bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification, partial characterization, and postembryonic levels of amylases from Sitophilus oryzae and Sitophilus granarius
Authors: Baker J. E. , Woo S. M.
Published: February 2005

Amylases from adults of Sitophilus oryzae (L.) and S. granarius (L.) were purified by using a sequential procedure of ammonium sulfate precipitation, glycogen‐complex formation, and ion exchange chromatography. Amylase of S. oryaze was purified 47.4‐fold to a specific activity of 478 units/mg protein. One amylase unit equals 1 mg maltose hydrate produced/min at 30°C. Amylase of S. granarius was purified 85.4‐fold to a specific activity of 453 units/mg protein. Amylase of S. oryzae had a Km of 0.173% for soluble starch and consisted of two anionic isozyrnes with isoelectric points of pH 3.70 and pH 3.76. Amylase of S. granarius had a Km of 0.078% for starch and was a single protein with an isoelectric point of pH 3.76. Purified amylases of both species had molecular weights of 56,000 estimated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, were activated by chloride, and had double energies of activation calculated from Arrhenius plots. Based on fresh weights of adults feeding on whole wheat through 10 weeks of age, S. oryzae contained three‐fold and eight‐fold more amylase than S. granarius and S. zeamais Motschulsky, respectively. High amylase levels in S. oryzae may provide this species with an adaptive advantage when feeding on cereals containing naturally occurring amylase inhibitors.
Interactions of charatoxins and nereistoxin with the nicotinic acetylcholine receptors of insect cns and Torpedo electric organ
Authors: Sherby Shebl M. , Eldefrawi Amira T. , David Jonathan A. , Sattelle David B. , Eldefrawi Mohyee E.
Published: February 2005

Interactions of charatoxin (4‐methylthio‐1,2‐dithiolane; ChTX) and four openchain analogs as well as nereistoxin (NTX) with acetylcholine (ACh) receptors were studied using biochemical assays on the Torpedo electric organ and honey bee brain receptors and using electrophysiological assays on the response of the cell body of the fast coxal depressor motoneuron (Df) of the cockroach Periplaneta americana to ACh. The actions of ChTXs were complex. Except for ChTX Xl, they all potentiated the ACh‐induced current in Periplaneta neurons, but at higher concentrations all ChTXs, except for ChTX XII, caused voltage‐dependent block of this current. All CHTXs inhibited binding of [3H]perhydrohistrionicotoxin in the presence of ACh to the highaffinity noncompetitive blocker site on the Torpedo receptor, but all, except for ChTX XI, potentiated its binding in absence of ACh. The actions of ChTXs on the honey bee brain receptor were quite different from those on the Torpedo receptor. They inhibited, or had no effect on, [125I]α‐bungarotoxin (α‐BGT) binding to the Torpedo receptor, but all ChTXs, except for ChTX I, potentiated its binding to the honey bee receptor. It is suggested that the action of ChTXs on nicotinic ACh‐receptors resulted from binding to lowaffinity noncompetitive blocker site. On the other hand, NTX was more potent than ChTXs on nicotinic ACh‐receptors, and some similarities were noted between the actions of NTX on Torpedo and honey bee receptors NTX had a weak agonistlike effect in both cases and possibly bound to the ACh binding sites as well as the high‐affinity noncompetitive blocker site. Thus the mechanisms of action of ChTXs and NTX on nicotinic ACh‐receptors are different, and there are also differences in the responses to these toxins between receptors of insect central nervous system and Torpedo electric organ.
Major hemolymph proteins from larvae of the black swallowtail butterfly, Papilio polyxenes
Authors: Ryan Robert O. , Wang Xiao‐Yu , Willott Elizabeth , Law John H.
Published: February 2005

Three major hemolymph proteins of Papilio polyxenes larvae were isolated and characterized. Density gradient ultracentrifugation of hemolymph resulted in flotation of the major lipoprotein, lipophorin. P. polyxenes larval lipophorin is composed of two apoproteins, apolipophorin‐I and apolipophorin‐II, plus a mixture of lipids, to give a density of 1.13 g/ml. Immunoblotting experiments using antisera directed against Manduca sexta apolipophorin‐I and apolipophorin‐II, respectively, revealed cross‐reactivity of apoLp‐I with Manduca sexta apoLp‐I, and apoLp‐II with M. sexta apoLp‐II.

Gel permeation chromatography of the subnatant obtained following density gradient ultracentrifugation revealed the presence of a major protein peak which was shown to contain three major serum proteins, two of which were isolated and characterized. One of these proteins was purified by lectin affinity chromatography. Both proteins have native molecular weights in the range of 450,000 and appear to be hexamers of a single subunit type. Major serum protein‐1 is nonglycosylated and has a subunit molecular weight of 75,000. Major serum protein‐2 is glycosylated and has a subunit molecular weight of 74,000. Amino acid analysis of this protein revealed a tyrosine plus phenylalanine content of 20 mole percent, characteristic of the arylphorin class of insect storage proteins. Using antibodies against M. sexta larval hemolymph proteins, both the P. polyxenes major serum proteins were shown to be immunologically related to serum proteins of other lepidopteran species.
Metabolism of 26‐[14C]hydroxyecdysone 26‐phosphate in the tobacco hornworm, Manduca sexta L., to a new ecdysteroid conjugate: 26‐[14C]hydroxyecdysone 22‐glucoside
Authors: Thompson Malcolm J. , Feldlaufer Mark F. , Lozano Ruben , Rees Huw H. , Lusby William R. , Svoboda James A. , Wilzer Kenneth R.
Published: February 2005

