bluuebunny/crossref_metadata_2025 huggingface dataset:
Yolk degradation in tick eggs: I. Occurrence of a cathepsin L‐like acid proteinase in yolk spheres
Authors: Fagotto François
Published: February 2005

In crude extracts of eggs of the soft tick Ornithodoros moubata, maximum degradation of vitellin is at pH 3–3, 3.5, whereas no proteolysis is detected at neutral or weakly acidic pHs. Acidic proteolysis is maintained at high level throughout embryonic development, and rapidly decreases in the larva, during the high phase of yolk degradation. Proteinase, acid phosphatase, and N‐acetylglucosaminidase are localized within the yolk spheres; these can be considered as lysosomal‐like organelles containing both substrate (vitellin) and the degradative machinery. Proteolytic activity has been essentially attributed to a cathepsin L‐like enzyme through substrate specificity and inhibitors. The molecular weight is 37,000 to 39,000 as shown using gelatin‐containing SDS‐PAGE activity gels. At neutral pH the enzyme binds to vitellin, as demonstrated by gel filtration and PAGE under nondenaturing conditions. Acid proteinase activity at pH 5–6 is undetectable both with proteins and synthetic substrates, but is strongly increased after preincubation at pH 3–4. Activation at low pH could be important in the regulation of yolk degradation.
Yolk degradation in tick eggs: II. Evidence that cathepsin L‐like proteinase is stored as a latent, acid‐activable proenzyme
Authors: Fagotto François
Published: February 2005

Cathepsin L–like proteinase found in the eggs of the tick Ornithodoros moubata is latent during embryogenesis, but can be activated by acid treatment. In crude extracts as well as in partially purified fractions, activation requires reducing conditions and is inhibited by leupeptin, which indicates that it is mediated by a thiol proteinase, probably by the cathepsin L itself. Latency disappears in vivo at the time of the acute phase of yolk digestion, which takes place during late embryonic development and larval life. When egg cathepsin L is localized through its gelatinolytic activity on SDS‐PAGE, the activated enzyme migrates as lower Mr bands than the latent form. Disappearance of the higher Mr bands corresponding to the latent form is directly related to appearance of the lower Mr bands characteristic of the active one; transition from one pattern to the other and enzymatic activation are in perfect agreement with regard to kinetics and sensitivity to inhibitors. The same pattern change occurs in vivo, parallel to latency removal and intense yolk degradation. These results strongly suggest that egg cathepsin L is stored in the yolk as a proenzyme which is activated by partial proteolysis at low pH.
In vitro study of lipid metabolism in the mealworm larval fat body
Authors: Bahjou Ahmed , Gourdoux Lucienne , Moreau Robert , Puiroux Jacques , Dutrieu Janine
Published: February 2005

Lipid metabolism in Tenebrio larval fat body has been studied in vitro. Lipid release required the presence of diluted hemolymph in the incubation medium. This time‐dependent release of lipid was strongly stimulated in a dose‐dependent manner by Tenebrio corpora cardiaca (CC) extracts or synthetic adipokinetic hormone (AKH I). Furthermore, some glycerol was released when larval fat body was incubated without hemolymph, and this phenomenon was also dose dependent for added CC extracts. Lipid synthesis was estimated in vitro by following the incorporation of radioactivity from [6‐14C] glucose into fatty acids. Lipogenesis occurred in the absence of added carbohydrates in the medium, but it was stimulated by the addition of glucose, and especially trehalose (10 mg ml−1). Intestinal insulin‐like peptide (ILP) also stimulated in vitro lipogenesis in a dose‐dependent fashion. We conclude that lipolytic and lipogenetic activities of larval mealworm fat body in vitro are effectively under hormonal control.
Metabolism of ecdysteroids by a chitin‐synthesizing insect cell line
Authors: Ward Gordon B. , Beydon Philippe , LaFont Réne , Mayer Richard T.
Published: February 2005

