bluuebunny/crossref_metadata_2025 huggingface dataset:
Repetitive DNA interspersion patterns in diptera
Authors: Cockburn Andrew F. , Mitchell Sharon E.
Published: February 2005

A wide spectrum of repetitive DNA amounts and interspersion patterns is seen in mosquitoes and other dipterans. Using a simple and rapid technique, we show that these range from a minimal amount in five species of Anopheles through moderate amounts in Culex quinquefasciatus to large amounts in Aedes aegypti and Stomoxys calcitrans. Although Culex and Aedes are closely related and both have a considerable amount of interspersed repetitive DNA, the repetitive sequences are different between the two genera. These results and previously published information show that the amount of repetitive DNA and sequences involved have changed many times during the evolution of the Diptera.
Isolation of the vitellogenin‐binding protein from locust ovaries
Authors: Roehrkasten Axel , Ferenz Hans‐Joerg , Buschmann‐Gebhardt Beate , Hafer Johannes
Published: February 2005

A rapid, efficient procedure for the isolation and purification of the vitellogenin binding protein from locust ovarian membranes is described. After solubilization with the nonionic detergent octyl‐β‐D‐glucoside and removal of the detergent, the binding protein is subjected to affinity chromatography on vitellogenin coupled covalently to Affi‐Gel 15. The binding protein is eluted with suramin and EDTA at low pH value. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis reveals a polypeptide with a molecular weight of 156,000 in the eluted fraction. By ligand blotting this polypeptide could be identified as the vitellogenin binding protein. It retains its high‐affinity binding properties. The specific binding of vitellogenin increases from 4.8 μg (intact ovarian membranes) to 170.9 μg (affinity purified binding protein) per mg membrane protein, which corresponds to a purification factor of 35.
Persistence and effects of α‐methyl‐substituted amino acids on 5‐hydroxytryptamine and dopamine concentrations in cockroach (Periplaneta americana) nervous tissue
Authors: Sloley B. Duff
Published: February 2005

The α‐methylated derivatives of tryptophan, tyrosine, and dihydroxyphenylalanine were injected into cockroaches (Periplaneta americana). The levels of these compounds and those of dopamine, 5‐hydroxytryptamine, tyrosine, and tryptophan in the nervous tissue, hemolymph, and fat body were measured at various times after drug administration. Levels of 5‐hydroxytryptamine and tryptophan in the nervous tissue are significantly reduced by α‐methyltryptophan administration. Concentrations of dopamine in nervous tissue are reduced by α‐methyltyrosine administration. This effect also persists for several weeks, and α‐methyltyrosine is observed in the nervous tissue 3 weeks after injection. Levels of dopamine and 5‐hydroxytryptamine in the nervous tissue are unaffected by α‐methyldihydroxyphenylalanine, and this compound is less persistent in nervous tissue than α‐methyltyrosine or α‐methyltryptophan demonstrates that these compounds can be absorbed and affect amine levels in the nervous tissue when included in the diet. Inhibition of tryptophan hydroxylation by crude enzyme preparations of cockroach nervous tissue was demonstrated with both α‐methyltryptophan and α‐methyltyrosine, with α‐methyltryptophan being the more effective inhibitor. Aromatic amino acid decarboxylase activity toward dihydroxyphenylalanine in crude enzyme preparations of cockroach nervous tissue was strongly inhibited by α‐methyldihydroxyphenylalanine and monofluoromethyldihydroxyphenylalanine, slightly inhibited by α‐methyltyrosine and unaffected by α‐methyltryptophan at concentrations up to 10−3 M. The results indicate that α‐methyltyrosine and α‐methyltryptophan, but not α‐methyldihydroxyphenylalanine, can selectively alter amine concentrations in insect nervous tissue and that insects are only poorly able to metabolize or excrete these compounds. The selective and long‐lasting depletion of dopamine or 5‐hydroxytryptamine by some of these compounds suggest that they may be useful in behavioral studies designed to elucidate the roles of these amines in insects.
Synthesis and nuclear magnetic resonance study of 3‐dehydroecdysteroids
Authors: Girault Jean‐Pierre , Blais Catherine , Beydon Philippe , Rolando Christian , Lafont René
Published: February 2005

Several 3‐dehydro‐ (or 3‐oxo‐) ecdysteroids have been prepared by enzymatic and/or chemical means. Methods for their purification using various high‐performance liquid chromatography systems are described. Proton and carbon nuclear magnetic resonance analyses show that 3‐dehydroecdysteroids when dissolved in water or methanol (but not in chloroform) present a temperature‐dependent equilibrium between two forms. The possible structure of these two forms is discussed.
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 th e tick Ornithodoros moubata convert ingested 20‐hydroxyecdysone (20E) to apolar conjugates AP2, which are then converted to th e more polar conjugates API. Only small quantities of free hormone were transferred to th e hemolymph and the carcass within t h e 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 API conjugates.