Following injection into female Manduca sexta pupae, [14C]cholesterol is converted to a radiolabeled C21 nonecdysteroid conjugate as well as ecdysteroid conjugates, which in ovaries and newly‐laid eggs consist mainly of labeled 26‐hydroxyecdysone 26‐phosphate. During embryogenesis, as the level of 26‐hydroxyecdysone 26‐phosphate decreases there is a concurrent increase in the amount of a new, labeled ecdysteroid conjugate. This conjugate, which is the major ecdysteroid conjugate (9.4 μg/g) in 0‐ to 1‐hour‐old larvae was identified as 26‐hydroxyecdysone 22‐glucoside by nuclear magnetic resonance and chemical ionization mass spectrometry. This is the first ecdysteroid glucoside to be identified from an insect. The disappearance of 26‐hydroxyecdysone 26‐phosphate in 0‐ to 1‐hour‐old larvae indicates that the 26‐hydroxyecdysone 22‐glucoside is derived from 26‐hydroxyecdysone 26‐phosphate. 3‐Epi‐26‐hydroxyecdysone was the major free ecdysteroid isolated from these larvae and 3‐epi‐20,26‐dihydroxyecdysone was the next most abundant ecdysteroid isolated. Interestingly, the 0‐ to 1‐hour‐old larvae contained the highest levels of 3α‐ecdysteroids per gram of insect tissue (8.7 μg/g) to be isolated from an insect, yet there was a complete absence of the corresponding free 3β‐epimers. The ecdysteroid conjugate profiles of ovaries and 0‐ to 1‐hour‐old larvae are discussed. Methodology is presented that permits the efficient separation of free and conjugated ecdysteroids and nonecdysteroid conjugates (C21‐steroid conjugates).
Partial purification and characterization of locust allatotropin I
Authors: Gadot Michal , Rafaeli Ada , Applebaum Shalom W.
Published: February 2005

Methanol extracts of locust brains, corpora cardiaca (CC), and suboesophageal ganglia (SOG) were separated by gradient and/or isocratic reverse‐phase high‐performance liquid chromatography (HPLC) and allatotropic activity monitored in the eluted fractions. A major peak of activity, separated by isocratic separation with 12% 2‐propanol, designated allatotropin I, exhibited identical retention times in the three tissue extracts. Doseresponse curves of allatotropin I indicate similar content in brain and CC‐equivalents, whereas optic lobes, similarly separated by isocratic HPLC, contain only one‐tenth of this amount of allatotropin. Allatotropin I is resistant to boiling and is susceptible to tryptic and chymotryptic digestion. Methanol extracts of thoracic muscle, Malpighian tubules, fat body or ovaries, similarly prepared and boiled, did not exhibit allatotropic activity at high doses of tissue equivalents.
Diet‐induced nonmelanized cuticle in workers of the imported fire ant Solenopsis invicta buren
Authors: Williams David F. , Vander Meer Robert K. , Lofgren Clifford S.
Published: February 2005

Nonmelanized cuticle development was induced in workers of Solenopsis invicta by feeding them an insect‐free diet. The nonmelanized workers weighed less and had smaller mean headwidths than workers from normal colonies. Although nonmelanized ant colonies appeared to function normally in the laboratory, their attempts at stinging were felt only as “pin pricks.” Chemical analysis of venom alkaloids and cuticular hydrocarbons indicated no qualitative differences between nonmelanized and normal workers. Tyrosine, an essential amino acid tanning precursor, was found in adequate quantities in the free amino acid pool of nonmelanized ants. The specific cause of the nonmelanized condition is not known.
Permethrin‐ and malathion‐induced macromolecular abnormalities in adult Tribolium castaneum (herbst)
Authors: Saleem Mushtaq A. , Shakoori A. R.
Published: February 2005

In Tribolium castaneum adults, sublethal doses of 1 and 2 ppm permethrin and 300 ppm malathion led to significant changes in amylase, trehalase, acid phosphatase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and isocitrate dehydrogenase activities. Malathion at 150 ppm did not affect phosphatases and lactate dehydrogenase. Both malathion and permethrin significantly increased cholinesterase activity. Mixing of the two insecticides resulted in antagonistic action with reference to various enzymatic activities. Glucose and glycogen contents were at first mobilized for energy supply under insecticidal stress conditions followed by lipid and cholesterol. Soluble protein, total protein, free amino acids, and urea contents remained unaltered under all experimental conditions.
Evidence for hormonal control of diuresis after a blood meal in the mosquito Aedes aegypti
Authors: Wheelock G. D. , Petzel D. H. , Gillett J. D. , Beyenbach K. W. , Hagedorn H. H.
Published: February 2005

In previous studies we have presented evidence for the role of peptides, isolated from heads of the mosquito Aedes aegypti, in stimulating fluid secretion by isolated Malpighian tubules. In the present study we conducted experiments to investigate whether these peptides are involved in hormone‐mediated diuresis after a blood meal. In vivo experiments showed that the head was required to maintain diuresis after the blood meal. Whereas feeding on blood triggered a prompt diuresis in the intact mosquito, subsequent decapitation caused a gradual, not an abrupt, decline in urine excretion rate. Hemolymph collected from mosquitoes fed blood significantly stimulated fluid secretion in vitro by isolated Malpighian tubules, whereas hemolymph from unfed or blood‐fed decapitated mosquitoes did not. These results indicate that a diuretic factor was released into the hemolymph after a blood meal. This factor was not present in the hemolymph of decapitated females. We identified the head as a source of diuretic factors. Peptides isolated from a head extract by high‐performance liquid chromatography, when injected into the hemocoel of blood‐fed decapitated mosquitoes, triggered diuresis in vivo and also stimulated fluid secretion in isolated Malpighian tubules. These studies support the hypothesis that the head is a storage site for diuretic peptides that may be released after a blood meal to control diuresis.
Isolation and characterization of lipophorin from Drosophila melanogaster larvae
Authors: Fernando‐Warnakulasuriya Germain J. P. , Wells Michael A.
Published: February 2005