A chitin‐synthesizing cockroach cell line (UMBGE‐4) previously shown to secrete ecdysteroids was analyzed for its ability to metabolize potential precursors of ecdysone (e.g., 2‐deoxyecdysone, 2,22‐dideoxyecdysone, 2,22,25‐trideoxyecdysone, and cholesterol). All, except cholesterol, were actively metabolized by UMBGE‐4 cells. However, all but 2‐deoxyecdysone were converted to polar and hydrolyzable metabolites, and not to ecdysone. Labeling with cholesterol was unsuccessful. Labeling experiments with molting hormones, i.e., ecdysone and 20‐hydroxyecdysone, confirmed that this cell line can metabolize ecdysteroids and allowed identification of some of the products. Molting hormones were converted into acetate conjugates and polar conjugates which were often double‐conjugates, i.e., polar conjugates of acetate conjugates. Labeling experiments with ecdysone demonstrated that this cell line possesses a low ecdysone 20‐hydroxylase activity. The capacity of UMBGE‐2 cells, which do not synthesize chitin or ecdysteroids, was also examined. Neither ecdysone nor 20‐hydroxyecdysone was significantly metabolized by UMBGE‐2 cells. 2‐Deoxyecdysone and 2,22‐dideoxyecdysone were very slowly metabolized respectively to more polar compounds.
Action of RH 5849, a non‐steroidal ecdysteroid mimic, on Plodia interpunctella (Hübner) in vivo and in vitro
Authors: Silhacek Donald L. , Oberlander Herbert , Porcheron Patrick
Published: February 2005

We have examined the effects of RH 5849, a non‐steroidal ecdysteroid mimic, on the growth and development of Plodia interpunctella. When RH 5849 was administered in the diet, larval growth was inhibited in a dose‐dependent manner, while concentrations of 15 ppm and greater were highly toxic. However, the deleterious effects of RH 5849 could be prevented, except at very high concentrations of RH 5849, by the simultaneous administration of the juvenile hormone mimic methoprene. Larvae simultaneously treated with both hormone mimics continued to grow until they attained a size about three times normal. This growth was accompanied by at least one and sometimes two supernumerary molts, whereas, only an occasional supernumerary molt occurred in larvae treated with methoprene alone. In larvae undergoing super numerary molts, wing imaginal discs produced a tanned pupal cuticle, but did not evaginate. When wing discs were cultured in vitro, RH 5849 stimulated evagination and chitin synthesis at concentrations of 10 and 1 μM, respectively. Likewise, RH 5849 stimulated GlcNAc uptake and inhibited cellular proliferation in IAL‐PID2 cells at similar concentrations. These in vitro effects of RH 5849 also were produced by 20‐hydroxyecdysone, but at lower concentrations. We conclude that RH 5849 exhibits molting hormone activity in vivo as well as in vitro. However, the toxicological effects in P. interpuncetella result from action on feeding and growth, rather than molting. Thus, RH 5849 represents a new class of IGR, which will have impact on our understanding of endocrine regulation and open up new avenues for pest control.
Efficient transfection of mosquito cells is influenced by the temperature at which DNA‐calcium phosphate coprecipitates are prepared
Authors: Kjer Karl M. , Fallon Ann Marie
Published: February 2005

Trnsient expression of a heat‐shock protein‐chloramphenicol acteyltrans‐Perase (hsp‐CAT) recombinant plasmid was used to define the parameters that influence transfection of cultured mosquito cells using DNA‐calcium phosphate coprecipitates. The efficiency of the calcium phosphate procedure was strongly influenced by the growth state of recipient cells, and by the temperature at which the coprecipitate was prepared. Under optimal conditions, which included formation of the DNA‐calcium phosphate coprecipitate at 50°C, transfection frequencies were up to tenfold higher than those obtained using the previously described polybrene procedure.
Demonstration of β‐N‐acetyl‐D‐glucosaminidase and β‐N‐acetyl‐D‐hexosaminidase in Drosophila Kc‐cells
Authors: Sommer Ulrich , Spindler Klaus‐Dieter
Published: February 2005