API obtained in second instar nymphs fed with [3H]ecdysone ([3H]E) remain stable throughout the following nymphal instars. The ecdysteroid moiety of APT 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., API 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 API, 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.
Changes in the titer of the female‐predominant storage protein (81K) during larval and pupal development of the waxmoth, galleria mellonella
Authors: Bean Daniel W. , Silhacek Donald L.
Published: February 2005

The levels of an 81K storage protein in the waxmoth, Galleria mellonella, were monitored during the course of development using rocket immunoelectrophoresis. During the fifth and sixth larval stadia, 81K protein levels increased during feeding and growth but sharply declined at each larval molt. During the fifth and sixth stadia hemolymph levels of the 81K protein increased to about 1 and 2.5 mg/ml, respectively, with no discernible differences between levels in males and females. Neither the fat body nor the remainder of the carcass contained the 81K protein, indicating that the accumulation of this protein during the intermolt period was exclusively in the hemolymph and redistribution of the 81K protein into other tissues does not occur at the final two larval molts. During the seventh (final) larval stadium the absolute quantities of the 81K protein increased from 23 μg per insect to over 1,600 μg in females and to 300 μg in males. The hemolymph concentration of the 81K protein reached 28 mg/ml in females and 6 mg/ml in males with only low levels found in the remaining tissues. Shortly after pupal apolysis, marked by eyespot retraction, the fat body in both sexes rapidly and quantitatively sequestered the 81K protein from the hemolymph. The 81K protein in the hemolymph of both males and females rapidly dropped to nearly zero concentration by pupation. The 81K storage protein remained localized in the fat body cells after uptake occurred, even though the fat body cells disaggregate and reaggregate during metamorphosis. During pharate adult development the 81K storage protein disappeared from the fat body without entering the hemolymph. At adult eclosion 81K was virtually absent from the tissues of both males and females.
Ecdysteroids from developing eggs of Pieris brassicae
Authors: Beydon Philippe , Permana Agus‐dana , Colardeau Jacqueline , Morinière Madeleine , Lafont René
Published: February 2005

Ecdysone metabolism in Pieris brassicae eggs was revealed after injection of [3H]ecdysone into eggs. Labeled ecdysteroids were analyzed by HPLC. The metabolic fate of [3H]ecdysone is similar to that observed during postembryonic development, i.e., the larval instars and pupal instar. The ecdysone molecule is affected by hydroxylations at C‐20 and at C‐26; 26‐hydroxyecdysteroids can be further oxidized into ecdysteroid acids. In an effort to identify egg ecdysteroids, we examined the metabolism of [3H]cholesterol injected into female pharate adults to achieve long‐term labeling. In eggs, [3H]cholesterol was converted to 20‐hydroxyecdysonoic acid, ecdysonoic acid, 20‐hydroxyecdysone, and ecdysone. The rates of [3H]cholesterol incorporation into ecdysteroids increase according to the age of the eggs (from 0.06% at laying to 0.12% at hatching), while the cholesterol level remains constant throughout embryogenesis, suggesting that the embryos of Pieris brassicae can produce ecdysteroids either from cholesterol or from apolar ecdysteroids not identified by the analytical method. Pieris eggs contain amounts of ecdysteroids which increase from 1.5 nmol/g at laying to 3.8 nmol/g at hatching.
Metabolism of dietary Δ8‐sterols and 9β, 19‐cyclopropyl sterols by Locusta migratoria
Authors: Corio‐Costet Marie F. , Charlet Maurice , Benveniste Pierre , Hoffman Jules
Published: February 2005

Adult female locusts were reared on wheat seedlings (experimental wheat) containing more than 97% of 9,10‐cyclopropyl sterols and Δ8‐sterols and less than 2% of Δ5‐sterols. These insects showed a dramatic decrease in their cholesterol, cholestanol and Δ7‐cholestanol content compared with control insects. These changes were more dramatic for steryl esters than for free sterols. Similar results were observed in eggs laid by the insects fed on experimental wheat. The decrease in Δ5‐, Δ0‐ and Δ7‐sterols in insects reared on experimental wheat was compensated by a marked accumulation of Δ8‐sterols and 9β, 19‐cyclopropyl sterols in the free sterol fraction and especially in the steryl ester fractions. The ecdysteroid content of eggs laid by experimental female insects was reduced by up to 80% compared with controls. These and other results suggest that the dietary 9β, 19‐cyclopropyl sterols and Δ8‐sterols cannot be used by Locusta in place of Δ5‐sterols for ecdysteroid biosynthesis. To give support to this hypothesis, the experimental wheat was supplemented with various sterols before being presented to the insects immediately after the first egg laying. When cholesterol, sitosterol, or cholestanol were used as supplements, there was a complete recovery of the ecdysteroid titer in the eggs, a disappearance of 9β, 19‐cyclopropyl sterols, and a restoration of the cholesterol content in both the animals and the eggs. Stigmasterol, stigmastanol, and ergosterol were much less efficient in reversing the effects of the experimental wheat. As expected, 9β, 19‐cyclopropyl sterols were totally ineffective. These results are discussed in the light of our information on the role played by sterols in insect development and on the structural features required by the sterols to fulfill their role.
Ecdysone metabolism in the host‐parasitoid‐system Trichoplusia ni/Chelonus sp
Authors: Grossniklaus‐Buergin Christa , Connat Jean‐Louis , Lanzrein Beatrice
Published: February 2005

