bluuebunny/crossref_metadata_2025 huggingface dataset:
Oostatic hormone inhibits biosynthesis of midgut proteolytic enzymes and egg development in mosquitoes
Authors: Borovsky Dov
Published: February 2005

Injection of partially purified oostatic hormone (0.7 μg) into female Aedes aegypti inhibited egg development, proteolytic enzyme activity, and blood digestion in the midgut, whereas control injections of saline or insulin chain A (0.7 μg) did not affect these processes. Oostatic hormone given by enema, on the other hand, did not inhibit proteolytic enzyme activity, indicating that the hormone acts outside the midgut. A single injection of oostatic hormone (0.7 μg) caused a 1.7–1.5‐fold reduction in activity of trypsinlike enzymes during blood digestion, with a 10‐h delay in peak activity.

Using [1,3‐3H]diisopropylfluorophosphate (DFP) in the presence of 8 mM tosylamide‐2‐phenylethyl chloromethyl ketone, the synthesis of trypsinlike derivatives was followed in the midgut of female A. aegypti. A 4‐fold reduction in [1,3‐3H]diisopropylphosphoryl‐trypsinlike derivatives was noted after oostatic hormone treatment. Several isozymes that are normally synthesized were absent in the presence of DFP, as assessed by polyacrylamide gel electrophoresis. Injection of oostatic hormone into decapitated and ovariectomized females that did not synthesize ecdysteroids inhibited trypsinlike enzyme synthesis and blood digestion in the midgut, indicating that oostatic hormone inhibits the midgut cells and not the ovary or the brain's endocrine system.

Comparison between oostatic hormone and soybean trypsin inhibitor indicated that the former inhibited trypsin synthesis whereas the latter inhibited trypsin activity. A. aegypti oostatic hormone is not species specific and injections of the hormone into Culex quinquefasciatus, Culex nigripalpus, and Anopheles albimanus caused inhibition of egg development, blood digestion, and synthesis of trypsinlike enzymes. A direct relation between oostatic hormone synthesis and the regulation of trypsinlike activity in the midgut is proposed.
Uptake of cholesterol and cholestanol by the intestine, hemolymph, and fat body of Heliothis zea
Authors: Kuthiala Arun , Ritter Karla S.
Published: February 2005

The effect of the concentration and structure of dietary sterol on its uptake and distribution in the intestine, hemolymph and fat body was studied in sixth‐instar larvae of Heliothis zea. When cholesterol (cholest‐5‐en‐3β‐ol) was inoculated per os into the foregut of larvae, it was rapidly taken up by the intestine. Some of the dietary sterol then passed into the hemolymph, primarily via the midgut, during at least the first 9 h after inoculation, while at least 7% of the dose remained associated with the intestine. The amount of dietary sterol per 0.10 g of hemolymph increased until it reached 3–6% of the dose after 9 h. The amount of sterol per 0.10 g of the fat body increased to as much as 5% of the dose after 10 h. As the concentration of sterol in the dose increased from 0.3 to 15 μg/4 μl, the amount of sterol associated with the intestine, hemolymph, and fat body also increased. When cholesterol was inoculated intrahemocoelically, instead of per os, the amount of sterol in the hemolymph decreased, for at least the first 8 h after inoculation, and may have been absorbed, at least in part, by the intestine. The absence of a double bond in cholestanol (5α‐cholestan‐3β‐ol) had no significant effect, at least 5 h after inoculation, on the uptake and distribution of this sterol in the intestine, hemolymph, and fat body of the larva. The results of this study indicate that although larvae of H. zea fed cholestanol have a slower rate of growth than those fed cholesterol, this may be due to differences in the utilization of the two sterols rather than to differences in their uptake by the tissues.
Esterification of cholesterol and cholestanol in the whole body, tissues, and frass of Heliothis zea
Authors: Kuthiala Arun , Ritter Karla S.
Published: February 2005

The quantity of free and esterified sterols in the whole body, intestine, hemolymph, fat body, and frass of 6th‐instar larvae of H. zea, fed cholesterol or cholestanol, was measured in order to determine if there was a difference in the utilization of these two molecules. The principal sterol in the tissues of the larvae was cholestanol or cholesterol, when they were fed diet containing these two molecules, respectively; there was little, if any, metabolism of dietary cholestanol to cholesterol. There was little or no difference in the amount of total sterol in the whole body, tissues, or frass of larvae fed the two different diets, indicating that the absence of a Δ5‐bond in cholestanol does not prevent the uptake or distribution of this sterol to various tissues. However, the relative percentage of steryl ester was significantly higher in prepupae reared on a diet containing cholestanol instead of cholesterol (6–7‐, 4‐, 13‐, 4‐, and 2‐fold increase, for the whole body, intestine, hemolymph, fat body, and frass, respectively). The average percentage of total sterol that was esterified in the tissues was greater in the fat body (10.8 ± 15.4 and 44.2 ± 12.3%, respectively, for larvae fed cholesterol and cholestanol) than in the hemolymph (0.5 ± 0.1 and 6.3 ± 0.8%) and intestine (1.2 ± 0.1 and 4.7 ± 1.1%). The percentage of sterol that was esterified in the frass of larvae was large (26.9 ± 3.7 and 48.2 ± 0.5%, respectively, for larvae fed cholesterol and cholestanol). Therefore, the fact that larvae of H. zea fed cholestanol, instead of cholesterol, contain this saturated molecule as their principal tissue sterol and preferentially esterify it may explain, at least in part, why their rate of growth on cholestanol is slower than on cholesterol.
20‐Hydroxyecdysone and ovarian maturation in the firebrat Thermobia domestica (Thysanura: Lepismatidae)
Authors: Bitsch C. , Bitsch J.
Published: February 2005

