 3,4‐dihydroxybenzoic acid occur in lesser quantities. Catecholamines occur mainly as acid labile conjugates in hemolymph. Dopamine, conjugated primarily as the 3‐sulfate ester, increases in hemolymph from 0.1 to 0.8 mM during the last instar. Concentrations decrease by 75% in pharate adults, partially because of an increase in hemolymph volume. A second smaller peak of DA sulfate occurs after ecdysis followed by a rapid disappearance as the cuticle tans. A conjugate of catechol (o‐dihydroxybenzene) is also present in relatively high concentrations at all ages examined. In cuticle, N‐β‐alanylnorepinephrine accumulates during the early period of adult tanning, while NBAD, NADA, N‐acetylnorepinephrine, and DA increase more slowly. The N‐β‐alanyl and N‐acetyl derivatives of DA and NE occure in relatively high concentrations in tanned cutical of P. americana and probably play an important role in the stablization process.
Platelet activating factor‐acetylhydrolase in insects: Identification and partial characterization of a 1‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine acetylhydrolase in a cell‐free system of Heliothis
Authors: Lambremont Edward N. , Malone Boyd , Snyder Fred
Published: February 2005

A specific acetylhydrolase that inactivates platelet activating factor (PAF; 1‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine), a potent cellular mediator in mammalian cells, by removal of the sn‐2 acetyl moiety, has been found in the cytosolic fraction of several postembryonic developmental stages and specific tissues of the corn earworm, Heliothis zea (Boddie). Effects of magnesium, calcium, EGTA, deoxycholate, dithiothreitol, diisopropylfluorophosphate, egg phosphatidylcholine, and an acylacetyl‐glycerophosphocholine show that hydrolysis of the acetate moiety is due to a specific acetylhydrolase for PAF. The activity does not appear to be due to a typical cellular phospholipase A2 that utilizes phospholipid substrates with a long‐chain acyl group at position sn‐2 of glycerol. Specific activities and properties of the acetylhydrolase from this insect match closely with those described from tissues of vertebrate animals.
Prophenoloxidase activation in the hemolymph of Sarcophaga bullata larvae
Authors: Saul Steven J. , Sugumaran Manickam
Published: February 2005

Phenoloxidase in the hemolymph of Sarcophaga bullata larvae is present as an inactive proenzyme form. Localization studies indicate that the majority of the prophenoloxidase is present in the plasma fraction whereas only a minor fraction (about 4%) is present in the cellular compartments (hemocytes). Inactive prophenoloxidase can be activated by zymosan, not by either endotoxin or laminarin. This activation process is inhibited by the serine protease inhibitors, benzamidine and p‐nitrophenyl‐p∼‐guanidobenzoate. Exogenously added proteases, such as chymotrypsin and subtilisin, also activated the prophenoloxidase in the whole hemolymph but failed to activate the partially purified proenzyme. However, an activating enzyme isolated from the larval cuticle, which exhibits trypsinlike specificity, activated the partially purified prophenoloxidase. Inhibition studies and activity measurements also revealed the presence of a similar activating enzyme in the hemolymph. Thus, the phenoloxidase system in Sarcophaga bullata larval hemolymph seems to be comprised of a cascade of reactions. An endogenous protease inhibitor isolated from the larvae inhibited chymotrypsin‐mediated prophenoloxidase activation but failed to inhibit the cuticular activating enzyme‐catalyzed activation. Based on these studies, the roles of prophenoloxidase, endogenous activating proteases, and protease inhibitor in insect immunity are discussed.
Metabolism of [14C]cholesterol in Manduca sexta pupae: Isolation and identification of sterol sulfates, free ecdysteroids, and ecdysteroid acids
Authors: Lozano Ruben , Thompson Malcolm J. , Lusby William R. , Svoboda James A. , Rees Huw H.
Published: February 2005