In unparasitized 4th and 5th‐instar larvae of Trichoplusia ni and in 4th‐instar larvae parasitized by Chelonus sp. 20‐hydroxyecdysone, 20,26‐dihydroxyec‐dysone, and 20‐hydroxyecdysonoic acid were the predominant metabolites formed 2 h after injection of [3H]ecdysone. Other unidentified metabolites were seen, but none seemed to be specific for either parasitized or unparasitized larvae. The major difference between parasitized and unparasitized larvae was seen with respect to the quantity of apolar (unidentified) and polar metabolites (20‐hydroxyecdysonoic acid and unidentified ones), which were produced to a greater extent in parasitized larvae. Ecdysone was rapidly converted into 20‐hydroxyecdysone and the other polar metabolites in all stages investigated, and the parasitoid seemed not to affect the conversion of ecdysone into 20‐hydroxyecdysone. When analyzing the fate of [3H]ecdysone in host and parasite separately, at a stage when the parasite drinks hemolymph of its host, we observed that 10–20% of the radioactivity was recovered from the parasitoid. Analysis of the parasitoid's ecdysteroids revealed that ecdysone and 20‐hydroxyecdysone represented only a small proportion of the recovered labeled ecdysteroids, the majority being apolar and polar metabolites. Our data suggest that the parasitoid takes up ecdysteroids from its host, converts them, and to some extent releases apolar metabolites into the host.
Juvenile hormone esterase activity from Manduca sexta corpora allata in vitro
Authors: Sparks Thomas C. , Allen L. Gregory , Schneider Frank , Granger Noelle A.
Published: February 2005

Juvenile hormone esterase (JHE) activity released by the corpora allata (CA) into incubation media (CA‐JHE) was titered daily during the course of the last (fifth [V]) larval stadium of Manduca sexta. This CA‐JHE activity was relatively low during the early last stadium up to the time of commitment (V4), then rose rapidly to a peak on V6. Activity declined sharply almost to precommitment levels by V8, before rising to a second peak on the first day of the pupal phase (P0). This pattern of activity is distinct from that of hemolymph JHE activity, which peaks just prior to wandering on V4 and again just prior to pupation (V9). Although the CA‐JHE and hemolymph‐JHE possess different temporal patterns of activity, isoelectric focusing, gel electrophoresis, and initial studies with selected inhibitors suggest that the enzymes responsible for the CA‐JHE and hemolymph‐JHE activities are similar, but not identical, in nature.

Exposure of the V6 CA in vitro to JH II (0.1 μM) or fluoromevalonolactone (FMev; 0.1 mM) produced an approximate fivefold increase and 60% decrease in JH acid synthesis, respectively. Conversely, the same treatments resulted in an inhibition (JH II) and stimulation (FMev) of CA‐JHE activity. These observations suggest that JH may be involved in the direct positive feedback regulation of postwandering larval CA and that the CA‐JHE may also be integrally related to this positive feedback mechanism.
Further studies on the mechanism of oxidation of N‐acetyldopamine by the cuticular enzymes from Sarcophaga bullata and other insects
Authors: Sugumaran Manickam , Kundzicz Heather , Bedell‐Hogan Debra , Schinkmann Karin
Published: February 2005

In accordance with our earlier results, quinone methide formation was confirmed to be the major pathway for the oxidation of N‐acetyldopamine (NADA) by cuticle‐bound enzymes from Sarcophaga bullata larvae. In addition, with the use of a newly developed HPLC separation condition and cuticle prepared by gentle procedures, it could be demonstrated that 1, 2‐dehydro‐NADA and its dimeric oxidation products are also generated in the reaction mixture containing a high concentration of NADA albeit at a much lower amount than the NADA quinone methide water adduct, viz., N‐acetylnorepinephrine (NANE). By using different buffers, it was also possible to establish the accumulation of NADA quinone in reaction mixtures containing NADA and cuticle. That the 1,2‐dehydro‐NADA formation is due to the action of a NADA desaturase system was established by pH and temperature studies and by differential inhibition of NANE production. Of the various cuticle examined, adult cuticle of Locusta migratoria, presclerotized cuticle of Periplaneta americana, and white puparial cases of Drosophila melanogaster exhibited more NADA desaturase activity than NANE generating activity, while the reverse was observed with the larval cuticle of Tenebrio molitor and pharate pupal cuticle of Manduca sexta. These studies indicate that both NADA quinone methide and 1, 2‐dehydro NADA are formed during enzymatic activation of NADA in insect cuticle. Based on these results, a unified mechanism for β‐sclerotization involving quinone methides as the reactive species is presented.
Chemical‐ and cuticular phenoloxidase‐ mediated synthesis of cysteinyl–catechol adducts
Authors: Sugumaran Manickam , Dali Hemalata , Semensi Victor
Published: February 2005

N‐Acetylcysteine adducts of o‐benzoquinones derived from catechol, 4‐methylcatechol, and N‐acetyldopamine were chemically synthesized and characterized by a combination of UV, IR, and NMR spectral studies. Oxidation of catechol, 4‐methylcatechol, and N‐acetyldopamine by cuticle‐bound phenoloxidase from Sarcophaga bullata in the presence of N‐acetylcysteine resulted in the formation of covalent adducts between catecholic compounds and N‐acetylcysteine. Structural identities of these adducts were established by comparison of their HPLC retention time and UV spectra with those of synthetic adducts and by cochromatography with authentic samples. Although insect cuticle is known to contain only trace amounts of cysteine, the in vitro synthesis of quinone cysteine adducts mediated by cuticular phenoloxidase strongly indicates the occurrence of similar reactions in vivo as well and is in support of Pryor's quinone tanning hypothesis.
Binding of juvenile hormone III to lipophorin from the american cockroach Periplaneta americana
Authors: De Kort Stan , Koopmanschap Bertha
Published: February 2005