The hemolymph lipoprotein lipophorin has been isolated from third‐instar Drosophila melanogaster larvae by a technique that involves homogenization of whole larvae in a medium containing protease inhibitors and purification of the lipoprotein by density gradient centrifugation. Drosophila lipophorin has a density of 1.16 g/ml and is composed of 62.5% protein, 23.1% phospholipid, 7.4% diacylglycerol, 5.4% triacylglycerol, 0.9% hydrocarbon, and 0.7% sterol. As is the case with other insect lipophorins, Drosophila lipophorin contains two apolipoproteins, apolipophorin‐I (Mr ≈ 275,000) and apolipophorin‐II (Mr ≈ 76,000). Drosophila apolipophorin‐I does not crossreact with antibodies prepared against apolipophorin‐I from Manduca sexta.
Apolar conjugates of ecdysteroids are not used as a storage form of molting hormone in the argasid tick Ornithodoros moubata
Authors: Connat Jean‐Louis , Dotson Ellen Marie , Diehl Peter Allan
Published: February 2005

Fifth (last) instar nymphs of the tick Ornithodoros moubata convert ingested 20‐hydroxyecdysone (20E) to apolar conjugates AP2, which are then converted to the more polar conjugates AP1. Only small quantities of free hormone were transferred to the hemolymph and the carcass within the first 2 days after the blood meal. The proportion of radiolabel in these two compartments was highest at the time of the endogenous ecdysteroid peak; however, no traces of free [3H]20E were detected. The conversion probably occurs principally in the intestinal cells. Eleven days after ingestion, 84% of the radiolabel is located in the digestive tract, mainly in the form of AP1 conjugates.

AP1 obtained in second instar nymphs fed with [3H]ecdysone ([3H]E) remain stable throughout the following nymphal instars. The ecdysteroid moiety of AP1 remained unchanged. The hydrolysis, although not complete, always yielded a peak comigrating with the reference E but never 20E or any other clearly distinct peaks that may have corresponded to metabolites of 20E. Less label per individual was present in adults, but its nature remained the same, viz., AP1 mainly located in the digestive tract. In females, 2.5% of the label was transferred to the progeny during the first ovipositional cycle.

Apolar products (mainly AP2) that accumulated in eggs of females injected with [3H]E or [3H]20E during vitellogenesis remained unchanged during the whole embryonic development. During the molting cycle of larvae, there was only a slight conversion of AP2 to AP1, but esterase hydrolysis of these products released the same percentages of E and 20E as in the freshly laid eggs.

We conclude that in this tick species apolar conjugates of ecdysteroids are inactivation metabolites that are not reutilized during the development of the animal. These metabolites are mainly retained in the tick, probably because of its peculiar blocked midgut. Several studies have shown that in other arthropod species (ticks, spiders, and insects), these apolar metabolites are excreted in the feces.
Vitellogenesis and oocyte development in azadirachtin‐induced fifth‐instar overage nymphs of Locusta migratoria (L.)
Authors: Shalom Uri , Applebaum Shalom W. , Pener M. Paul
Published: February 2005

Injection of azadirachtin into females of Locusta migratoria at the beginning of the last nymphal instar prevented molting to the adult stage, and many of these locusts survived for long periods as overage fifth‐instar nymphs. Overage female nymphs synthesized vitellogenin; maximum vitellogenin content in their hemolymph was 6–7 times higher than that found in normal adult females. The overage female nymphs developed vitellogenic oocytes, but development was retarded to some extent: although vitellogenin did accumulate in the proximal oocytes, their maximum average length was only about 2.8 mm (compared to 6.2 mm in normal adult females) and extensive oocyte resorption was observed. Thus, attainment of adult competence of the organs and processes involved in female reproduction is independent to a considerable extent from the process of overt adult morphogenesis.
Chelonus sp. near curvimaculatus venom proteins: Analysis of their potential role and processing during development of host Trichoplusia ni
Authors: Leluk Jacek , Jones Davy
Published: February 2005

The venom that Chelonus sp. near curvimaculatus injects into each parasitized Trichoplusia ni egg is entirely injected within the first 8 s of the 19‐s oviposition period, before deposition of the parasitoid egg that is injected during the final 1‐2 s of the oviposition. The parasitization factor, causing precocious metamorphosis of the host, is injected after the venom, but before the parasite egg. The venom by itself does not cause developmental redirection of the host. Chelonus venom proteins are very stable in the host egg during the first 2 days of egg development. Then, on the last day before hatching, they are rapidly degraded by the proteolytic enzymes appearing in 3‐day‐old T. ni eggs. Among those that degrade the venom proteins are serine‐type proteinases, and at least one seems to be a trypsin‐like enzyme.
On the oxidation of 3,4‐dihydroxyphenethyl alcohol and 3,4‐dihydroxyphenyl glycol by cuticular enzyme(s) from Sarcophaga bullata
Authors: Sugumaran Manickam , Semensi Victor , Saul Steven J.
Published: February 2005