The effects of 20‐hydroxyecdysone on ovary maturation in the firebrat Thermobia domestica were investigated by in vivo injections of different doses of the hormone on various days of the reproductive cycle. Several females were neck‐ligated or treated with precocene II before injection. Experiments were also performed on ovarioles incubated in vitro. It was demonstrated that 20‐hydroxyecdysone has direct and dose‐dependent effects on the ovary, inducing growth of all the previtellogenic follicles whatever the day of the reproductive cycle, except at times when the ovaries contain follicles undergoing choriogenesis. The major effect of the hormone is the stimulation of young‐follicle formation in the anterior part of the previtellarium and the accelerated growth of a set of basal previtellogenic follicles, which reach the critical size required for yolk deposition. However, 20‐hydroxyecdysone did not induce cellular differentiation, in particular, the enlargement of the perioocyte space and the development of oocyte microvilli, which normally occur before yolk precursor incorporation. The present results give a better understanding of the temporal relationships between molting and reproductive cycles and also explain the periodicity of ovarian maturation.
Yolk hydrolase activities associated with polypeptide and oligosaccharide processing of Blattella germanica vitellin
Authors: Purcell John P. , Kunkel Joseph G. , Nordin John H.
Published: February 2005

Various aspects of the processing of Blattella germanica vitellin (Vt) in the oocyte and egg have been investigated. Employing subunit specific antibodies, the precursor product relationships among the subunits of this Vt have been determined. After endocytosis of Vt by the oocyte, the Mr 160,000 subunit Vt is cleaved to products of Mr 95,000 and Mr 50,000. In association with an unprocessed Mr 102,000 peptide, these form the subunits of the Vt of freshly ovulated eggs. Between 4 and 5 days post ovulation (at 30°C), all three subunits of Vt are again processed proteolytically before use by the embryo. Although Vt's high mannose‐type oligosaccharides are trimmed during embryogenesis, their modification occurs subsequent to the day 4–5 proteolysis, precluding the possibility that changes in oligosaccharide content or structure contribute to regulating this second proteolytic event. Although the predominant oligosaccharide of Vt is Man9GlCNAc2, the Mr 50,000 subunit of egg‐borne Vt contains a much higher proportion of Man6GlCNAc2 than the other two subunits; therefore, this portion of the precursor vitellogenin must be more accessible to the processing mannosidases of the endoplasmic reticulum during its biosynthesis. A microtechnique for aspirating the yolk from individual eggs in an oothecapermits its isolation free of contamination by embryonic tissue. With this procedure, the specific activity profiles of exo‐α‐mannosidase, exp‐β‐N‐acetylglucosaminidase, α‐glucosidase and acid phosphatase were monitored during the first 6 days after ovulation, and some of their properties were also determined. Expression of the acid phosphatase and exo‐β‐N‐acetyl‐glucosaminidase activities coincide with the day 4–5 proteolysis, while α‐mannosidase remains relatively constant throughout the first 6 days. Functions for these enzymes and the oligosaccharides of Vt during Vt storage and utilization are proposed.
Quinone methides—and not dehydrodopamine derivatives—as reactive intermediates of β‐sclerotization in the puparia of flesh fly Sarcophaga bullata
Authors: Sugumaran Manickam
Published: February 2005

Cuticular phenoloxidase(s) from Sarcophaga bullata larvae oxidized a variety of o‐diphenolic compounds. While catechol, 3,4‐dihydroxybenzoic acid, dopa, dopamine, and norepinephrine were converted to their corresponding quinone derivatives, other catechols such as 3,4‐dihydroxyphenylacetic acid, 3,4‐dihydroxyphenethyl alcohol, 3,4‐dihydroxyphenyl glycol, 3,4‐dihy‐droxymandelic acid, and N‐acetyldopamine were oxidized to their side‐chain oxygenated products. In addition, the enzyme‐catalyzed oxidation of the latter group of compounds accompanied the formation of colorless catecholcuticle adducts consistent with the operation of β‐sclerotization. Radioactive trapping experiments failed to support the participation of 1,2‐dehydro‐N‐acetyldopamine as a freely formed intermediate during phenoloxidase‐mediated oxidation of N‐acetyldopamine. When specifically tritiated substrates were provided, cuticular enzyme selectively removed tritium from [7‐3H]N‐acetyldopamine and not from either [8‐3H] or [ring‐3H]N‐acetyldopamine during the initial phase of oxidation. The above results are consistent with the generation and subsequent reactions of quinone methides as the initial products of enzyme‐catalyzed N‐acetyldopamine oxidation and confirm our hypothesis that quinone methides and not 1,2‐dehydro‐N‐acetyldopamine are the reactive intermediate of β‐sclerotization of sarcophagid cuticle. Quinone methide sclerotization resolves a number of conflicting observations made by previous workers in this field.
Inhibition of ecdysteroid production in nymphs of Rhodnius prolixus treated with ethoxyprecocene II
Authors: Garcia Eloi S. , De Azambuja Patricia , Feder Denise , Bowers William S.
Published: February 2005

Rhodnius prolixus nymphs fed 7‐ethoxy‐6‐methoxy‐2,2‐dimethylchromene (ethoxyprecocene II, EPII) show a variety of responses, including precocious molting to diminutive adults, severe retardation of molting, or a condition of permanent ecdysial stasis. The latter two conditions are reversible by subsequent treatment with 20‐hydroxyecdysone. Ecdysteroid titers in the hemolymph of individual insects, determined by radioimmunoassay (RIA), show that the ecdysteroid cycle in nymphs undergoing precocious metamorphosis is similar to that of untreated fifth stage nymphs during normal imaginal molting. Nymphs in ecdysial stasis, following EPII treatment, were found to have very low ecdysteroid titers. Analysis of ecdysteroid synthesis by the prothoracic glands (PG), cultured in vitro, showed that: 1) only traces of ecdysteroid were detectable in PG from nymphs treated in vivo with EPII; 2) the PG from untreated nymphs incubated in culture medium with EPII possessed significantly lower ecdysteroid synthesis compared with controls. These studies sought to determine if the inhibition of ecdysteroid biosynthesis observed in Rhodnius, following exposure to EPII in vivo and in vitro, is due to a direct action on the PG or result as an indirect effect perhaps mediated by the neuroendocrine system.
Peptidoglycan fragments elicit antibacterial protein synthesis in larvae of Manduca sexta
Authors: Kanost Michael R. , Dai Wei , Dunn Peter E.
Published: February 2005