[14C]Cholesterol was injected into fifth‐instar larvae of Manduca sexta, and the metabolites were isolated and identified from 8‐day‐old male and female pupae. A major portion of the metabolized cholesterol was esterified either with a sulfate group or with fatty acids. The predominant ecdysteroid metabolites were 20‐hydroxyecdysone, 20,26‐dihydroxyecdysone, 20‐hydroxyecdysonoic acid, and 3‐epi‐20‐hydroxyecdysonoic acid. Smaller amounts of ecdysteroids were identified as conjugates of 26‐hydroxyecdysone, 3‐epi‐20‐hydroxyecdysone, 20,26‐dihydroxyecdysone, and its 3α‐epimer. The metabolic profiles were similar for both male and female pupae. The two ecdysteroid acids were identified by nuclear magnetic resonance spectroscopy and chemical ionization mass spectrometry and by mass spectral analyses of their methyl esters. Detection of 3‐epi‐20‐hydroxyecdysonoic acid as a major metabolite is significant, as its occurrence has been scarcely reported. 3‐Epiecdysteroid acid formation is discussed as a possible ecdysteroid‐inactivating pathway that may be operating specifically in lepidopterous insects or in particular developmental stages such as eggs or pupae.
Ecdysteroid profiles for hemolymph and testes from larvae, pupae, and pharate adults of the European corn borer, Ostrinia nubilalis hubner
Authors: Gelman Dale B. , Woods Charles W. , Borkovec Alexej B.
Published: February 2005

Total ecdysteroid levels as well as concentrations of several individual ecdysteroids were determined for hemolymph and testes of fifth instars, pupae, and pharate adults of the European corn borer, Ostrinia nubilalis (Hubner). For total levels, the patterns of fluctuation in hemolymph and testes were similar, but the concentrations in testes were lower than those in hemolymph. In both hemolymph and testes there were two ecdysteroid peaks: the first just prior to the formation of the pharate pupa, the second just prior to the formation of the pharate adult.

An examination of ecdysteroid profiles revealed some important differences. Ecdysone was either absent or present at extremely low levels in larval testes, whereas in hemolymph there was a premolt ecdysone peak. In pupal testes, ecdysone was present, but levels of 26‐hydroxyecdysone were much lower than those in hemolymph. Thus, in regard to ecdysteroids, testes have the ability to control their own internal milieu.
Novel assay for determining the metabolic fate of juvenile hormone III: A study with Drosophila melanogaster
Authors: Ottea J. A. , Harshman L. G. , Hammock Bruce D.
Published: February 2005

A thin‐layer chromatographic assay was developed for the resolution of hydrolytic and conjugative catabolites of juvenile hormone (JH). A single‐dimension, dual‐development thin‐layer system allowed complete resolution of the catabolites. Thus, this system provided a means for the rapid and economic analysis of JH hydrolysis even when different hydrolytic activities were present concurrently. Purified hydrolytic enzymes were found to be superior to chemical methods for the generation of small amounts of standards of JH catabolites. The relative levels of activities of an epoxide hydrolase and an esterase toward JH III were found to be similar in microsomal preparations from three lines of adult Drosophila melanogaster isolated from a field population. However, selection of flies by exposure to cut orange resulted in the elevation of levels of epoxide hydrolase activities, whereas esterase levels were not affected to the same extent. The formation of the JH acid‐diol was not detected under the conditions of this study, suggesting that the JH acid and diol were not good substrates for epoxide hydrolase and juvenile hormone esterase, respectively.
On the nature of nonenzymatic and enzymatic oxidation of the putative sclerotizing precursor, 1,2‐dehydro‐N‐acetyldopamine
Authors: Sugumaran Manickam , Hennigan Brian , Semensi Victor , Dali Hemalata
Published: February 2005