The catabolic fate of 3,4‐dihydroxyphenethyl alcohol (DHPA) and 3,4‐dihydroxyphenylethyl glycol (DHPG) in insect cuticle was determined for the first time using cuticular enzyme(s) from Sarcophaga bullata and compared with mushroom tyrosinase‐medicated oxidation. Mushroom tyrosinase converted both DHPA and DHPG to their corresponding quinone derivatives, while cuticular enzyme(s) partly converted DHPA to DHPG. Cuticular enzyme(s)‐mediated oxidation of DHPA also accompanied the covalent binding of DHPA to the cuticle. Cuticle‐DHPA adducts, upon pronase digestion, released peptides that had bound catechols. 3,4‐Dihydroxyphenyl‐acetaldehyde, the expected product of side chain desaturation of DHPA, was not formed at all. The presence of N‐acetylcysteine, a quinone trap, in the reaction mixture containing DHPA and cuticle resulted in the generation of DHPA‐quinone‐N‐acetylcysteine adduct and total inhibition of DHPG formation. The insect enzyme(s) converted DHPG to its quinone at high substrate concentration and to 2‐hydroxy‐3′,4′‐dihydroxyacetophenone at low concentration. They converted exogenously added DHPA‐quinone to DHPG, but acted sluggishly on DHPG‐quinone. These results are consistent with the enzymatic transformations of phenoloxidase‐generated quinones to quinone methides and subsequent nonenzymatic transformation of the latter to the observed products. Thus, quinone methide formation in insect cuticle seems to be caused by the combined action of two enzymes, phenoloxidase and quinone tautomerase, rather than the action of quinone methide‐generating phenoloxidase (Sugumaran: Arch Insect Biochem Physiol 8, 73–88, 1988). It is proposed that DHPA and DHPG in combination can be used effectively to examine the participation of (1) quinone, (2) quinone methide, and (3) dehydro derivative intermediates in the metabolism of 4‐alkylcatechols for cuticular sclerotization.
Storage proteins of the fall webworm, Hyphantria cunea drury
Authors: Kim Hak Ryul , Kang C. S , Mayer Richard T.
Published: February 2005

Two storage proteins, storage protein‐1 (SP1) and storage protein‐2 (SP2), were found in hemolymph and fat body during the development of Hyphantria cunea, the fall webworm. Both storage proteins show similiar quantitative changes during development in males and females; however, SP1 is more abundant. The hemolymph of last instar larvae contains high concentrations of the storage proteins. However, following pupation, the storage proteins accumulate in fat bodies. SP1 peaks in the hemolymph of males and females late in last instar larvae (8‐day‐old 7th instar larvae).

SP1 has a native molecular weight of 460,000 and consists of six identical subunits (Mr = 76,700), while SP2 has a molecular weight of 450,000 and is composed of two different subunits (Mr = 74,100 and 72,400). Both SP1 and SP2 are hexamers and are phosphorylated glycolipoproteins. The pl values of SP1 and SP2 were determined to be 5.70 and 5.50, respectively.

Antibodies raised against SP1 react positively with vitellogenin and ovary extract, as well as with proteins in the hemolymph from last instar larvae and proteins in pupal fat bodies. Storage protein synthesis starts in fat bodies of a 4‐day‐old 7th instar larvae and in female peaks at 6–8 days of the 7th instar.
Juvenile hormone biosynthesis and oocyte development in adult female Blattella germanica: Effects of grouping and mating
Authors: Gadot Michal , Burns Edina , Schal Coby
Published: February 2005

The roles of grouping and mating in modulating the activity of the corpora allata (CA) in adult female cockroaches were investigated using the in vitro radiochemical assay of juvenile hormone (JH) biosynthesis. Isolated virgin females have longer, asynchronous cycles of CA activity and oocyte maturation than do isolated females mated on day 8. Three factors were identified as the major contributors to this difference: (1) an experimental artifact of selection for sexually receptive females, (2) a positive effect of grouping on JH synthesis and oocyte maturation, and (3) a positive effect of copulation on oviposition and retention of the ootheca. Mated females constitute a subpopulation of receptive females that differ significantly from other females by having higher rates of JH synthesis prior to mating. The relative importance of such selection is substantial when the rate of mating is low, as in experiments with isolated females that are exposed to males for a short period of time. Long‐term exposure of females to males introduces a grouping effect, which obscures any additional effect of mating on CA activity and oocyte development. However, mating influences ootheca formation and its retention. The effect of grouping can be mimicked in isolated females by transection of the nerves connecting the CA–corpora cardiaca complex to the brain, suggesting that in this insect isolation causes brain inhibition of the CA, and grouping provides disinhibitory stimuli that release the CA from brain inhibition.
Some macromolecular abnormalities developed by the interaction of malathion and permethrin and subsequent refeeding in Tribolium castaneum larvae
Authors: Shakoori Abdul Rauf , Saleem Mushtaq A.
Published: February 2005

The effects of sublethal treatments of malathion and malathion + permethrin combinations on activities of acetylcholine esterase (ChE), carbohydrate‐metabolizing enzymes (amylase; lactate dehydrogenase, LDH), protein‐metabolizing enzymes (alanine and aspartate aminotransferase, ALAT, ASAT), as well as acid and basic phosphatases (AcP and AkP, respectively), and Kreb's cycle enzymes (isocitrate dehydrogenase, ICDH) were studied on sixth instar larvae of the red flour beetle, Tribolium castaneum. In addition, the levels of lipid, cholesterol, glucose, glycogen, proteins, free amino acids (FAA), urea, and nucleic acids (DNA and RNA) were determined. Malathion (400 ppm) increased the activity of LDH (53%), as well as the concentrations of FAA (31%) and urea (39%). Malathion treatments of 400 ppm decreased the glucose (20%) and glycogen (24%) content but did not affect other enzymes and biochemical components. Permethrin (200 ppm) and malathion (20 ppm) mixtures increased the activities of ChE (708%) and LDH (55%), and raised the concentrations of FAA (26%), urea (24%), glucose (23%), lipid (14%), cholesterol (21%), DNA (24%), and RNA (8%). The decrease in AcP activity and in glycogen concentration observed with malathion was sustained by permethrin in the mixture. Permethrin + malathion mixtures also depressed the levels of AkP (30%), ICDH (24%), and glycogen (36%). The intensity of effects commensurated with the dose and duration of insecticide exposure. Refeeding showed tendencies towards normalization of various biochemical parameters.
Alterations in the ecdysteroid and juvenile hormone esterase profiles of Trichoplusia ni parasitized by the polyembryonic wasp Copidosoma floridanum
Authors: Strand Michael R. , Dover Barry A. , Johnson Jena A.
Published: February 2005