In several insect species, serum lysozyme and antibacterial peptide concentration increases after injection of bacteria and other foreign substances. The purpose of this study was to characterize the specificity of this induction in the tobacco hornworm, Manduca sexta. By 48 h after injection of killed bacteria, lysozyme activity was approximately tenfold greater than in untreated insects. This maximal response was observed after injection of every bacterial species tested and after injection of purified cell walls of Micrococcus luteus. A variety of acellular particles, soluble molecules, and bacterial cell wall components were either poor lysozyme inducers or elicited no change in lysozyme concentration. The polysaccharide zymosan from yeast cell walls was a moderate lysozyme inducer. Peptidoglycan from M. luteus cell walls was found to induce lysozyme to a level as great or greater than whole cell walls. Small fragments of peptidoglycan generated by hen egg white lysozyme digestion were isolated, partially characterized, and shown to be good inducers of lysozyme as well as other antibacterial peptides. It appears that peptidoglycan provides a signal that initiates antibacterial responses in the insect.
Immunohistochemical and electrochemical detection of serotonin in the nervous system of the blood‐feeding bug, Rhodnius prolixus
Authors: Lange Angela B. , Orchard Ian , Lloyd Robert J.
Published: February 2005

The distribution of serotonin throughout the nervous system of the blood‐feeding bug Rhodnius prolixus has been studied using immunohistochemistry and reversed‐phase high‐performance liquid chromatography coupled to electrochemical detection.

Approximately 150 serotonin‐like immunoreactive neurons are distributed throughout the central nervous system. These neurons are distributed over both ventral and dorsal surfaces and are found in all ganglia. Several of these neurons appear to be homologous to previously described serotonin‐like immunoreactive neurons in insects. These include a large pair of bilaterally symmetrical neurons that project axons out of the suboesophageal ganglion, and some serially homologous neurons which project contralaterally and appear to be interneurons.

Immunoreactive branches and varicosities are found in the neuropile of all ganglia, and immunoreactive axons are found in all interganglionic connectives. Several of the peripheral nerves are covered in a plexus of immunoreactive processes. These processes result in a meshwork of fine varicose branches lying superficially over the peripheral nerves, which resemble neurohemal areas.

The central nervous system of Rhodnius contains about 5.5 pmol serotonin unequally distributed between the ganglia. The highest content is found in the brain and optic lobes, and the lowest content is found in the prothoracic ganglion. Substantial amounts of serotonin are present in the corpus cardiacum and in the peripheral nerves possessing the serotonin‐like neurohemal areas. Serotonin is released from these latter neurohemal areas in a calcium‐dependent manner in response to high‐potassium saline.

It is concluded that serotonin plays an important central and peripheral role in this insect.
Gene structure, cDNA sequence, and developmental regulation of a low molecular weight hemolymph protein from Locusta migratoria
Authors: Kanost M. R. , Bradfield J. Y. , Cook K. E. , Locke J. , Wells M. A. , Wyatt G. R.
Published: February 2005

From a Locusta migratoria genomic DNA library, a gene has been isolated that codes for a previously unrecognized hemolymph protein of Mr = 19,000, designated 19k protein. The gene has at least five exons, extending over about 9 kb of DNA. Its polypeptide product, obtained by cell‐free translation of mRNA selected from adult fat body RNA by hybridization with the cloned DNA, is precipitated by antiserum against a low molecular weight hemolymph protein fraction. The mature protein product has been purified from locust hemolymph, and an N‐terminal sequence of 20 amino acids has been determined. In polyacrylamide gel electrophoresis, this protein comigrates with apolipophorin III, from which it was previously not distinguished, but it is clearly distinct by amino acid composition and sequence. The genomic clone was used as a probe to isolate a fat body cDNA clone of the 19k protein mRNA. The 938‐base pair cDNA clone contains a 516‐base pair open reading frame. The deduced 172‐amino acid polypeptide includes an apparent signal peptide, a sequence of four amino acids that may represent a prosegment, and a sequence identical (with a single exception, which may reflect polymorphism) with the N‐terminal sequence of the hemolymph protein. Its mRNA occurs at a low level in late larval fat body, is abundant in the newly eclosed adult, then declines to a low level, and rises again at days 8–10; it is greatly reduced after destruction of the corpora allata with precocene and then is elevated after treatment with methoprene, suggesting stimulation by juvenile hormone. The biological role of 19k protein is unknown.
Purification, characterization, and synthesis of vitellin from the cabbage butterflyPieris rapaeL.
Authors: Kim Hak R. , Ko Y. G. , Mayer Richard T.
Published: February 2005