The mode of oxidation of 1,2‐dehydro‐N‐acetyldopamine by nonenzymatic and enzymatic systems was examined. At acidic and near neutral pH, this compound is fairly stable; however, even at slightly alkaline pH it is highly labile and undergoes spontaneous, nonenzymatic aerobic oxidation. Borate, which is known to chelate with catechols, prevented such nonenzymatic reaction, indicating the participation of the o‐dihydroxyphenolic group in the oxidation process. The product of nonenzymatic oxidation was found to be not the expected o‐benzoquinone derivative, but a benzodioxan‐type dimer. Although mushroom tyrosinase also catalyzed this reaction (Sugumaran et al.: Journal of Biological Chemistry 262:10546–10549, 1987), cuticular phenoloxidase(s) from Sarcophaga bullata failed to mediate this conversion. Rather, the cuticular phenoloxidase(s) oxidized the parent compound to a reactive intermediate which got bound to cuticle irreversibly through covalent linkage. Proteolytic digests of dehydro‐N‐acetyldopamine‐treated cuticle released peptide‐bound catechols. Such cuticle which on acid hydrolysis yielded ketocatechols consistent with the binding of dehydro‐N‐acetyldopamine to the cuticle through its side chain. Based on these results, the mechanisms of oxidation of 1,2‐dehydro‐N‐acetyldopamine by nonenzymatic and enzymatic systems are discussed.
Juvenile hormone metabolism during embryogenesis in the tobacco hornworm, Manduca sexta (L.)
Authors: Share Marla R. , Venkatesh Krishnappa , Jesudason Princy , Roe R. Michael
Published: February 2005

Juvenile hormones (JH)‐I and ‐III were metabolized in egg homogenates by two primary routes, ester hydrolysis and epoxide hydration, during embryogenesis of the tobacco hornworm, Manduca sexta (L.). The duration of embryogenesis was 3.5 days at 27°C. Preovipositional and newly oviposited eggs had the highest rate of JH metabolism, which was reduced by day 1 and remained unchanged thereafter. The decline in JH metabolism was the result of a decrease by one‐half in the JH esterase activity. JH epoxide hydrolase activity remained unchanged throughout embryogenesis. Ester hydrolysis averaged 1.9 times faster for JH‐I than JH‐III and epoxide hydration 6.6 times faster for JH‐III than JH‐I. There was a sixfold increase in the α‐naphthyl acetate (α‐NA) esterase activity during the time in embryogenesis when the JH esterase activity was declining or at low levels. Developmental, inhibitor, substrate specificity, gel filtration, and isoelectric focusing studies indicated that the egg JH esterase has some specificity for JH, compared with α‐NA, and in part is similar to the hemolymph JH esterase of the adult female. The possible functional role of JH metabolism during embryogenesis is discussed.
Developmental arrest induced by juvenile hormone in larvae of Bombyx mori
Authors: Sakurai Sho , Imokawa Hiroshi
Published: February 2005

Injection of the juvenile hormone analog (JHA) methoprene into day 3, fifthinstar larvae of Bombyx mori induced developmental arrest. Feeding activity declined, and the larvae remained as larvae for more than 2 weeks, after which they died. After JHA injection, the hemolymph ecdysteroid titer was low, and the prothoracic glands were almost inactive for 7 days. During this period, prothoracic glands were stimulated by prothoracicotropic hormone (PTTH) in vitro, indicating that JHA did not inhibit the competence of the glands to respond to PTTH. When brain‐corpora cardiaca‐corpora allata complexes were removed from intact fifth‐instar larvae on day 4, the prothoracic glands became autonomously active and produced enough ecdysone for pupation. When PTTH injections were given to larvae previously injected with JHA (7 days before), the larvae recovered feeding activity, purged their guts, and pupated. Injections of 20‐hydroxyecdysone into larvae that had been injected with JHA 7 days earlier induced larval molting. These results suggest that JHA affects both the brain and the prothoracic gland.
Differential mechanism of oxidation of N‐acetyldopamine and N‐acetylnorepinephrine by cuticular phenoloxidase from Sarcophaga bullata
Authors: Sugumaran Manickam , Hennigan Brian , Semensi Victor , Mitchell Wayne , Rivera Theresa
Published: February 2005