Trichoplusia ni parasitized by Copidosoma floridanum weighed more in the fourth and fifth instar than unparasitized controls. While parasitized and unparasitized fourth‐instar larvae molted at the same time, parasitized fifth‐instar larvae ceased feeding and initiated wandering 1 day later than unparasitized fifth‐instar larvae. Measurement of hemolymph ecdysteroid, juvenile hormone esterase (JHE), and protein titers suggested that the delay in wandering had an endocrinological basis. Ecdysteroid titers of parasitized and unparasitized fourth‐instar larvae were similar, but the ecdysteroid titer in parasitized fifth‐instar larvae rose about 24 h later than in unparasitized fifth‐instar larvae. Two major peaks of JHE activity occurred in unparasitized fifth‐instar larvae, but only one occurred in parasitized fifth‐instar larvae. JHE activity coincided on the first day of the fifth instar (L5D1) in both parasitized and unparasitized fifth‐instar larvae; however, activity peaked and fell later in parasitized individuals. Few differences were noted in the hemolymph protein levels of parasitized and unparasitized hosts. Ligation of parasitized T. ni between the head and prothorax adversely affected C. floridanum development. Ligation before or immediately after ecdysis to the fourth instar inhibited differentiation of the C. floridanum morulae. C. floridanum ecdysed to the second instar or pupated in hosts ligated prior to wandering in the fifth instar, but the number of progeny that pupated and emerged as adult wasps was reduced relative to unligatured controls.
Evaluation of teratocyte functions: An overview
Authors: Dahlman Douglas L.
Published: February 2005

Teratocytes, derived from extra‐embryonic tissues of parasitic Braconidae, Trichogrammatidae, and Scelionidae, play several important roles in the parasitoid‐host interaction. It is clear from the literature that the specific role (s) vary among species. Only recently have the biochemical and endocrinological roles of these cells been considered. This overview examines the recent literature on teratocytes and stresses the importance of in vitro procedures to elucidate the functional roles (trophic, immunosuppression, secretory) of teratocytes in the parasitoid‐host relationship.
Qualitative and quantitative analyses of juvenile hormone and ecdysteroids from the egg to the pupal molt in Trichoplusia ni
Authors: Grossniklaus‐Bürgin Christa , Lanzrein Beatrice
Published: February 2005

A method was developed to determine in the same extract juvenile hormone and various types of ecdysteroids in precisely staged eggs and larvae of Trichoplusia ni. Ecdysteroids were tentatively identified on the basis of their retention time in ion suppression reversed‐phase HPLC and their cross‐reactivity with two relatively non‐specific, complimentary antibodies, whereas juvenile hormone was identified using reversed‐phase HPLC combined with Galleria bioassay. Freshly laid eggs contained low levels of immunoreactive ecdysteroids. Mid‐polar ecdysteroids increased in the phase of segmentation (14–18 h) and 1st larval cuticle formation (36–44 h), when 20‐hydroxyecdysone and 20,26‐dihydroxyecdysone were found to be predominant. Only traces of ecdysone and 26‐hydroxyecdysone were seen. Toward hatching ecdysteroids decreased and represented mainly compounds more polar than 20,26‐dihydroxyecdysone. In larval development ecdysteroids were low at the beginning of the feeding phases, increased toward cessation of feeding, and reached highest levels 12–15 h before ecdysis. In feeding stages ecdysone and 20‐hydroxyecdysone were predominant, whereas in molting stages they were seen together with 20,26‐dihydroxyecdysone and 20‐hydroxyecdysonoic acid. The juvenile hormone titer was very low in freshly laid eggs and was high (approximately 25 ng/g) in embryos at the stage of 1st larval cuticle formation and eye pigmentation. In eggs we tentatively identified juvenile hormones I and II, whereas in larval stages juvenile hormone II appeared to be the predominant or exclusive juvenile hormone. Its titer fluctuated rapidly and was high in early 1st‐instar larvae and again before the molts into the 3rd, 4th, and 5th instar. Highest titers were reached concomitant with the peak in 20‐hydroxyecdysone 12–15 h before ecdysis.
Conversion of 3‐dehydroecdysone by a ketoreductase in post‐diapause, pre‐hatch eggs of the gypsy moth Lymantria dispar
Authors: Kelly Thomas J. , Thyagaraja Belgaum S. , Bell Robert A. , Masler Edward P. , Gelman Dale B. , Borkovec Alexej B.
Published: February 2005

The prothoracic glands (PGs) of Lymantria dispar (day‐5 female, last‐stage larvae) produce both ecdysone and an ecdysteroid which has the same retention time on reverse‐phase liquid chromatography (RPLC) as a known standard of 3‐dehydroecdysone. The latter ecdysteroid can be converted by a heat‐labile factor in extracts of post‐diapause, pre‐hatch L. dispar eggs to an ecdysteroid which has the same retention time on RPLC as ecdysone. Purified 3‐dehydroecdysone, similarly treated with egg extract, also gives the same retention time on RPLC as ecdysone. Taken together, these data suggest that, like Manduca sexta, a major product of the PGs in L. dispar is 3‐dehydroecdysone. Furthermore, these data suggest that L. dispar eggs, which contain mature embryos, possess ecdysteroid ketoreductase activity capable of converting 3‐dehydroecdysone to ecdysone. This is the first report of ecdysteroid ketoreductase activity in embryonated eggs.
Purification and characterization of honey bee vitellogenin
Authors: Wheeler Diana E. , Kawooya John K.
Published: February 2005