Kc‐cells from Drosophila melanogaster, grown under serum‐free conditions, produce two β‐hexosaminidases and secrete these enzymes into the medium. The two enzymes were separated by DEAE‐exchange chromatography. According to their substrate specificities one enzyme is a β‐N‐acetyl‐D‐glucosaminidase (E.C.3.2.1.30), the other one a β‐N‐acetyl‐D‐hexosaminidase (E.C.3.2.1.52). The β‐N‐acetyl‐D‐glucosaminidase is predominant in the medium, the β‐N‐acetyl‐D‐hexosaminidase within the cells. The Km values for the substrates pNP‐GlcNAc, pNP‐GalNAc, and (GlcNAc)2 are 0.8, 16.73, and 1.67 mM for the β‐N‐acetyl‐D‐glucosaminidase and 0.24, 0.44, and 0.2 mM for the β‐N‐acetyl‐D‐hexosaminidase. Both enzymes are inhibited by the products and the β‐N‐acetyl‐D‐glucosaminidase is also inhibited stereospecifically by the substrates pNP‐GlcNAc and (GlcNAc)2. Both enzymes are inhibited in a partial competitive way by acetamidolactones, the Kis being as low as 0.1 μM.
Lysozyme in the midgut of Manduca sexta during metamorphosis
Authors: Russell Virginia W. , Dunn Peter E.
Published: February 2005

Low levels of lysozyme were found in the midgut epithelium of the tobacco hornworm, Manduca sexta, during the early part of the fifth larval stadium. This was observed in control insects as well as in bacterially challenged insects. No lysozyme was detected in the gut contents of either group of insects which were actively eating or in the early stages of metamorphosis. However, high levels of lysozyme activity were detected in homogenates of midgut tissue collected from insects later in the stadium. Immunocytochemical studies demonstrated that lysozyme accumulates in large apical vacuoles in regenerative cells of the midgut during the larval‐pupal molt. These cells, initially scattered basally throughout the larval midgut epithelium, multiply and form a continuous cell layer underneath the larval midgut cells. At the larval/pupal ecdysis the larval midgut epithelium is sloughed off and the regenerative cells, now forming the single cell layer of the midgut, release the contents of their vacuoles into the midgut lumen. This release results in high lysozyme activity in the lumen of the pupal midgut and is thought to confer protection from bacterial infection. This is the first indication that the lysozyme gene may be developmentally regulated in a specific tissue in the absence of a bacterial infection.
3‐oxoecdysteroid 3β‐reductase in various organs of the European corn borer, Ostrinia nubilalis (Hubner)
Authors: Gelman Dale B. , Demilo Albert B. , Thyagaraja Belgaum S. , Kelly Thomas J. , Masler Edward P. , Bell Robert A. , Borkovec Alexej B.
Published: February 2005

Extracts of brains, subesophageal ganglia, Malpighian tubules, mandibular glands, proctodaea, salivary glands, testes, and fat body from mature fifth‐stage European corn borer larvae contain a 3‐oxoecdysteroid 3β‐reductase that converts 3‐dehydroecdysone to an RIA‐detectable ecdysteroid that has the same retention time (on HPLC and TLC) as ecdysone. Enzyme activity is destroyed by boiling, exposure to organic solvents, and treatment with trypsin. The reductase has a molecular weight in the range of 24–37 kD and is dependent upon the presence of NADPH for activity. Under the conditions utilized here, subesophageal ganglia, proctodaea, and brain extracts exhibited the highest specific activity; mandibular glands, testes, salivary glands, and Malpighian tubules had moderate specific activity; and fat body had the least. Based on total organ activity, however, fat body and salivary glands had the greates activity; testes, Malpighian tubules, mandibular glands, and proctodaeum had moderate activity; and brain and subesophageal ganglion had the least.
Stimulation of juvenile hormone biosynthesis by analogues of a Manduca sexta allatotropin: In vitro studies
Authors: Unni Balagopalan G. , Bhaskaran Govindan , Dahm Karl H. , Hayes Timothy K.
Published: February 2005