Whole hemolymph from the American cockroach, Periplaneta americana, efficiently binds juvenile hormone (JH) III and to a lesser extent JH‐I and 10, 11‐epoxyfarnesyl diazoacetate (EFDA). The dissociation constants for racemic JH‐III and EFDA are 30 ± 2 nM and 1.0 μM, respectively. Isolated lipophorin also binds [3H]JH‐III and to a lesser extent JH‐I. Other proteins from the hemolymph do not bind JH‐III. Binding of JH‐III to lipophorin is enantioselective. The dissociation constant, measured with a 92% 10R and 8% 10S mixture, is 21 ± 2 nM. Each lipophorin molecule contains one specific binding site for JH‐III. It is concluded that lipophorin is the JH‐III‐specific transport protein in the hemolymph of the American cockroach. By a combination of photoaffinity labelling and gradient electrophoresis with sodium dodecyl sulphate on polyacrylamide gel, we showed that the JH‐III‐specific binding site is probably located on apolipophorin I.
Purification and characteristics of a specific alkaline phosphatase from the integument of the mediterranean fruit fly, Ceratitis capitata
Authors: Psachoulia Christina , Bourtzis Kostas , Marmaras Vassilis J.
Published: February 2005

A specific alkaline phosphatase (ALPase) from the integument of white pupae has been purified 500‐fold. The purification procedure included solubilization with Triton X‐100, butanol extraction, fractionation with ammonium sulfate, and chromatography on concanavalin A‐Sepharose, Sephadex G‐200, and Sepharose 6B. Two peaks with enzyme activity were observed. The major peak had a molecular weight of approximately 180,000, while the minor peak, which had identical kinetic parameters and substrate specificity as those of the major one, was eluted in a high molecular weight form (about 900,000), probably cross‐linked with chitin, since the enzyme was separated from the chitin only by lysozyme treatment. The enzyme hydrolyzes only tyrosine phosphate and β‐glycerophosphate, with apparent Kms of 0.35 mM and 0.22 mM, respectively, but not serine phosphate, threonine phosphate, ATP, and AMP. The optimum pH was in the alkaline range, with a peak at pH 9.4. The divalent cations Mn2+, Mg2+, and Ba2+ had stimulatory actions, while Cu2+ exerted a very strong inhibitory action on the enzyme activity. The ALPase was inhibited by L‐tyrosine in a dose‐dependent fashion. At a concentration of 2 mM, L‐tyrosine totally inhibited the enzyme activity, while L‐phenylalanine inactivated the enzyme about 25%. The accumulated evidence that ALPase is involved in the sclerotization process of insect integument is discussed.
Distribution, properties, and functions of midgut carboxypeptidases and dipeptidases from Musca domestica larvae
Authors: Jordão Beatriz P. , Terra Walter R.
Published: February 2005

Dipeptidase and carboxypeptidase A activities were determined in cells and luminal contents of the fore‐, mid‐, and hind‐midgut of Musca domestica larvae. Dipeptidase activity was found mainly in hind‐midgut cells, whereas carboxy‐peptidase activity was recovered in major amounts in both cells and in luminal contents of hind‐midguts. The subcellular distribution of dipeptidase and part of the carboxypeptidase A activities is similar to that of a plasma membrane enzyme marker (aminopeptidase), suggesting that these activities are bound to the microvillar membranes. Soluble carboxypeptidase A seems to occur both bound to secretory vesicles and trapped in the cell glycocalyx. Based on density‐gradient ultracentrifugation and thermal inactivation, there seems to be only one molecular species of each of the following enzymes (soluble in water or solubilized in Triton X‐100): membrane‐bound dipeptidase (pH optimum 8.0; Km 3.7 mM GlyLeu, Mr 111,000), soluble carboxypeptidase (pH optimum 8.0; Km 1.22 mM N‐carbobenzoxy‐glycyl‐L‐phenylalanine (ZGlyPhe), Mr45,000) and membrane‐bound carboxypeptidase (pH optimum 7.5, Km 2.3 mM ZGlyPhe, Mr58,000). The results suggest that protein digestion is accomplished sequentially by luminal trypsin and luminal carboxypeptidase, by membrane‐bound carboxypeptidase and aminopeptidase, and finally by membrane‐bound dipeptidase.
Microplitis croceipes teratocytes cause developmental arrest of Heliothis virescens larvae
Authors: Zhang Deqing , Dahlman D. L.
Published: February 2005

Microplitis croceipes teratocytes placed into nonparasitized Heliothis virescens larvae survived in the absence of a parasitoid larva and caused developmental changes in the host. Expressions of these changes included delayed larval mortality, incomplete larval‐pupal ecdysis, or delayed pupation. Two day old 4th stadium H. virescens larvae were more sensitive to injected teratocytes than were 5th stadium larvae. Three day old teratocytes were more effective than were 6 day old teratocytes. The degree of response was related to the number of injected teratocytes. For example, 750 three day old teratocytes (the approximate number from a single parasitoid egg) caused delayed larval mortality in 96% of the treated larvae whereas 175 three day old teratocytes caused delayed larval mortality in only 33% of the treated larvae. Even a dose of 80 teratocytes resulted in 15% incomplete larval‐pupal ecdysis compared to 0% for controls. Treatment with hemocyte‐and teratocyte‐free hemolymph from parasitized larvae, hemocytes from nonparasitized H. virescens, unfertilized M. croceipes eggs, Cotesia congregata teratocytes, or Micrococcus lysodeikticus cells all had very little effect either on larval growth or development time.
Juvenile hormone‐like effects of a thiazolylurea on lepidopteran larvae
Authors: Loeb Marcia J. , DeMilo Albert B. , Jones Grace , Jones Davey
Published: February 2005