A protocol has been developed for the purification of vitellogenin from the honey bee, Apis mellifera. Purification allows for the first characterization of a vitellogenin from the large order Hymenoptera. Hymenopteran vitellogenins are unusual among insect vitellogenins in that they contain only one type of apoprotein. The honey bee vitellogenin was isolated from hemolymph of honey bee queens by a combination of density gradient ultracentrifugation, ion‐exchange chromatography, and affinity chromatography. The native vitellogenin particle is a very high density glycolipoprotein containing approximately 91% protein, 7% lipid, and 2% carbohydrate. Phospholipid and diacylglycerol are the major lipid components. The equilibrium density (1.28 g/ml) is the same as that for Manduca sexta vitellogenin, which contains a much higher proportion of lipid. The covalently bound carbohydrate moiety of the particle is high in mannose. The amino acid composition of vitellogenin is similar to those of vitellogenins from other insect species. The N‐terminal amino acid sequence of the apoprotein was determined, the first such sequence for any insect vitellogenin. When analyzed by sodium dodecyl sulfate (SDS)‐gel electrophoresis A. mellifera vitellogenin resolved into a single band with an apparent Mr of 180,000. Gel filtration under reducing and native conditions yielded estimated Mr values of about 300,000.
On the mechanism of side chain oxidation of N‐β‐alanyldopamine by cuticular enzymes from Sarcophaga bullata
Authors: Sugumaran Manickam , Saul Steven J. , Dali Hemalata
Published: February 2005

The metabolism of N‐β‐alanyldopamine (NBAD) by Sarcophaga bullata was investigated. Incubation of NBAD with larval cuticular preparations resulted in the covalent bindings of NBAD to the cuticle and generation of N‐β‐alanylnorepinephrine (NBANE) as the soluble product. When the reaction was carried out in presence of a powerful quinone trap viz., N‐acetylcysteine, NBANE formation was totally abolished; but a new compound characterized as NBAD‐quinone‐N‐acetylcysteine adduct was generated. These results indicate that NBAD quinone is an obligatory intermediate for the biosynthesis of NBANE in sarcophagid cuticle. Accordingly, phenylthiourea—a well‐known phenoloxidase inhibitor—completely inhibited the NBANE production even at 5 μM level. A soluble enzyme isolated from cuticle converted exogenously supplied NBAD quinone to NBANE. Chemical considerations indicated that the enzyme is an isomerase and is converting NBAD quinone to its quinone methide which was rapidly and nonenzymatically hydrated to form NBANE. Consistent with this hypothesis is the finding that NBAD quinone methide can be trapped as β‐methoxy NBAD by performing the enzymatic reaction in 10% methanol. Moreover, when the reaction was carried out in presence of kynurenine, two diastereoisomeric structures of papiliochrome II‐{Nar‐[α‐3‐aminopropionyl amino methyl‐3,4‐dihydroxybenzyl]‐L‐kynurenine} could be isolated as byproducts, indicating that the further reactions of NBAD quinone methide with exogenously added nucleophiles are nonenzymatic and nonstereoselective. Based on these results, it is concluded that NBAD is metabolized via NBAD quinone and NBAD quinone methide by the action of phenoloxidase and quinone isomerase respectively. The resultant NBAD quinone methide, being highly reactive, undergoes nonenzymatic and nonstereoselective Michael‐1,6‐addition reaction with either water (to form NBANE) or other nucleophiles in cuticle to account for the proposed quinone methide sclerotization.
Identification, accumulation, and biosynthesis of the cuticular hydrocarbons of the southern armyworm, Spodoptera eridania (cramer) (lepidoptera: Noctuidae)
Authors: Guo Lin , Blomquist Gary J.
Published: February 2005

The accumulation and biosynthesis of cuticular and internal hydrocarbons in the Southern armyworm, Spodoptera eridania, were examined at closely timed intervals during larval and pupal development. Gas chromatography‐mass spectrometry (GC‐MS) was used to identify n‐alkanes, monomethylalkanes, and dimethylalkanes ranging in chain length from 23 to 35 carbons. The amount of cuticular hydrocarbon stayed relatively constant during each stadium, while the amount of internal hydrocarbon increased dramatically during the first half of each larval stadium, presumably to replace the cuticular hydrocarbon lost on the shed cast skin with each molt. The accumulation of internal hydrocarbon was mirrored by large increases in the rate of incorporation of labeled acetate into the hydrocarbon fraction. Hydrocarbon production fell to very low rates during the latter part of the fourth and fifth larval stadia. Relatively high rates of hydrocarbon production were observed during the first and last one‐third of the pupal stage and essentially all of the hydrocarbons produced during this stage remained internal. These data document large changes in the rates of hydrocarbon production during development in S. eridania and suggest that most of the hydrocarbon produced during each stage was stored internally and then transported to the cuticle of the next stage.
Oxidation of 3,4‐dihydroxybenzyl alcohol: A sclerotizing precursor for cockroach ootheca
Authors: Sugumaran Manickam , Semensi Victor , Dali Hemalata , Nellaiappan Kaliappan
Published: February 2005

The oxidation of 3,4‐dihydroxybenzyl alcohol, one of the sclerotizing precursors for the tanning of the ootheca of cockroach Periplaneta americana, is reported for the first time. Mushroom tyrosinase catalyzed oxidation of 3,4‐dihydroxybenzyl alcohol generated the corresponding quinone which was found to be unstable and readily transformed to produce 3,4‐dihydroxybenzaldehyde as the stable product probably through the intermediary formation of a quinone methide. Phenoloxidase isolated from the left colleterial gland of P. americana also catalyzed this new reaction. When the enzymatic oxidation of 3,4‐dihydroxybenzyl alcohol was performed in the presence of a test protein such as lysozyme, the reactive species formed, caused the oligomerization of test protein. Similar studies with colleterial gland proteins, failed to generate oligomers, but produced insoluble polymeric proteins. The probable fate of 3,4‐dihydroxybenzyl alcohol for the tanning of cockroach ootheca is discussed.
PCR‐generated conspecific sodium channel gene probe for the house fly
Authors: Knipple Douglas C. , Payne Lori L. , Soderlund David M.
Published: February 2005