Vitellin from the cabbage butterflyPieris rapaeL. was purified and characterized by electrophoresis. Vitellin fromP. rapaeis a phosphorylated glycolipoprotein of 380,000 ± 10,000 molecular weight as determined by nondenaturing polyacrylamide gel electrophoresis. Two subunits with an Mrof 150,000 and 40,000 were obtained from vitellin. The native molecule is thought to be a tetramer composed of two molecules of each of these subunits. The isoelectric point, as determined by isoelectric focusing on polyacrylamide gels, is 6.10. Vitellin and vitellogenin were indistinguishable by immunological methods such as double diffusion and tandem‐crossed immunoelectrophoresis. Vitellogenin from the hemolymph and vitellin from the ovary were quantified by rocket immunoelectrophoresis. Vitellogenin and vitellin were first detected in 6‐day‐old pupae, and their levels increased continuously during ovarian development. Vitellogenin synthesis by the fat body in 4‐day‐old female pupae could be induced by juvenile hormone I.
Identification and analysis of the major yolk polypeptide from the Caribbean fruit fly, Anastrepha suspensa (loew)
Authors: Handler Alfred M. , Shirk Paul D.
Published: February 2005

A single major yolk polypeptide (YP) having a molecular mass of approximately 48,000 daltons (Da), was identified in the ovaries and oviposited eggs of the Caribbean fruit fly, Anastrepha suspensa. The polypeptide was partially purified from oviposited eggs using gel permeation and ion‐exchange chromatography. Analysis of YP synthesis in vivo and in tissues cultured in vitro indicated that the ovary was the major site of synthesis with very low levels of YP derived from the adult fat body. Using a monospecific polyclonal antiserum to 48 kDa YP in an immunoblot assay, low levels of vitellogenin were found in female hemolymph; slightly lower levels of an immunoreactive 48‐kDa polypeptide were detectable in male hemolymph. Although YP synthesis was detectable within 12 h after eclosion, the major increase in YP accumulation occurred at 3–4 days posteclosion coincident with the initiation of observable yolk deposition. The physical characteristics of YP from A. suspensa were similar to YPs from other dipterans in terms of molecular mass and antigenicity, yet the tissue‐ and sex‐specific regulation of the YP differed from other dipterans as well as most other insects.
Vitellogenin mRNA from Dacus oleae: Characterization, cDNA cloning, and dependence on sex and developmental stage
Authors: Levedakou Eleni N. , Konialis Christophoros P. , Sekeris Constantine E.
Published: February 2005

Poly(A)+ RNA was isolated from 5‐day‐old females of the olive fruitfly Dacus oleae. In vitro translation of mRNA, followed by immunoprecipitation with anti‐vitellin antiserum, demonstrated the synthesis of two vitellogenin polypeptides of 54,000 and 56,000 daltons. In sucrose gradient centrifugation, the vitellogenin mRNA sediments in the 14.4S region that corresponds to an RNA size of approximately 1,600 nucleotides. Translation of poly(A)+ RNA isolated from male flies revealed the presence of small quantities of vitellogenin mRNA, consistent with the finding of vitellogenin in male hemolymph of Dacus. Using the female poly(A)+ RNA population as template, we constructed a cDNA expression library in a lambda gt11 vector, which was screened with anti‐vitellin antiserum. Four recombinant clones were isolated containing inserts of 550–800 bp*, which were identified as vitellogenin cDNA clones by hybrid‐selected translation. All four cDNA clones showed crossreaction with vitellogenin genes of Drosophila melanogaster. The Dacus clones were used as probes to study vitellogenin expression during postembryonic development of Dacus. In females, vitellogenin mRNA appeared at the time of eclosion with increasing accumulation up to the 5th day of adult life; then it reached a plateau. On day 10, the mRNA titer had already started to decrease and on day 25, no vitellogenin mRNA was detectable. In males, vitellogenin mRNA was found up to the 8th day after eclosion, in very low concentrations. No vitellogenin mRNA was present during larval and pupal stages.
Action of antioxidant enzymes and cytochrome P‐450 monooxygenases in the cabbage looper in response to plant phototoxins
Authors: Lee Keywan , Berenbaum May R.
Published: February 2005

Effects of two biosynthetically distinct plant phototoxins—xanthototoxin, a furanocoumarin, and harmine, a β‐carboline alkaloid, which are known to produce toxic oxygen species—on the food utilization efficiencies and enzymatic detoxification systems of the polyphagous cabbage looper. Trichoplusia ni (Lepidoptera: Noctuidae), were studied. Newly molted fifth‐instar larvae were allowed 36 h to ingest diets containing these two phototoxins at 0.15% wet weight in the presence of near ultraviolet (UVA). The growth and development of the larvae, as well as the corresponding activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX), and glutathione reductase (GR) and the detoxification enzyme cytochrome P‐450, were measured. Xanthotoxin reduced rates of relative growth and consumption and efficiencies of conversion of ingested and digested food to biomass. Harmine reduced rates of growth and consumption without affecting efficiencies of conversion. Specific activities of SOD, CAT, GPOX, and GR of whole‐body homogenates in the absence of compounds were 0.88 units, 153μmol H2O2 decomposed·mg protein−1·min—1, 38.3 nmol NADPH oxidized·mg protein−1·min−1, and 0.56 nmol NADPH oxidized·mg protein−1·min−1, respectively. SOD activity was induced 2.9‐fold and 3.8‐fold by dietary xanthotoxin and harmine, respectively. CAT and GPOX activities were induced 1.2‐fold by harmine only, and GR activity was not changed by either chemical. The P‐450 activity toward xanthotoxin in the microsomal fraction of midguts was low (0.15 nmol xanthotoxin metabolized·mg protein−1·min−1) and was not induced by xanthotoxin ingestion. These studies indicate that P‐450 and antioxidant enzyme systems may be independent but consequential, the induction of antioxidant enzymes by phototoxins occurring when low P‐450 activity toward the phototoxin permits the accumulation of oxidative stress from unmetabolized phototoxin, which in turn induces antioxidant enzymes.
Possible role for covalent modification in the reversible activation of ecdysone 20‐monooxygenase activity
Authors: Hoggard Nigel , Fisher Michael J. , Rees Huw H.
Published: February 2005