Heliothis virescens maintained on a diet containing 50 ppm of thiazolylurea (5‐[[[2‐thiazolylamino]amino]carbonyl]‐1,3‐benzenedicarboxylic acid dimethyl ester) showed symptoms of juvenile hormone overdose in the last larval instar. Increases in development time and weight, as well as larval color changes, were similar in animals fed thiazolylurea or topically treated with the juvenile hormone mimic methoprene. The juvenile hormone esterase titer profile in hemolymph of animals fed thiazolylurea was much broader than in controls, and peaked 2 days later than in controls; the premolt ecdysteroid peak in hemolymph of animals fed thiazolylurea appeared 2 days earlier than in controls. These events are characteristic of high hemolymph JH titers throughout the last larval instar.
Relationship between the fatty acids of fat body triacylglycerol and lipophorin diacylglycerol of non‐diapause and diapause larvae of the southwestern corn borer, Diatraea grandiosella
Authors: Kuthiala Arun , Chippendale G. Michael
Published: February 2005

The fatty acids of the triacylglycerol reserves in the fat body and of the diacylglycerol of lipophorin in the hemolymph of non‐diapause and diapause larvae of D. grandiosella were compared. For both non‐diapause and diapause larvae palmitate, palmitoleate, oleate, and linoleate were the predominant fatty acids present in fatty body triacylglycerol, and palmitate, oleate, and linoleate were the predominant fatty acids present in lipophorin diacylglycerol. However, differences were detected in the relative amounts of oleate and linoleate present in lipophorin diacylglycerol of non‐diapause and diapause larvae. The relative amount of linoleate in lipophorin diacylglycerol declined during diapause, whereas that of oleate remained relatively high during diapause. The fatty acid profile of lipophorin diacylglycerol from non‐diapause larvae treated with a juvenile hormone analog to induce a diapause‐like state more closely matched that of diapause larvae than that of non‐diapause larvae. The differences detected in the fatty acid composition of lipophorin diacylglycerol in non‐diapause and diapause larvae appear to be due mainly to the different physiological states rather than to the different rearing temperatures employed. The results are discussed in relation to the essential role fatty acids, especially oleate, play in the survival of diapause larvae.
Catecholamines in the cuticles of four strains of the german cockroach Blattella germanica (L.) during sclerotization and melanization
Authors: Czapla Thomas H. , Hopkins Theodore L. , Kramer Karl J.
Published: February 2005

Catecholamines were extracted from the cuticles of four strains of the cockroach Blattella germanica at different times 48 h after adult ecdysis and analyzed by reverse phase HPLC with electrochemical detection. The wild (VPl), black (Bl), orange (or), and yellow (y) phenotypes differ in cuticular pigmentation, particularly in the extent of melanization. N‐β‐Alanyldopamine (NBAD) and N‐β‐alanylnorepinephrine (NBANE) were major o‐diphenolic compounds in extracts from cuticle of all strains during the main period of sclerotization. N‐Acetyldopamine (NADA) and N‐acetylnorepinephrine (NANE) were minor the first day after ecdysis, but accumulated to higher levels thereafter. Dopamine (DA) concentrations were higher in the darker pigmented cuticles of strains Bl and or than in the lighter‐colored cuticles of strains VPl and y. Extractable DA rapidly increased in VPl, Bl, and or cuticles shortly after ecdysis, reached peak levels 6–24 h later, and then decreased after melanization. Only small amounts of DA were detected in strain y cuticle, whereas NBANE concentrations were very high. Therefore, high DA levels in cuticle are correlated with melanization that occurs during the first few hours after adult ecdysis, whereas sclerotization is correlated with high levels of the N‐β‐alanylcatecholamines. Sclerotization appears to be delayed in strain Bl, since only low concentrations of the N‐acylated catecholamines accumulate until after melanization is completed.
Characterization of quinone tautomerase activity in the hemolymph of Sarcophaga bullata larvae
Authors: Saul Steven J. , Sugumaran Manickam
Published: February 2005

The hemolymph of Sarcophaga bullata larvae was activated with either zymosan or proteolytic enzymes such as chymotrypsin or subtilisin and assayed for phenoloxidase activity by two different assays. While oxygen uptake studies readily attested to the wide specificty of activated phenoloxidase, visible spectral studies failed to confirm the accumulation of quinone products in the case of 4‐alkyl substituted catechols such as N‐acetyldopamine and N‐β‐alanyldopamine. Sepharose 6B column chromatography of the activated hemolymph resolved phenoloxidase activity into two fractions, designated as A and B. Peak A possessed typical o‐diphenoloxidase (o‐diphenol, oxygen oxidoreductase EC 1.10.3.1) activity, while peak B oxidized physiologically important catecholamine derivatives such as N‐acetyldopamine, N‐acetylnorepinephrine, and N‐β‐alanyldopamine into N‐acetylnorepinephrine, N‐acetylarterenone, and N‐β‐alanylnorepinephrine, respectively, and converted 3,4‐dihydroxyphenylacetic acid, 3,4‐dihydroxymandelic acid, and 3,4‐dihydroxyphenylglycol into 3,4‐dihydroxymandelic acid, 3,4‐dihydroxybenzaldehyde, and 2‐hydroxy‐3′,4′‐dihydroxyacetophenone, respectively. These transformations are consistent with the conversion of phenoloxidase‐generated quinones to quinone methides and subsequent non‐enzymatic transformations of quinone methides. Accordingly, Peak B contained both o‐diphenoloxidase activity and quinone tautomerase activity. Sepharose 6B column chromatography of unactivated hemolymph resulted in the separation of quinone tautomerase from prophenoloxidase. The tautomerase rapidly converted both chemically made and mushroom tyrosinase‐generated quinones to quinone methides. Thus the failure to observe the accumulation of quinones with N‐acyl derivatives of dopamine and related compounds in the whole hemolymph is due to the rapid conversion of these long lived toxic quinones to short lived quinone methides. The latter, being unstable, undergo rapid non‐enzymatic transformations to form side‐chain‐oxygenated products that are non‐toxic. The possible roles of quinone isomerase and its reaction products—quinone methides—as essential components of sclerotization of cuticle and defense reaction of Sarcophaga bullata are discussed.
Ecdysone oxidase and 3‐oxoecdysteroid reductases in Manduca sexta midgut: Kinetic parameters
Authors: Weirich Gunter F. , Thompson Malcolm J. , Svoboda James A.
Published: February 2005