A segment of the house fly (Musca domestica) homologue of the para (paralytic) sodium channel gene of Drosophila melanogaster was isolated by using mixed sequence oligonucleotide primers in the polymerase chain reaction (PCR). The specificity of the procedure was demonstrated by genomic Southern analysis using the housefly PCR amplification product as a probe and by DNA sequence analysis. The latter showed structural homology to the para gene, but not to the corresponding region of DSC1, another D. melanogaster gene with structural similarity to vertebrate sodium channel genes.
Purification of ribosomal subunits from Ceratitis capitata and comparison of their proteins from two different developmental stages
Authors: Pilali Maria M. , Fragoulis Emmanuel G.
Published: February 2005

Ribosomal subunits have been prepared from larvae of Ceratitis capitata at two different developmental stages. The subunits are stable and pure on the basis of recentrifugation experiments and characterization of the RNA contained in them. When ribosomal subunits were allowed to combine, they reassociated to form an 80S ribosome, which is active in poly(U)‐directed polyphenylalanine synthesis. The proteins derived from these subunits were characterized by one and two dimensional electrophoresis and the patterns obtained from 6‐day old larvae and white prepupae were compared.
Temporal expression of ornithine decarboxylase in developing embryos of Musca domestica
Authors: Sparks Robert B. , Vaske David , Lilleberg Stan , Leopold Roger A.
Published: February 2005

The objective of this study was to partially characterize and follow the temporal expression of the enzyme ornithine decarboxylase (ODC) throughout embryonic and early larval development of Musca domestica. Enzymatically active ODC was shown to be present at detectable levels in the embryos only during the latter stages of embryogenesis. This temporally expressed enzyme displayed maximum activity at the time of hatching, and the activity rapidly declined in the newly hatched larvae. The half‐life of ODC activity in extracts at the time of hatching and 30 min after hatching was 57 min and 12 min, respectively. The subunit molecular weight of the embryonic ODC was determined to be 46,000, and the apparent native molecular weight was determined to be 276,000. The concentrations of the polyamines putrescine, spermidine, and spermine also were determined throughout embryogenesis. Spermidine was found to be present in the embryos at about 10‐fold higher concentrations than spermine and at about 100‐fold higher concentrations than putrescine. These polyamines did not undergo major changes in concentration throughout development of the embryos.
Digestive proteinases of larvae of the corn earworm, Heliothis zea: Characterization, distribution, and dietary relationships
Authors: Lenz Cynthia J. , Kang Jaesun , Rice William C. , McIntosh Arthur H. , Chippendale G. Michael , Schubert Karel R.
Published: February 2005

Proteinases and peptidases from the intestinal tract of fifth‐instar larvae of Heliothis ( = Helicoverpa) zea (Boddie) (Lepidoptera:Noctuidae) were characterized based on their substrate specificity, tissue of origin, and pH optimum. Activity corresponding to trypsin, chymotrypsin, carboxypeptidases A and B, and leucine aminopeptidase was detected in regurgitated fluids, midgut contents, and midgut wall. High levels of proteinase activity were detected in whole midgut homogenates, with much lower levels being observed in foregut and salivary gland homogenates. In addition, enzyme levels were determined from midgut lumen contents, midgut wall homogenates, and regurgitated fluids. Proteinase activites were highest in the regurgitated fluids and midgut lumen contents, with the exception of leucine aminopeptidase activity, which was found primarily in the midgut wall. Larvae fed their natural diet of soybean leaves had digestive proteinase levels that were similar to those of larvae fed artificial diet. No major differences in midgut proteinase activity were detected between larvae reared under axenic or xenic conditions, indication that the larvae are capable of digesting proteins in the absence of gut microorganisms. The effect of pH on the activity of each proteinase was studied. The pH optima for the major proteinases were determined to be pH 8.0‐8.5 for trypsin, when tosyl‐L‐arginine methyl ester was used as the substrate; and pH 7.5‐8.0 for chymotrypsin, when benzoyl‐L‐tyrosine ethyl ester was used as the substrate.
Hemolymph lipids and flight activity in Helicoverpa zea (Boddie) (lepidoptera: Noctuidae)
Authors: Judge David N. , Mullins Donald E. , Eaton John L.
Published: February 2005

In this study the flight activity of female and male Helicoverpa zea (Boddie) moths was observed and compared to hemolymph lipid concentrations. The major male and female H. zea flight activity occurred between simulated dusk (1700) and dawn (0300). Male flight activity was up to 7 times greater than females through 6 days after eclosion except for the 1st day (0.8 times). Females had a unimodal pattern of flight activity, peaking between dusk and 2 h later. Males had a bimodal pattern; one between dusk and 2 h later, and another 3 h after dusk, continuing for h. Prior to dusk, total neutral hemolymph lipids (neutral) of H. zea day 4 moths was 64 μg/μl for males and 48 μg/μl for females. Typical lipid composition in day 4 males prior to flight was 1,2‐diacylglycerides (DG) (50% w/w), triacylglycerides (TG) (35%), cholesterol esters (2%), and less than 1% monoacylglycerides and cholesterol. The remainder consisted of free fatty acids (<0.5 μg/μl), and various uncharacterized phospholipids and lipophilic compounds. Hemolymph DG concentration patterns were similar between day 4 males and females, were highest in both sexes prior to, during, and after flight (approximately 32 μg/μl), and then decreased steadily throughout the flight period to approximately 16 μg/ml as flight ceased. Hemolymph TG were lower than DG, but followed the same pattern except at 2100 and 2300. In day 4 males between 2100 and 2300, TG increased to 33 μg/μl which was when DG was lowest (15 μg/μl) and their flight activity was highest. Hemolymph DG decreased (26 to 20 μg/μl) in day 4 females between 2100 and 2300 as TG remained fairly constant (18 μg/μl).
Fat body changes in a locust, Chortoicetes terminifera (Walker) (Orthoptera: Acrididae), parasitized by a nemestrinid fly
Authors: Horwood Martin A. , Hales Dinah F.
Published: February 2005