Evidence is presented for the reversible activation‐inactivation of the microsomal ecdysone 20‐monooxygenase from fat body of the cotton leafworm, Spodoptera littoralis, in a manner commensurate with reversible changes in its phosphorylation state. The activity of the monooxygenase was higher following preincubation with fluoride (an inhibitor of phosphoprotein phosphatases) than in its absence. Preincubation with alkaline phosphatase or with cAMP‐dependent protein kinase resulted in appreciable diminution or enhancement, respectively, in monooxygenase activity. Activation of ecdysone 20‐monooxygenase activity could also be effected by incubation with a cytosolic fraction in the presence of cAMP, ATP, and fluoride; this activation was prevented by a cAMP‐dependent protein kinase inhibitor. Similarly, inactivation of the monooxygenase was achieved by preincubation with cytosol, the effect being enhanced by Ca2+‐calmodulin or by Mg2+ ions. The combined results provide indirect evidence that the microsomal ecdysone 20‐monooxygenase exists in an active phosphorylated form and an inactive dephosphorylated form, interconvertible by a cAMP‐dependent protein kinase and a phosphoprotein phosphatase.
Intestinal amino acid absorption in tobacco hornworm larvae is stimulated by potassium and sodium but not rubidium or lithium
Authors: Hennigan Brian B. , Wolfersberger Michael G.
Published: February 2005

We used rapid filtration assays to determine the ion selectivity of ion gradientdriven phenylalanine uptake by brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm (Manduca sexta). Phenylalanine uptake by these vesicles is stimulated by both potassium and sodium. Phenylalanine uptake by larval M. sexta midgut brush border membrane vesicles is voltage sensitive and shows little selectivity for potassium over sodium. However, phenylalanine uptake by these vesicles is stimulated by neither rubidium nor lithium.
Stimulating action of methyl 12, 12, 12‐trifluorofarnesoate on in vitro juvenile hormone III biosynthesis in blattella germanica
Authors: Bellés Xavier , Camps Francisco , Casas Josefina , Mauchamp Bernard , Piulachs Maria‐Dolors , Messeguer Angel
Published: February 2005

Methyl 12, 12, 12‐trifluorofarnesoate (MTFF) at a dose of 10 μM, stimulated in vitro juvenile hormone (JH) release in corpora allata (CA) from 6‐day‐old, freshly ecdysed, and 8‐day‐old (period of ootheca transport) adult virgin females of Blattella germanica. In addition, MTFF also induced intraglandular accumulation of JH and MF in treated CA. Trifluorofarnesoic acid (TFFA) and trifluorofarnesol (TFF) exhibited the same properties, although to a lesser extent than MTFF. The detection of MTFF in TFFA‐treated CA suggested that TFFA and TFF were biotransformed into MTFF by the CA enzymatic system and that this ester might be responsible for the activity observed. Equivalent experiments carried out with farnesoic acid (FA) resulted in a more significant stimulation of JH production. This is not surprising, because exogenous FA is readily epoxidized at C10‐C11 double bond and methylated to afford JH. Conversely, analytical data have shown that the C6‐C7 double bond of MTFF is epoxidized by the CA enzymatic system, whereas that at C10‐C11 remains practically unaltered.
Juvenile hormone identification, titer, and degradation during the last larval stadium of Spodoptera littoralis
Authors: Zimowska Grazyna , Rembold Heinz , Bayer Gabrielle
Published: February 2005

Juvnile hormone (JH) was identified and titers determined during the last larval stadium of the cotton leafworm Spodoptera littoralis using combined gas chromatography‐selected ion monitoring‐mass spectrometry. JH was almost exclusively limited to JH II, occurring at two major peaks: the first at the beginning of the stadium and the second at days 4 and 5 just after wandering. Degradation of JH and α‐naphthyl acetate (α‐NA) in hemolymph and fat body was determined during the same developmental periods. JH degradation in hemolymph occurred initially as a broad bimodal peak during the second half of feeding, with peaks at days 2 and 3. The second peak occurred at the end of the stadium, subsequent to the second JH II titer peak. Three α‐NA esterase peaks in hemolymph were detected when JH degradation was at a very low level. Fat body JH degradation was resolved as four activity peaks throughout the stadium, with the first three peaks occurring just previous to the JH degradation peaks in the hemolymph. The highest levels of JH and α‐NA degradation activities in fat body were related to the wandering stage.
Biosynthesis of sex pheromone components from linolenic acid in arctiid moths
Authors: Rule Geoffrey S. , Roelofs Wendell L.
Published: February 2005

Sex pheromone components of two species of arctiid moths, Estigmene acrea and Phragmatobia fuliginosa, were shown to be derived from linolenic acid. Female pupae were injected with radiolabeled malonic acid or an 18‐, 20‐, 21‐, or 22‐carbon triunsaturated fatty acid, and the pheromone components from emerged adults analyzed for radioactivity. The data support a biosynthetic pathway in which the 21‐carbon pheromone component,(Z, Z)‐3,6‐cis‐9,10‐epoxyheneicosadiene, of these moths is produced by chain elongation of linolenic acid to docosatrienoic acid with subsequent reductive decarboxylation. The 18‐carbon aldehyde components,(Z, Z)‐9,12‐octadecadienal and (Z, Z, Z)‐9,12,15‐octadecatrienal, of E. acrea are produced from linoeic and linolenic acids directly. No detectable amounts of intermediate 20‐, 21‐, or 22‐carbon fatty acid precursors were found in the gland of E. acrea.
JH III levels in larvae and pharate pupae of Anastrepha suspensa (Diptera: Tephritidae) and in larvae of the parasitic wasp Biosteres longicaudatus (Hymenoptera: Braconidae)
Authors: Lawrence Pauline O. , Baker Fred C. , Tsai Leslie W. , Miller Christine A. , Schooley David A. , Geddes Lloyd G.
Published: February 2005