Two analogues of a Manduca sexta allatotropin (Mas‐AT) were synthesized. They correspond to the active fragment, amino acids 5–13, of the natural Mas‐AT with substitution of norleucine for methionine. ATANA has the structure Val‐Glu‐Nle‐Nle‐Thr‐Ala‐Arg‐Gly‐Phe‐NH2, ATAA is acetylated at the N‐terminus. Allatotropic potency was evaluated by measuring the in vitro rates of juvenile hormone (JH) biosynthesis in corpora allata (CA) of M. sexta.

At a concentration of 20 nM, ATANA and ATAA increased JH production in day 0, day 1, and day 3 adult female CA by a factor of 3–8. Larval CA were not affected. These results correspond to activities reported for the natural Mas‐AT. ATANA did not stimulate pharate adult female CA to produce JH. Stimulation of female CA with ATANA was reversed when the CA were transferred to fresh medium while stimulation with ATAA under the same condition persisted.

Exogenous farnesoate was converted to JH‐III at a rate exceeding the highest ATANA‐stimulated rate. ATANA in addition to farnesoate did not increase JH‐III production, but increased JH‐II production in addition to the already high production rate for JH‐III. It is inferred that Mas‐AT stimulates a rate‐limiting enzyme in the biosynthesis of farnesoate and its homologues but does not affect epoxidation and methylation.
Influence of light on plant allelochemicals: A synergistic defense in higher plants
Authors: Downum Kelsey R. , Swain Lee A. , Faleiro Lavina J.
Published: February 2005

Plant phototoxins are broad‐spectrum biocides which adversely affect an array of potential plant enemies, including among others disease‐causing pathogens, nematodes, insect herbivores, and competing plant species. Thus far, plants which contain these broad‐spectrum allelochemicals have been found to occur in open habitats (i.e., in full sunlight) where a defensive mechanism mediated by light would seem to operate most effectively. The levels of available light in shaded environments, although considerably lower than full sun (1–10% of full sun), are equivalent to the intensities of light used to kill phototoxin‐treated insects in laboratory studies. This suggests that phototoxic reactions might mediate important organismal interactions in shaded environments as well. In this study, more than 230 Costa Rican rainforest plants were bioassayed for phototoxic metabolites in an effort to ascertain their prevalence among plants growing in moderate to extreme shade. Microbial bioassays, employing Bacillus cereus (a gram positive bacterium), Escherichia coli (a gram negative bacterium), and Saccharomyces cerevisiae (a yeast) were used to rapidly and sensitively indicate phototoxic action and potential for insecticidal action. Tissue extracts from 12 plant families tested positive for phototoxins. This is the first report of phototoxins occurring in eight of those families (Acanthaceae, Campanulaceae, Gesnariaceae, Loganiaceae, Malpigaceae, Phytolaccaceae, Piperaceae, and Sapotaceae). The presence of phototoxins in rainforest plants suggests that phototoxic plant allelochemicals may function as important defenses in low‐light, as well as high‐light, environments.
Spotted alfalfa aphid, Therioaphis maculata, fatty acids relative to the condition and susceptibility of its host
Authors: Bergman Douglas K. , Dillwith Jack W. , Berberet Richard C.
Published: February 2005

Fatty acids of spotted alfalfa aphids, Therioaphis maculata (Buckton), were compared to determine what effects host condition and susceptibility to aphids have on fatty acid metabolism. Myristic acid was the predominant fatty acid in aphids that were feeding on newly infested, green alfalfa tissues. In contrast, linoleic acid was the predominant fatty acid in aphids that were feeding on chlorotic leaves 14 days after the plants were infested. Amounts of myristic acid per mg of aphid, examined 14 days after the plants were infested, were ca. 71% less than that for aphids collected from alfalfa 4 days after the plants were infested. Amounts of linoleic acid per mg of aphid live weight did not change over this period. These changes in spotted alfalfa aphid fatty acids were not due to variations in either fatty acid compositions of aphid life stages or alfalfa fatty acids.