Ecdysone and 20‐hydroxyecdysone are converted to their 3‐epimers by enzymes in the midgut cytosol of Manduca sexta larvae. A partially purified cytosol preparation has been used to analyze the nature of and the interaction between these enzymes. The cytosol was shown to contain ecdysone oxidase, one or more 3‐oxoecdysteroid 3α‐reductase(s), and one or more 3‐oxoecdysteroid 3β‐reductase(s). The reductases reacted at different velocities with NADH and NADPH. With NADH, 3α‐reduction was the major reaction; with NADPH, 3β‐reduction was the major reaction. The apparent kinetic parameters for the enzymes support the assumed two‐step mechanism for the 3‐epimerization with a 3‐oxoecdysteroid as intermediate.
Two hypolipidemic agents, cholestyramine and niacin, affect the development and lipid content of Heliothis zea
Authors: Ritter Karla S. , Smith V. Blair
Published: February 2005

Two vertebrate hypolipidemic agents, cholestyramine and niacin, affected the growth and development of Heliothis zea as well as the quantity of acylglyceride or sterol present in the larva. As the concentration of cholestyramine in the diet increased to 6.0%: the number of larval molts increased from 5 or 6 to as many as 7 or 8, the time required for the onset of pupation increased from 11 or 12 to 20 days, and the number of adults that emerged decreased from at least 70 to 0%. The growth and development of the insect may have slowed, at least in part, because this agent reduced the quantity of sterol and glyceride in the tissues of the larva. Niacin also affected the growth and development of the insect. As the concentration of niacin in the diet increased to 5.0%: the number of larval molts increased by 1, the time required for the onset of pupation increased to 21 days, pupal weight decreased significantly, but adult emergence was normal. The growth and development of the insect may have slowed, at least in part, because this agent caused sterol to accumulate in the hemolymph of the larva. A water‐soluble component in the hemolymph also increased in the presence of niacin, but there was little or no change in the glyceride content. Further studies are warranted to determine the mode of action of these hypolipidemic agents in H. zea.
Contributing factors to reduced ecdysteroid titers in Heliothis virescens parasitized by Microplitis croceipes
Authors: Dahlman Douglas L. , Coar Danise L. , Noah Koller C. , Neary Todd J.
Published: February 2005

Heliothis virescens parasitized by Microplitis croceipes stop development at a predictable point prior to parasite emergence. The objectives of this study were to examine several factors which might contribute to the syndrome by comparing parasitized and nonparasitized larvae at specific and correspondingly similar physiological points in their development. Fifth stadium nonparasitized larvae experience a small pupal commitment burst of ecdysone followed by a large burst. In contrast, ecdysteroid titers in parasitized larvae remained at the pupal commitment level during the entire 5th stadium. Data shows that ecdysone 20‐monooxygenase and sterol precursors are not limiting factors, with the possible exception of limited hemolymph sterol at the end of parasitoid development. In addition, isolated prothoracic glands from parasitized larvae produce amounts of ecdysteroid comparable to controls when stimulated with a crude Manduca sexta prothoracicotropic hormone preparation. Juvenile hormone esterase titers in parasitized larvae are low throughout the 5th stadium. They do not show the major rapid increase in activity normally associated with the latter part of the active feeding period in the nonparasitized 5th instar. Possible explanations for the low ecdysteroid titers in parasitized larvae include failure to synthesize and release prothoracic gland stimulatory factor by the poorly developed fat body, insufficient sterol precursors at the critical time of ecdysteroid synthesis, inhibition of the release of PTTH, failure of the prothoracic gland to respond to PTTH, and the consequences of abnormally high juvenile hormone titers.
Developmental responses of Trichoplusia ni (Lepidoptera: Noctuidae) to parasitism by the ectoparasite Euplectrus plathypenae (Hymenoptera: Eulophidae)
Authors: Coudron Thomas A. , Kelly Thomas J. , Puttler Benjamin
Published: February 2005

Parasitism by Euplectrus plathypenae inhibits larval–larval ecdysis in Trichoplusia ni by injecting nonparalytic factor(s) into the host during the stinging process via the parasitoid's ovipositor. The parasitized host moves freely, feeds, and gains weight prior to the time of the normal ecdysis but does not molt. Parasitoid development is not required for the expression of molt arrest in the host. Parasitism during the first three fourths of the larval stadium results in molt arrest. Arrestment of molting is independent of 20‐hydroxyecdysone and juvenile hormone. The arrestment factor(s) affect the epidermal tissue of the thorax and abdomen in ligated hosts without apparent interaction from other areas of the body. Cuticle and epidermal tissue of parasitized insects do not show signs of apolysis or ecdysis.
Arylphorin of Trichoplusia ni: Characterization and parasite‐induced precocious increase in titer
Authors: Kunkel Joseph G. , Grossniklaus‐Buergin Christa , Karpells Sharon T. , Lanzrein Beatrice
Published: February 2005