This is the first report of a juvenile hormone (JH) in the Caribbean fruit fly, Anastrepha suspensa (Diptera: Tephritidae). JH III was identified in whole body extracts of L3 (final instar) larvae and pharate pupae (PhP) by using a physicochemical method. JH III was also found in PhP superparasitized (up to 11.3 ± 3.5 S.D. parasites/host) by the solitary wasp Biosteres longicaudatus (Hymenoptera: Braconidae) and in first instars of the parasite. The levels of JH III in hosts were up to 15 × higher than those of the controls. The JH esterase (JHE) levels in the hemolymph of 1‐day‐old PhP controls were significantly (P < 0.05) higher than those of superparasitized individuals. These results suggest that the elevated JH III levels induced by superparasitism may arise from JH buildup due either to decreased JHE activity to continued synthesis of JH by the ring gland or to secretion of JH III by the parasites into the host's hemocoel.
Sex pheromone biosynthesis in the processionary moth Thaumetopoea pityocampa by delta‐13 desaturation
Authors: Arsequell Gemma , Fabriàs Gemma , Camps Francisco
Published: February 2005

In vivo treatments of female sex pheromone glands of the processionary moth, Thaumetopoea pityocampa, with mass‐labeled fatty acids showed that (Z)‐13‐hexadecen‐11‐ynyl acetate, the main sex pheromone component, is biosynthesized from palmitic acid by the combined action of delta‐11 and delta‐13 desaturases. The involvement of this unusual delta‐13 has been proven by application of [16,16,16‐2H3] [1,2‐13C2]‐hexadecanoic acid to the glands with a resultant incorporation of all labeled atoms into the pheromone and each one of the corresponding intermediates. These results seem to exclude alternative biosynthetic pathways, such as chain shortening and elongation combined with delta‐11 desaturation. The delta‐11 desaturase responsible for the formation of the triple bond in both the 11‐hexadecynoyl and (Z)‐13‐hexadecen‐11‐ynoyl intermediates is also an unusual enzyme not previously reported in lepidopteran sex pheromone biosynthesis.
Activity of the corpora allata of adult female Leucophaea maderae: Effects of mating and feeding
Authors: Aclé Dadié , Brookes Victor J. , Pratt Grahame E. , Feyereisen René
Published: February 2005

Juvenile hormone III biosynthesis by corpora allata of adult female Leucophaea maderae was measured by an in vitro radiochemical assay. In fed females, JH III synthesis increases more than 20‐fold after mating to a peak of 55 pmol/pair/h on day 9 and then rapidly declines. This increase in JH III synthesis concomitant with rapid oocyte growth in mated females is not observed in virgin females. The corpora allata from starved, virgin females appear to be inactive. The addition of 150 μM 2E,6E‐farnesol (a JH III precursor) to the incubation medium stimulates the corpora allata from starved, virgin females less than the corpora allata from starved, mated females. Both feeding and mating are necessary for the expression of a normal cycle of JH III synthesis in this cockroach.
20‐hydroxyecdysone triggers the resumption of imaginal wing disc development after diapause in the European corn borer, Ostrinia nubilalis
Authors: Peypelut Lionel , Beydon Philippe , Lavenseau Lucien
Published: February 2005

During the last‐larval instar, Ostrinia may display a facultative larval diapause. Diapausing larvae contain very low ecdysteroid titers and exhibit poorly differentiated imaginal wing discs. After diapause, development resumes when larvae are placed in favorable post‐diapause conditions. After a few days in these conditions, a small and transient peak of 20‐hydroxyecdysone was observed in hemolymph before any visible resumption of imaginal wing disc development. [3H]Cholesterol‐labeling experiment confirmed this result. Conversion of [3H]Cholesterol into [3H]ecdysteroids also began before any visible resumption of wing disc development. These data suggest that the resumption of wing disc development is induced by very low concentrations of molting hormone. After this small first peak, ecdysteroid concentrations increase until a second peak, which precedes ecdysis. Increase of titers was correlated with an increase of biosynthetic activity from [3H]cholesterol. Unexpectedly, biosynthetic activity remained very high after the second peak, but the synthesized ecdysone was immediately inactivated into 20‐hydroxyecdysonic acid.
Genetic changes in methotrexate‐resistant mosquito cells
Authors: Shotkoski Frank A. , Fallon Ann Marie
Published: February 2005

A stepwise selection procedure was used to obtain from Mtx‐5011 Aedes albopictus cells, variants with increased resistance to methotrexate (mtx). On the basis of growth, the Mtx‐5011 derivatives were 270‐ to 3,000‐fold more resistant to mtx than wild‐type mosquito cells. Properties associated with mtx resistance in these cells were consistent with amplification of the dihydrofolate reductase (DHFR) gene. The cells overproduced DHFR protein, were enriched with DHFR mRNA, and DNA from resistant cells was enriched for a band that likely contained the DHFR coding sequence. Karyotype analysis indicated that high levels of resistance were accompanied by a conversion to tetraploidy, chromosome rearrangements, and an apparent duplication of one of the mosquito chromosomes.
Polyploidy in the nuclei of ovarian nurse and follicle cells of the silk moth, Hyalophora cecropia
Authors: Cardoen Johan , Watson Cornelius , Deloof Arnold , Berry Spencer J.
Published: February 2005

The ploidy of ovarian nurse cells of Hyalophora cecropia was determined at three arbitrarily chosen stages of follicle development. C‐values ranged from 8,192 to 65,536 for the nurse cells, and increased in orderly geometric fashion with the age of the follicle. The ploidy of accompanying follicle cells increased from 8 to 512C during the same development sequence.
Blood digestion in the mosquito, Anopheles stephensi liston (diptera: Culicidae): Partial characterization and post‐feeding activity of midgut aminopeptidases
Authors: Billingsley Peter F.
Published: February 2005

Aminopeptidase activity was partially characterized from midguts of Anopheles stephensi Liston which had been dissected 30 h after blood feeding. In crude midgut homogenate supernatants the aminopeptidases showed optimum activity at pH 8.0 and preferentially hydrolyzed alanine‐ and leucine‐terminal amino acid substrates. Methionine, proline, lysine, and arginine terminal substrates were hydrolysed, but not glutamic acid. Activity was stimulated by Mg2+, EDTA, and low Ca2+ concentrations, while Mn2+, Tris, 1,10 phenanthroline, and higher Ca2+ concentrations were inhibitory.