Ratios of myristic acid to linoleic acid varied in aphids removed from plants 4 days after infestation compared to aphids with which the plants were originally infested. These ratios increased in aphids feeding on alfalfa plants that were susceptible to spotted alfalfa aphids and decreased in aphids feeding on plants that exhibited resistance characteristics of antibiosis and/or antixenosis.
Physical properties of β‐N‐acetyl‐D‐glucosaminidase and β‐N‐acetyl‐D‐hexosaminidase from Drosophila Kc‐cells
Authors: Sommer Ulrich , Spindler Klaus‐Dieter
Published: February 2005

Kc‐cells from Drosophila produce two different β‐D‐hexosaminidases, a β‐N‐acetyl‐D‐glucosaminidase (E.C.3.2.1.30) and a β‐N‐acetyl‐D‐hexosaminidase (E.C.3.2.1.52), which are also secreted into the medium. The Mr of both enzymes is about 126,000 ± 9,700; the S‐values are 8.37 ± 0.44. Both enzymes have about the same pH optima at 5.5 and the same thermal stability. The temperature optima are identical (50°C) for both enzymes if p‐nitrophenyl‐N‐acetylglucosaminide is used as a substrate. However, when p‐nitrophenyl‐N‐acetylgalactoseaminide is used as the substrate the β‐N‐acetyl‐D‐hexosaminidase has a temperature optimum about 10°C higher. With higher salt concentrations, the activity of the β‐N‐acetyl‐D‐glucosaminidase increases, whereas β‐N‐acetyl‐D‐hexosaminidase is inhibited. Both enzymes also differ in their sensitivity to urea, the β‐N‐acetyl‐D‐hexosaminidase being less sensitive than the β‐N‐acetyl‐D‐glucosaminidase.
Development of continuous cell lines from the egg parasitoids Trichogramma confusum and T. exiguum
Authors: Lynn Dwight E. , Hung Akey C. F.
Published: February 2005

Although numerous insect cell lines have been developed over the past three decades, few of these have been from the order Hymenoptera. This report describes two new continuous cell lines from trichogrammid wasps. The extremely small size of these insects has made physiological and biochemical studies difficult. Now, with the development of the cell lines, a limitless supply of biologically active material is available for a wide variety of basic biological studies. The Trichogramma confusum and T. exiguum cell lines (designated IPLB‐Tcon1 and IPLB‐Tex2) were characterized by chromosome and isozymes techniques. Evidence of their utility is shown by morphological response to the developmental hormone, 20‐hydroxyecdysone. The morphological change in IPLB‐Tex2 is accompanied by an induction of highly contractile cells which indicates this cell line may be composed of myoblast cells.
Acidification of yolk granules in Blattella germanica eggs coincident with proteolytic processing of vitellin
Authors: Nordin John H. , Beaudoin Esther L. , Liu Xiaodong
Published: February 2005

In eggs of the cockroach Blattella germanica, vitellin (Vt) utilization by the embryo is initiated at day 4 postovulation by the proteolytic processing of its three subunits to a specific set of peptides. A report from our laboratory (Nordin et al.: Archives of Insect Biochemistry and Physiology 15:119, 1990) described a yolk proteinase, activated at days 3–4, which processes the Vt. Further investigation of this event has focused on the yolk granules. Granules from eggs 4–6 days postovulation contained a significant subpopulation which accumulated high concentrations of the dye acridine orange (AO), a fluorescent probe of vesicle acidification, while those from eggs 0–3 days postovulation did not. AO accumulation was caused by proton translocation and was not due to dye binding or a Donnan equilibrium. The temporal correlation of granule acidification with Vt processing suggests a role for this event in yolk proteinase activation in B. germanica. This hypothesis was supported by the finding that incubation of yolk from freshly ovulated eggs in vitro at pH of 5 and below resulted in Vt processsing. Yolk granules of the blowfly Phormia regina also became acidified but this occurred in the oocyte prior to egg deposition.
Structure‐activity studies on adipokinetic hormones in Manduca sexta
Authors: Ziegler Rolf , Eckart Klaus , Jasensky Ronald D. , Law John H.
Published: February 2005