Arlyphorin (Ap) is the principal protein of the last larval instar hemolymph of Trichoplusia ni. It was shown to be homologous with the Aps of Manduca sexta and Lymantria dispar by Western blot and quantitative immunoelectrophoresis. Another hemolymph storage protein in T. ni of lesser titer was shown to be homologous with larval hemolymph protein (LSP) of M. sexta. Ap titer increased dramatically in the last larval instar of T. ni, as in other holometabolous insects studied. Parasitization by Chelonus sp. caused the Ap titer to rise prematurely in the penultimate larval instar of T. ni. This rise in Ap in the fourth instar is one of the earliest diagnostic signs of parasitization. Among the suite of behaviors of the Chelonus larva on exiting the host is depletion of the host cadaver of most remaining protein. The T. ni Ap titer in the alimentary tract of Chelonus peaks at that time and declines to zero in the first 24 h after parasitoid emergence, prior to its pupation. Aps are a source of phenolic storage compounds. Hence, premature induction of T. ni is advantageous for the parasitoid's own pupation and adult development.
Metabolism of parasitized Manduca sexta examined by nuclear magnetic resonance
Authors: Thompson Stewart N. , Lee Robert W.‐K. , Beckage Nancy E.
Published: February 2005

Metabolism of fifth instar Manduca sexta larvae was significantly altered during parasitization by Cotesia congregata. The in vivo 13C natural abndance spectrum and histological examination of the fat body demonstrated significantly reduced fat levels and increased deposition of glycogen in parasitized insects. 1H NMR analysis demonstrated that the free amino acid level was reduced in the hemolymph of parasitized larvae, but control and parasitized insects had similar levels of trehalose. [13C]2‐pyruvate injected into the dorsal vessel was incorporated into numerous metabolites including amino acids, Krebs cycle intermediates, trehalose, and fat. In control larvae pyruvate was incorporated primarily into fat following oxidation to CoASAc. Pyruvate was metabolized slower in parasitized larvae and following decarboxylation to oxaloacetate was metabolized by Krebs cycle and incorporated into amino acids. Only small amounts of trehalose were synthesized from pyruvate in control and parasitized larvae and glycogen synthesis was not observed. Analysis of hemolymph indicated no net synthesis of glucose by gluconeogenesis. Control and parasitized larvae incorporated [13C]1‐glucose primarily into trehalose. Control larvae also synthesized fat from gulcose. In contrast, fat synthesis was not observed in parasitized insects which incorporated glucose into glycogen. Differences in glucose metabolism, therefore, appeared to explain the difference in storage metabolite levels between control and parasitized larvae.
Serosal cells of Biosteres longicaudatus (Hymenoptera: Braconidae): Ultrastructure and release of polypeptides
Authors: Lawrence Pauline O.
Published: February 2005

As a prelude to a study in vitro of the function of the embryonic serosa of the parasitic wasp Biosteres longicaudatus (Braconidae), the ultrastructure of serosas of different ages reared in vivo and in vitro were compared. The evidence suggests that the serosal capsule consists of one to three cell layers. The innermost (internal cells) which line the serosal capsule and the outermost (external) cells which are bathed by the host's hemolymph are secretory. Large, coated vesicles in the internal cells increase in number and size with age and, likely, take up and transport molecules into the serosa. Multivesicular bodies, Known for their enzyme‐degradative function, occur in external cells and are eventually extruded into the surrounding environment. Distinctive electrondense, rod‐shaped particles appear in external cells within 8 h after larvae hatch, increase in number with larval age, and occur at the bases of microvilli. The latter appear electron dense with age and eventually they and the lobulated microvilli in internal cells fragment into the surrounding environment. To determine whether parasites and/or their serosas release substances into the host Anastrepha suspensa (Diptera: Tephritidae), hemolymph from unparasitized and superparasitized (> 1 parasite/host) pharate pupae was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Serosas and/or parasite larvae were incubated in artificial media and each of these was subjected to SDS‐PAGE. A polypeptide, approximately 24 kilodaltons (Kd) occurred in the hemolymph of 24‐h‐old superparasitized pharate pupal hosts but not in the control. A similar polypeptide was observed in medium cultured with parasite larva and serosa as well as serosa alone, but was not in their respective control media. This approximately 24‐Kd band in SDS‐PAGE gels corresponds to a band in the serosa homogenate and may be identical to it. Serosas and parasite larvae in vivo and in vitro have similar protein profiles. Based upon these ultrastructural and electrophoretic studies, it appears that the serosa of B. longicaudatus has a synthetic function, as has been reported for the extra‐embryonic membrane of other parasitic Hymenoptera. It may sequester and degrade molecules from the host hemolymph and likely release newly synthesized as well as degraded products into the host.
Eretmocerus penetration and immature development: A novel approach to overcome host immunity
Authors: Gerling Dan , Orion Tamar , Delarea Yaakov
Published: February 2005

The mode of penetration of parasitoids belonging to the genus Eretmocerus into whitefly larvae and their immature development were examined. Examination included scanning electron microscopy and light microscopy of stained and unstained whole mounts and sections.