The mechanism of oxidation of two related sclerotizing precursors—N‐acetyldopamine and N‐acetylnorepinephrine—by the cuticular phenoloxidase from Sarcophaga bullata was studied and compared with mushroom tyrosinase‐mediated oxidation. While the fungal enzyme readily generated the quinone products from both of these catecholamine derivatives, sarcophagid enzyme converted N‐acetyldopamine to a quinone methide derivative, which was subsequently bound to the cuticle with the regeneration of o‐dihydroxy phenolic function as outlined in an earlier publication [Sugumaran: Arch Insect Biochem Physiol, 8, 73 (1988)]. However, it converted N‐acetylnorepinephrine to its quinone and not to the quinone methide derivative. Proteolytic digests of N‐acetyldopamine‐treated cuticle liberated peptides that had covalently bound catechols, while N‐acetylnorepinephrine‐treated cuticle did not release such peptides. Acid hydrolysis of N‐acetyldopamine‐treated cuticle, but not N‐acetylnorepinephrine‐treated cuticle liberated 2‐hydroxy‐3′,4′‐dihydroxyacetophenone and arterenone. These results further confirm the unique conversion of N‐acetyldopamine to its corresponding quinone methide derivative and N‐acetylnorepinephrine to its quinone derivative by the cuticular phen‐oloxidase. Significance of this differential mechanism of oxidation for sclerotization of insect cuticle is discussed.
Quantitative determination of trypsinlike and chymotrypsinlike enzymes in insects
Authors: Borovsky Dov , Schlein Yosef
Published: February 2005

A quantitative and highly specific assay for the determination of trypsinlike and chymotrypsinlike enzymes in insects has been developed. The assay is based on the specific binding of [1,3‐3H]diisopropylfluorophosphate to trypsinlike and chymotrypsinlike enzymes. Trypsinlike enzymes can be determined specifically in the presence of 10 mM TPCK (tosylamide‐2‐phenylethyl chloromethyl ketone; chymotrypsin inhibitor) and chymotrypsinlike enzymes can be determined in the presence of 10 mM TLCK (tosyl‐L‐lysine chloromethyl ketone HCI; trypsin inhibitor). The assay can easily detect 65 ng of either trypsinlike or chymotrypsinlike enzymes in midgut homogenates or whole extracts of many insect species. Using this assay, we have determined the amount of trypsinlike equivalents in Phlebotomus papatasi, Pediculus humanus, Stomoxys calcitrans, Musca domestica, Leishmania major promastigotes, Aedes aegypti, Culex nigripalpus, Culex quinquefasciatus, and Culicoides variipennis. No trypsinlike equivalents were found in Rhodnius prolixus or Ornithodoros moubata. The assay is useful for comparative studies and can be expanded for use as an electrophoretic fluorographic tool in the study of trypsinlike and chymotrypsinlike isozymes.
Disruptive effects of azadirachtin on development of Cotesia congregata in host tobacco hornworm larvae
Authors: Beckage Nancy E. , Metcalf Jeanette S. , Nielsen Barbara D. , Nesbit Dorothy J.
Published: February 2005

Injection of the insect growth regulator (IGR) azadirachtin into Manduca sexta larvae that were parasitized by Cotesia congregata adversely affected subsequent endoparasite development when the compound was administered prior to the first larval ecdysis of the wasps. Injection of 2.5–10 μg of azadirachtin into newly ecdysed fourth or fifth instar hosts partially inhibited or totally suppressed the first larval ecdysis of the parasites. Instead of initiating rapid growth similar to parasites developing in hosts injected with the solvent carrier (ethanol) only, parasites in azadirachtin‐treated hosts never grew beyond the size normally attained in early terminal stage hosts. The host larvae survived for 2 weeks or longer following injection of azadirachtin, but their parasites never recovered and died encased within one or two exuvial cuticles. The parasitism‐specific hemolymph polypeptides previously shown to be characteristic of terminal stage hosts were undetectable in larvae in which the parasites failed to ecdyse to the second instar. The observed effects of azadirachtin on development of C. congregata differ significantly from those induced by other IGRs in parasitized M. sexta, suggesting azadirachtin has a unique, though as yet undefined, mode of action. Compared to other IGRs, this compound is extremely potent in terms of the minimum doses required to prevent emergence, but affects only the L1‐L2 ecdysis and not the L2‐L3 transition. In contrast, other compounds such as juvenoids and the JH esterase inhibitor BEPAT (S‐benzyl‐0‐ethyl phosphoramidothiolate) permit the parasite to grow and develop normally until the wasps become pharate third instar larvae, then inhibit the second larval ecdysis and emergence from the host.
On the intracellular localization of the ecdysone 20‐monooxygenase in Musca domestica. L
Authors: Agosin Moises , Srivatsan Jayanthi , Weirich Margaret
Published: February 2005