Supernatants from midguts homogenized in 1% Triton X‐100 showed a twofold increase in activity. Differential centrifugation of midgut homogenates demonstrated 45% of the total activity in a putative microvillar pellet and 32% in a soluble fraction. More than 92% of the total activity was solubilized after homogenization in Triton X‐100.

Activity in homogenate supernatants was restricted to one major peak (Mr = 552,000) with a higher molecular weight shoulder. Three distinct peaks of aminopeptidase activity were observed forllowing Triton X‐100 treatment: a minor high molecular weight peak (Mr = 552,000), and two major peaks at Mr = 123,000 and Mr = 32,000 respectively.

The activity of aminopeptidase increased after a blood meal, in parallel to the post‐feeding changes in trypsin activity, indicating its important role in secondary digestion of blood meal proteins.
Biosynthesis of dehydro‐N‐acetyldopamine by a soluble enzyme preparation from the larval cuticle of Sarcophaga bullata involves intermediary formation of N‐acetyldopamine quinone and N‐acetyldopamine quinone methide
Authors: Saul Steven J. , Sugumaran Manickam
Published: February 2005

The enzymes involved in the side chain hydroxylation and side chain desaturation of the sclerotizing precursor N‐acetyldopamine (NADA) were obtained in the soluble form from the larval cuticle of Sarcophaga bullata and the mechanism of the reaction was investigated. Phenylthiourea, a well‐known inhibitor of phenoloxidases, drastically inhibited both the reactions, indicating the requirement of a phenoloxidase component. N‐acetylcysteine, a powerful quinone trap, trapped the transiently formed NADA quinone and prevented the production of both N‐acetylnorepinephrine and dehydro NADA. Exogenously added NADA quinone was readily converted by these enzyme preparations to N‐acetylnorepinephrine and dehydro NADA. 4‐Alkyl‐o‐quinone:2‐hydroxy‐p‐quinone methide isomerase obtained from the cuticular preparations converted chemically synthesized NADA quinone to its quinone methide. The quinone methide formed reacted rapidly and nonezymatically with water to form N‐acetylnorepinephrine as the stable product. Similarly 4‐(2‐hydroxyethl)‐o‐benzoquinone was converted to 3,4‐dihydroxyphenyl glycol. When the NADA quinone‐quinone isomerase reaction was performed in buffer containing 10% methanol, β‐methoxy NADA was obtained as an additional product. Furthermore, the quinones of N‐acetylnorepinephrine and 3,4‐dihydroxyphenyl glycol were converted to N‐acetylarterenone and 2‐hydroxy‐3′,4′‐dihydroxyacetophenone, respectively, by the enzyme. Comparison of nonenzymatic versus enzymatic transformation of NADA to N‐acetylnorepinephrine revealed that the enzymatic reaction is at least 100 times faster than the nonezymatic rate. Resolution of the NADA desaturase system on Benzamidine Sepharose and Sephacryl S‐200 columns yielded the above‐mentioned quinone isomerase and NADA quinone methide:dehydro NADA isomerase. The latter, on reconstitution with mushroom tyrosinase and hemolymph quinone isomerase, catalyzed the biosynthesis of dehydro NADA from NADA with the intermediary formation of NADA quinone and NADA quinone methide.

The results are interpreted in terms of the quinone methide model elabrated by our group [Sugumaran: Adv. Insect Physiol. 21 :179–231, 1988; Sugumaran et al.: Arch. Insect Biochem. Physiol. 11 :109,1989] and it i s concluded that the two enzyme p‐sclerotization model [Andersen: Insect Biochem. 19:59–67,375–382,1989] is inadequate to account for various observations made on insect cuticle.
Quinone and quinone methide as transient intermediates involved in the side chain hydroxylation of N‐acyldopamine derivatives by soluble enzymes from Manduca sexta cuticle
Authors: Saul Steven J. , Dali Hemalata , Sugumaran Manickam
Published: February 2005

Proteins solubilized from the pharate cuticle of Manduca sexta were fractionated by ammonium sulfate precipitation and activated by the endogenous enzymes. The activated fraction readily converted exogenously supplied N‐acetyldopamine (NADA) to N‐acetylnorepinephrine (NANE). Either heat treatment (70°C for 10 min) or addition of phenylthiourea (2.5 μM) caused total inhibition of the side chain hydroxylation. If chemically prepared NADA quinone was supplied instead of NADA to the enzyme solution containing phenylthiourea, it was converted to NANE. Presence of a quinone trap such as N‐acetylcysteine in the NADA‐cuticular enzyme reaction not only prevented the accumulation of NADA quinone, but also abolished NANE production. In such reaction mixtures, the formation of a new compound characterized as NADA‐quinone‐N‐acetylcysteine adduct could be readily witnessed. These studies indicate that NADA quinone is an intermediate during the side chain hydroxylation of NADA by Manduca cuticular enzyme(s). Since such a conversion calls for the isomerization of NADA quinone to NADA quinone methide and subsequent hydration of NADA quinone methide, attempts were also made to trap the latter compound by performing the enzymatic reaction in methanol. These attempts resulted in the isolation of β‐methoxy NADA (NADA quinone methide methanol adduct) as an additional product. Similarly, when the N‐β‐alanyldopamine (NBAD)‐Manduca enzyme reaction was carried out in the presence of L‐kynurenine, two diastereoisomers of NBAD quinone methide‐kynurenine adduct ( = papiliochrome IIa and IIb) could be isolated. The above results are in agreement with our hypothesis that N‐acylnorepinephrine formed in Manduca cuticle is biosynthesized by an indirect route involving intermediary formation of N‐acyldopamine quinone and N‐acyldopamine quinone methide as established in the case of Sarcophaga bullata and is not produced by the action of a β‐hydroxylase.
Alkanes of four related moth species, Helicoverpa and Heliothis
Authors: Carlson David A. , Milstrey Susan K.
Published: February 2005