The fat body of Chortoicetes terminifera (Walker) (Orthoptera: Acrididae) parasitized by Trichopsidea oestracea (Westwood) (Diptera: Nemestrinidae) was hypertrophied and showed extensive cytological and functional changes. Lipid content (as a percentage of dry weight) was significantly higher in parasitized locusts. Regression of lipid content against parasitoid weight was quadratic in female hosts and linear (negative) in male hosts. The fatty acid composition of C. terminifera was not affected by parasitization. Parasitized male locusts had lower body weight than nonparasitized males and contained less soluble protein. Results indicated that egg development was inhibited in parasitized female locusts. These conditions may be related to changes in the host's fat body.
Pharmacologic characterization of muscarinic receptors of insect brains
Authors: Abdallah Elsayed A. M. , Eldefrawi Mohyee E. , Eldefrawi Amira T.
Published: February 2005

Muscarinic receptors in brain membranes from honey bees, houseflies, and the American cockroach were identified by their specific binding of the non‐selective muscarinic receptor antagonist [3H]quinuclidinyl benzilate ([3H]QNB) and the displacement of this binding by agonists as well as subtype‐selective antagonists, using filtration assays. The binding parameters, obtained from Scatchard analysis, indicated that insect muscarinic receptors, like those of mammalian brains, had high affinities for [3H]QNB (KD = 0.47 nM in honey bees, 0.17 nM in houseflies and 0.13 nM in the cockroach). However, the receptor concentration was low (108, 64.7, and 108 fmol/mg protein for the three species, respectively). The association and dissociation rates of [3H]QNB binding to honey bee brain membranes, sensitivity of [3H]QNB binding to muscarinic agonists, and high affinity for atropine were also features generally similar to muscarinic receptors of mammalian brains.

In order to further characterize the three insect brain muscarinic receptors, the displacement of [3H]QNB binding by subtype‐selective antagonists was studied. The rank order of potency of pirenzepine (PZ), the M1 selective antagonist 11‐((2‐((dimethylamino)‐methyl)1‐ piperidinyl)acetyl)‐5, 11‐dihydro‐6H‐pyrido(2,3‐b)‐(1,4)‐benzodiazepin‐6 one (AF‐DX 116), the M2‐selective antagonist, and 4‐DAMP (4‐diphenylacetoxy‐N‐methylpiperidine methiodide) the M3‐selective antagonist, was also the same as that of mammalian brains, i.e., 4‐DAMP > PZ > AF‐DX 116. The three insect brain receptors had 27–50‐fold lower affinity for PZ (Ki 484–900 nM) than did the mammalian brain receptor (Ki 16 nM), but similar to that reported for the muscarinic receptor subtype cloned from Drosophila. Also, the affinity of insect receptors for 4‐DAMP (Ki 18.9–56.6 nM) was much lower than that of the M3 receptor, which predominates in rat submaxillary gland (Ki of 0.37 nM on [3H]QNB binding). These drug specificities of muscarinic receptors of brains from three insect species suggest that insect brains may be predominantly of a unique subtype that is close to, though significantly different from, the mammalian M3 subtype.
Assembly and secretion of lipophorin by the larval fat body of the southwestern corn borer, Diatraea grandiosella: An in vitro study
Authors: Shelby Kent S. , Chippendale G. Michael
Published: February 2005

The synthesis, processing, and secretion of lipophorin by the larval fat body of the southwestern corn borer, Diatraea grandiosella, was examined using in vitro techniques. Pulse‐labeling of lipophorin with [35S]methionine showed that apolipophorin‐I and ‐II were each synthesized and secreted from the fat body into Grace's medium with an intracellular transit time of about 45 min. Secretion of the apolipoproteins from the fat body became insensitive to the presence of monensin, which disrupts protein processing in the Golgi complex, at 30 min, indicating that most of the pulse‐labeled apolipoprotein has transited the Golgi complex by this time. Three inhibitors of protein processing, carbonylcyanide m‐chlorophenyl hydrazone, monensin, and brefeldin A, inhibited secretion of lipophorin into medium. Puromycin treatment did not appear to result in the secretion into the medium of lipophorin particles containing incomplete translation products of apolipophorin‐I or ‐II. Incubation of fat bodies with [3H]oleate resulted in the secretion of lipophorin containing [3H]glycerides, a process that was inhibited by cycloheximide, puromycin, and monensin, indicating that apolipoprotein synthesis is required for secretion of [3H]glyceride on nascent lipophorin particles. In contrast, suramin, which has been shown to block the binding of lipophorin to plasma membrane receptors, inhibited the synthesis and secretion of lipophorin, but it did not appear to inhibit the transfer of [3H]lipid from the fat body to lipophorin. Inhibitors of protein synthesis and processing, therefore, can be used to distinguish between secretion of lipophorin‐associated lipids and secretion of lipids mediated by the lipid‐transfer particle outside the plasma membrane of the fat body.