The Eretmocerus larva pierces the venter of its host shortly after hatching, and subsequently enters the host through the same hole. The host reacts by forming a cellular capsule around the Eretmocerus larva. This capsule is incomplete, with an opening in its ventral side opposite the penetration hole. The capsule remains intact during most of the second instar of the parasitoid. It then disintegrates, but its remnants are still visible around the third instar. Whenever two Eretmocerus larvae penetrate, they are surrounded by two capsules. The capsule does not prevent parasitoid development, but it apparently precludes contact of cellular elements of the host's blood with the developing parasitoid larva.

Adaptive features of Eretmocerus larval biology include the early contact with the host's internal medium that permits host regulation; and the delay of contact with the host's blood cells that may preclude the need to confront host immunological systems.
Hydrolysis of sphingomyelin and phosphatidylcholine by midgut homogenates of the stable fly
Authors: Spates George E. , Bull Don L. , Chen Andrew C.
Published: February 2005

Qualitative and quantitative analyses were made to characterize the enzymatic degradation of sphingomyelin and phosphatidylcholine by midgut homogenates of the adult stable fly, Stomoxys calcitrans (L.). The results indicated that sphingomyelin was hydrolyzed by an enzyme with sphingomyelinase‐like properties, and that phosphatidylcholine was hydrolyzed by an enzyme with properties similar to phospholipase C. The optimum pH for the sphingomyelinase was 7.6, and the rate of hydrolysis of sphingomyelin at that pH was linear from 1 to 4 nmol of substrate and 5 to 25 μg of enzyme preparation. Dialysis of the homogenates against Tris‐HCI and imidazole buffers resulted in a decrease of sphingomyelinase activity by 59% and 98%, respectively, and the original activity was not restored with the addition of Ca++, Mg++, or Mn++.
Cloning of the mitochondrial genome of Anopheles quadrimaculatus
Authors: Cockburn Andrew F. , Mitchell Sharon E. , Seawright Jack A.
Published: February 2005

The entire 15 kilobase (kb) Anopheles quadrimaculatus mitochondrial DNA (mtDNA) was cloned as three EcoRI fragments in a bacteriophage vector and then subcloned into plasmid vectors. The cloned DNA was physically mapped with restriction endonucleases, and the maps were compared to the restriction patterns of native A. quadrimaculatus mtDNA. Several genes were mapped by sequencing the ends of A. quadrimaculatus mtDNA subclones and by hybridization with the previously characterized Aedes albopictus mtDNA clones. These portions of the genetic map were identical in gene order to those of Drosophila yakuba. The predicted amino acid sequence of the protein coding regions that were sequenced were between 72% and 98% homologous to D. yakuba. The cloned mtDNA will be useful as a probe for population genetic analysis of mosquitoes.
Modulation of tension production by proctolin in the dorsal longitudinal muscles of the cricket, Teleogryllus oceanicus
Authors: A. O'Gara Bruce
Published: February 2005

High‐frequency electrical stimulation (∼20 Hz) of the lateral nerve in abdominal segments of the cricket, Teleogryllus oceanicus, caused an increase in tonus of the abdominal dorsal longitudinal muscle (DLM). This effect persisted for 1–5 min following stimulation. Application of the pentapeptide proctolin (threshold 1–10 nM) mimicked the increase in muscle tonus produced by electrical stimulation. Individual twitches were unaffected or slightly reduced by proctolin. Low‐frequency electrical stimulation (<7 Hz) of the lateral nerve counteracted a previously induced increase in muscle tonus, apparently by activation of an inhibitory motoneuron. γ‐Aminobutyric acid (GABA) mimicked the effect of low‐frequency stimulation and reduced muscle tonus. Octopamine, in concentrations of ≤0.1 mM, was inactive on the abdominal DLM when stimulated at low frequencies (0.5–2 Hz). Application of proctolin to the metathoracic DLM caused an increase in twitch amplitude but had little effect on basal tonus. In conjunction with the previously described responses of the metathoracic DLM to octopamine, these results show that the serially homologous abdominal and metathoracic DLMs have dissimilar responses to the modulators proctolin and octopamine.
Lipid biosynthesis in adult Acyrthosiphon pisum: Effect of age and symbiont population
Authors: de Renobales Mertxe , Cripps Colleen , Kinsey Marvin
Published: February 2005

The incorporation of [1‐14C]acetate into total lipids in 1‐day‐old adult pea aphids is 3.3‐fold higher than in 20‐, 22‐, and 24‐day old adults. The polar lipid fraction was the main lipid class synthesized at any age and contained primarily eighteen carbon fatty acids. While the relative mass of 18:0, 18:1 and 18:2 decreased in older aphids, the relative amount of label incorporated into these fatty acids remained constant. Myristic acid was the main fatty acid of the triacylglycerol fraction, and the relative amount of radioactivity incorporated into this fatty acid decreased in older aphids. Twenty‐day‐old aphids had 60% fewer mycetocytes than did 1‐day‐old insects. We conclude that symbionts within the mycetocytes do not appear to be involved in the synthesis of linoleic acid, while their role in the synthesis of myristic acid is less clear.
A simple and rapid technique for identification of large numbers of individual mosquitoes using DNA hybridization
Authors: Cockburn Andrew F.
Published: February 2005

A general method for obtaining species‐specific repetitive DNA sequences is described. The method is based on the detection of recombinant DNA clones containing repetitive sequences using labeled total genomic DNA. These repetitive DNA sequences can be used to identify individual mosquito adults, pupae, and larvae squashed on filter membranes (squash blots). This technique was used to distinguish individuals of the four sibling species of the Anopheles quadrimaculatus complex. Repetitive DNA sequences and squash blots can be of use for rapid identification of other insect species in field collections.