The cytochrome P‐450‐dependent 20‐monooxygenation of ecdysone is catalyzed both by mitochondria and microsomes isolated from Musca domestica (L.) larvae; however, about 50% of the activity is associated with mitochondria, and 37% is associated with microsomes. Pretreatment of larvae with ecdysone results in an increase in Vmax and a decrease in Km values in mitochondria but not in microsomes. Phenobarbital, a known cytochrome P‐450 inducer, increases the cytochrome P‐450 levels in microsomes without affecting the 20‐monooxygenase activity, but both the cytochrome P‐450 levels and monooxygenase activity are depressed in mitochondria from phenobarbital‐pretreated larvae. The ecdysone 20‐monooxygenase activity is equally distributed between mitochondria and microsomes in adult insects. Pretreatment of the insects with ecdysone does not significantly modify the 20‐monooxygenase activity of either mitochondrial or microsomal fractions, but the cytochrome P‐450 levels are reduced in mitochondria. Phenobarbital also depresses the mitochondrial cytochrome P‐450 levels while markedly increasing the microsomal cytochrome P‐450 levels. However, no significant changes in ecdysone 20‐monooxygenase activity are produced by phenobarbital pretreatment. The effects of ecdysone on adult cytochrome P‐450 are mostly evidenced in mitochondria isolated from females, whereas in males the changes are not statistically significant. It is concluded that the mitochondrial ecdysone 20‐monooxygenase is under regulatory control by ecdysone in the larval stage, which suggests that only the mitochondrial activity has a physiological role during insect development in M. domestica. In adults, both the mitochondrial and microsomal ecdysone 20‐monooxygenase activities are not responsive to ecdysone, which, coupled to their high Km values, indicates that the reaction may not be of physiological importance in adult insects and that the mitochondrial cytochrome P‐450 species being depressed by ecdysone in females are possibly not involved in ecdysone metabolism.
Juvenile hormone regulates the titer of a hemolymph factor that enhances ecdysone synthesis by insect (Manduca sexta) prothoracic glands in vitro
Authors: Watson R. Douglas , Haire Mary E. , Bollenbacher Walter E.
Published: February 2005

The hemolymph of last instar Manduca sexta larvae contains a protein factor that enhances ecdysone synthesis by prothoracic glands in vitro. The titer of the factor fluctuates during development in a pattern that suggests that it is regulated by juvenile hormone (JH). In untreated control larvae, the titer drops from 2.17 U ml−1 on day 1 to 0.27 U ml−1 on day 3. When larvae were treated with (7S)‐hydroprene (a JH analog), the titer remained elevated (2.09 U ml−1 on day 3). JH I, however, was ineffective in preventing the precommitment drop in the titer of the factor. After pupal commitment, the titer of the factor increases in untreated larvae from 0.84 U ml−1 on day 5 to 1.62 U ml−1 on day 7. This increase was blocked when the sources of JH (the corpora allata) were removed on day 5 by head ligation. When head‐ligated day 5 larvae were treated with either (7S)‐hydroprene or JH I, the titer of the factor was driven to a level (1.88 U ml−1 and 2.05 U ml−1, respectively) that was not significantly different from that found in untreated day 7 larvae (1.62 U ml−1). The combined results indicate the titer of the hemolymph factor is regulated by JH.
Production of a monoclonal antibody to the arylphorin of Heliothis zea
Authors: Lenz Cynthia J. , Greenstone Matthew H.
Published: February 2005