Comparison of the presence and quantities of cuticular hydrocarbons has been used successfully for identifying sibling species and races of several groups of insects. This approach has been extended to four species of moths previously regarded as belonging to the same genus, Heliothis. Gas chromatography was used to quantify the numerous high‐molecular weight alkanes found on the cuticle of two pairs of closely related species: Helicoverpa zea and Helicoverpa armigera, and Heliothis virescens and Heliothis subflexa. Both sexes of H. zea and H. armigera contained different quantities of several alkanes that could be used for unambiguous identification. Similar comparisons of H. subflexa and H. virescens showed four peak ratios that were different for each species. Sexual dimorphism was minor in H. subflexa and H. virescens.
Ecdysteroid‐stimulated synthesis and secretion of an N‐acetyl‐D‐glucosamine‐rich glycopeptide in a lepidopterna cell line derived from imaginal discs
Authors: Porcheron Patrick , Morinière Madeleine , Coudouel Noëlline , Oberlander Herbert
Published: February 2005

Hormone‐regulated processing of N‐acetyl‐D‐glucosamine was studied in an insect cell line derived from imaginal wing discs of the Indian meal moth, Plodia interpunctella (Hübner). The cell line, IAL‐PID2, responded to treatment with 20‐hydroxyecdysone with increased incorporation of GlcNAc into glycoproteins. Cycloheximide and tunicamycin counteracted the action of the hormone. In particular, treatment with 20‐hydroxyecdysone resulted in the secretion of a 5,000 dalton N‐acetyl‐D‐glucosamine‐rich glycopeptide by the IAL‐PID2 cells. Accumulation of this peptide was prevented by the use of teflubenzuron, a potent chitin synthesis inhibitor. A glycopeptide of similar molecular weight was observed in imaginal discs of P. interpunctella treated with 20‐hydroxyecdysone in vitro, under conditions that induce chitin synthesis. Although the function of the 5,000 dalton glycopeptide is not known, we believe that the PID2 cell line is a promising model for molecular analysis of ecdysteroid‐regulated processing of aminosugars by epidermal cells during insect development.
Control of the pheromone biosynthetic pathway in Helicoverpa zea by the pheromone biosynthesis activating neuropeptide
Authors: Jurenka Russell A. , Jacquin Emmanuelle , Roelofs Wendell L.
Published: February 2005

Hormonal control of the production of the main sex pheromone component, Z11‐hexadecenal (Z11‐16:Ald), of female Helicoverpa zea was investigated using labeled precursors to monitor the pheromone biosynthetic pathway. The pathway originates with the biosynthesis of palmitic acid followed by δ11 desaturation to form the intermediate Z11‐16:Acid, which is then reduced to a primary alcohol and then oxidized to Z11‐16:Ald. Incubation of glands either in vivo or in an isolated gland culture with the deuterium labeled compounds, D3‐16:Acid or D9‐Z11‐16:Acid, yielded no differences in label incorporation into pheromone in the presence or absence of synthetic PBAN (Pheromone biosynthesis activity neuropeptide). However, unlabeled pheromone titers increased about tenfold above controls in the presence of PBAN in both in vivo and in vitro cultures. When isolated glands were incubated with [1‐14C]acetate, about a thirtyfold increase in the incorporation of radiolabel into Z11‐16:Ald above controls was observed in the presence of PBAN. Also, about a tenfold increase in incorporation was observed in saturated and monounstaturated fatty acids found in the gland upon stimulation with PBAN. These results indicate that the hormone PBAN controls pheromone biosynthesis in H. zea by regulating a step in or prior to fatty acid biosynthesis.
Plant biochemistry and aphid populations: Studies on the spotted alfalfa aphid, Therioaphis maculata
Authors: Dillwith Jack W. , Berberet Richard C. , Bergman Douglas K. , Neese Paul A. , Edwards Rose M. , McNew Ronald W.
Published: February 2005

Feeding by the spotted alfalfa aphid, Therioaphis maculata (Buckton), on susceptible alfalfa, Medicago sativa L., results in dramatic changes in plant biochemistry that in turn have profound effects on aphid physiology. These aphids select older leaves on the plant as feeding sites. One component of this selection process may be the amount and composition of plant epicuticular lipids, which vary with leaf age. Feeding aphids induce a senescence‐like state in the leaf that is characterized by loss of chlorophyll, decreased levels of soluble protein and fatty acids, and increased production of ethylene. This process involves lipid peroxidation and, like senescence, is probably free‐radical‐mediated. Leaves of alfalfa having resistance to spotted alfalfa aphid contain higher activities of catalase than do susceptible leaves. This enzyme may function in concert with other antioxidant enzymes to quench aphid‐induced free radical damage and thus impart resistance. Aphid fatty acid metabolism is altered by changes in plant metabolism and thus reflects the close relationship between aphid and plant biochemistry.