A species‐specific monoclonal antibody was produced to whole plasma of fifth instar larvae of the corn earworm, Heliothis zea (Boddie) (Lepidoptera:Noctuidae). This antibody did not cross‐react with proteins from the plasma of any of the other lepidopteran larvae tested, including other Heliothis spp. The antigen recognized by this antibody was characterized and found to be arylphorin, a prominent larval storage protein. This conclusion was based on the electrophoretic as well as immunological characteristics of the antigen. Polyacrylamide gel electrophoresis indicated that the antigen had an apparent native molecular weight of 460,000, and that it was composed of subunits having apparent molecular weights of 76,000. The isoelectric point of the antigen was 5.9, with some microheterogeneity being seen. Western blotting of arylphorin against the monoclonal antibody clearly identified the antigen as arylphorin. This protein was not found in egg homogenates or early instar larval plasma, but it was present in large quantities in pupal homogenates and, at trace levels, in adult homogenates. The efficient production and selection of hybridomas producing antibodies specific to H. zea arylphorin using unfractionated plasma as immunogen illustrates the fact that monoclonal antibody technology can produce highly specific antibodies using crude antigen preparations. We discuss the tradeoffs one must accept when choosing this strategy over one using purified immunogens.
Stimulation of sex pheromone production by head extract in Spodoptera littoralis at different times of the photoperiod
Authors: Martínez Teresa , Camps Francisco
Published: February 2005

Adult females of Spodoptera littoralis (Lepidoptera: Noctuidae) showed a cyclic pattern of sex pheromone production, and high titers of (Z,E)‐9,11‐tetradecadienyl acetate, the major component of its pheromone blend, were only detected during scotophase. Maximal amounts of pheromone were extracted approximately 2 h into second scotophase. Decapitation before the beginning of darkness inhibited normal production of pheromone, and no calling behavior was observed. Injection of brain‐suboesophageal ganglion (Br‐SOG) homogenate at the onset of scotophase restored pheromone production in decapitated females to the levels characteristic of second scotophase. Pheromone biosynthesis was also stimulated in decapitated females during photophase. The response to Br‐SOG homogenate injection was dose‐dependent. The pheromonotropic activity of Br‐SOG extract was the same when females were injected during photophase or scotophase.
On the mechanism of formation of N‐acetyldopamine quinone methide in insect cuticle
Authors: Sugumaran Manickam , Saul Steven J. , Semensi Victor
Published: February 2005

The mechanism of formation of quinone methide from the sclerotizing precursor N‐acetyldopamine (NADA) was studied using three different cuticular enzyme systems viz. Sarcophaga bullata larval cuticle, Manduca sexta pharate pupae, and Periplaneta americana presclerotized adult cuticle. All three cuticular samples readily oxidized NADA. During the enzyme‐catalyzed oxidation, the majority of NADA oxidized became bound covalently to the cuticle through the side chain with the retention of o‐diphenolic function, while a minor amount was recovered as N‐acetylnorepinephrine (NANE). Cuticle treated with NADA readily released 2‐hydroxy‐3′,4′‐dihydroxyacetophenone on mild acid hydrolysis confirming the operation of quinone methide sclerotization. Attempts to demonstrate the direct formation of NADA‐quinone methide by trapping experiments with N‐acetylcysteine surprisingly yielded NADA‐quinone‐N‐acetylcysteine adduct rather than the expected NADA‐quinone methide‐N‐acetylcysteine adduct. These results are indicative of NADA oxidation to NADA‐quinone and its subsequent isomerization to NADA‐quinone methide. Accordingly, all three cuticular samples exhibited the presence of an isomerase, which catalyzed the conversion of NADA‐quinone to NADA‐quinone methide as evidenced by the formation of NANE—the water adduct of quinone methide. Thus, in association with phenoloxidase, newly discovered quinone methide isomerase seems to generate quinone methides and provide them for quinone methide sclerotization.
Vitellogenesis in the cockroach Nauphoeta cinerea: Separation of two classes of ovarian binding sites and calcium effects on binding and uptake
Authors: König Rolf , Kindle Helmut , Kunkel Joseph G. , Lanzrein Beatrice
Published: February 2005

Two classes of vitellogenin binding sites with Kd‐values of 7.3 nM and 290 nM were observed in follicle‐membrane preparations of the cockroach Nauphoeta cinerea using a membrane‐binding assay at pH 8. Separation of follicle cells and basal laminae from oocyte membranes prior to binding studies showed that the fraction consisting of follicle cells and basal laminae (FC/BL) contained high‐affinity binding sites for vitellogenin (Kd=16.6 nM), whereas loweraffinity binding sites (Kd=200 nM) were found in the oocyte membrane fraction. The concentration of Ca2+ had a distinct effect on vitellogenin binding and uptake: maximal binding to the oocyte membrane fraction was observed at 0.3 mM Ca2+ and to the FC/BL fraction at 10 mM, whereas uptake of vitellogenin by oocytes in vitro was highest at 4 mM Ca2+. The calcium ionophore A23187 decreased vitellogenin uptake. This effect of A23187 could be counteracted by the calcium chelator Quin2. A hypothetical model for the uptake of vitellogenin into follicles of Nauphoeta cinerea is suggested.
Alteration of hemolymph polypeptides in Manduca sexta larvae parasitized by Cotesia congregata: A two‐dimensional electrophoretic analysis and comparison with major bacteria‐induced proteins
Authors: Beckage Nancy E. , Nesbit Dorothy J. , Nielsen Barbara D. , Spence Kemet D. , Barman Miel A. E.
Published: February 2005

Analyses using one‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) previously demonstrated that parasitization by the braconid wasp Cotesia congregata significantly alters the normal hemolymph polypeptide profile of host Manduca sexta larvae. In the present study two‐dimensional gel analyses corroborated our earlier findings and provided additional evidence that multiple parasitism‐specific polypeptides were induced, which varied according to the stage of development of the wasps. Parasitization additionally elicited changes in the total protein concentration detected in the blood. Initially an elevation was observed, with newly parasitized larvae exhibiting a twofold elevation in hemolymph protein concentration by 12–24 h postoviposition. In contrast, terminal‐stage hosts with second instar parasites had significantly less protein in the hemolymph, likely due to reduced growth and inhibition of arylphorin synthesis by the fat body during the final stages of parasitism. Comparison of the array of hemolymph polypeptides produced in unparasitized larvae injected with 106cells of the gram‐negative bacterium Enterobacter cloacae with those proteins induced by parasitization indicated the two classes are different. Our findings confirm that the hostresponse to parasitism is a specific one, and not mimicked by bacterial challenge. Duringshort‐term in vitro culture of wasp larvae dissected from the host hemocoel, several proteins were detected in the medium using SDS ‐ PAGE, with their appearance in vitro suggestive of secretion by the wasps in vivo. Moreover, hemolymph from the parasites had significant amounts of putative host proteins, including an arylphorin ‐ like polypeptide and a protein with a mobility similar to that of insecticyanin. Thus, a dynamic interchange of proteins may occur, with the parasites accumulating host proteins while simultaneously secreting a variety of factors into the host hemocoel.
Binding of octopamine to membranes from the white‐eyed and wild‐type housefly heads
Authors: Hashemzadeh‐Gargari Hossein , Wierenga Joel M.
Published: February 2005

In attempting to develop an octopamine (OA) receptor preparation with ready access to large amounts of tissue, we examined the binding of OA to membranes from the heads of white and red houseflies (Musca domestica L.). Binding was dependent on the presence of L‐ascorbic acid in the medium. However, equilibrium was reached only over 24–36 h at 4°C and reversal of binding was also slow and incomplete. Scatchard analysis revealed at least two binding sites in the white‐eyed housefly. A high‐affinity site (Kd = 13.9 nM and Bmax = 3.9 pmol/mg protein) was present, but the majority of the binding had low affinity (Kd = 1130 nM and Bmax = 165 pmol/mg protein). Scatchard analysis revealed a low affinity in the red‐eyed housefly (Kd = 240 nM and Bmax = 12 pmol/mg protein). Catecholamines were the best competitors for OA binding followed by phenolamines such as OA and synephrine. 5‐Hydroxytryptamine was less effective. Phentolamine and mianserin, which are good antagonists of the ability of OA to stimulate adenylate cyclase in housefly head membranes, and formamidine and imidazolines, which are potent partial agonists of this adenylate cyclase, were poor competitors of OA binding. The slow kinetics, low affinity, large amount, and unconventional pharmacological profile of this binding is not congruent with it being a neuroreceptor. When the brain was dissected free from the head, less than 10% of the total specific binding of OA was found in the brain membrane fraction. This suggests that most of the binding of OA may be to cuticular sites that possibly are associated with the metabolism of catecholamines in cuticular synthesis. Thus, binding studies made with labeled catecholamines and phenolamines on insect tissues containing significant cuticular elements should be interpreted with caution.