Structure‐activity studies were performed for adipokinetic hormone (AKH) in Manduca sexta. Seven naturally occurring and four synthetic peptides of the red pigment concentrating hormone (RPCH)/AKH family were tested in larvae of M. sexta for activation of glycogen phosphorylase in fat body. pGlu at the N‐terminal was found to be important for activity of peptides; however, Manduca AcGly1 AKH is partially active. The amino acids at all positions appear to be of importance for activity, with the possible exception of the two serine residues in positions six and seven. Generally, the more amino acids are exchanged, the less the peptide will bind to the receptor. In M. sexta a β‐bend appears not to be important for the binding of peptides. Peptides ten amino acids long appear to be more active than shorter ones.
Expression of heat shock proteins in response to high and low temperature extremes in diapausing pharate larvae of the gypsy moth, Lymantria dispar
Authors: Yocum George D. , Joplin Karl H. , Denlinger David L.
Published: February 2005

Diapausing pharate first instars of the gypsy moth, Lymantria dispar, respond to high temperature (37–41°C) by suppressing normal protein synthesis and synthesizing a set of seven heat shock proteins with Mrs of 90,000, 75,000, 73,000, 60,000, 42,000, 29,000, and 22,000 as determined by SDS‐PAGE. During recovery at 25°C from heat shock, synthesis of the heat shock proteins gradually decreases over a period of 6 h, while normal protein synthesis is restored. A subset of these same heat shock proteins is also expressed during recovery at 4°C or 25°C from brief exposures to low temperature (‐10 to 20°C), and its expression is more intense with increased severity of cold exposure. During recovery at 4°C after 24 h at −20°C, both 90,000 and 75,000 Mr heat shock proteins are expressed for more than 96 h. While normal protein synthesis is suppressed during heat shock and recovery from heat shock, normal protein synthesis coincides with synthesis of the heat shock proteins during recovery from low temperatures, thus implying that expression of the heat shock proteins is not invariably linked to suppression of normal protein synthesis. Western transfer, using a monoclonal antibody that recognizes the inducible form of the human 70,000 Mr heat shock protein, demonstrates that immunologically related proteins in the gypsy moth are expressed at 4°C and during recovery from cold and heat shock.
Characterization and regulation of HMG‐Coa synthase in Locusta migratoria corpora allata
Authors: Couillaud Franck , Rossignol Françoise
Published: February 2005

The conversion of acetyl‐coenzyme A (CoA) to 3‐hydroxy‐3‐methylglutaryl (HMG)‐CoA was measured by a radioenzymatic assay in locust corpora allata (CA) which produce juvenile hormone (JH‐III). The conversion of acetyl‐CoA to HMG‐CoA was limited to the cytosolic fraction of the CA with an apparent KM = 400 μM. The HMG‐CoA synthase activity measured during the first 15 days of the female adult life did not follow the pattern of JH‐III biosynthesis. No relationship was found between JH‐III release and HMG‐CoA synthase activity when measured sequentially on the same CA pair. HMG‐CoA synthase activity did not reflect the asymmetry in spontaneous rates of JH biosynthesis observed in left and right corpora allata. Decline of JH release after in situ transection of the nervus corporis allati 1 (NCA‐1) was not associated with a decline in HMG‐CoA synthase activity. The results suggest that HMG‐CoA synthase activity does not have a prime influence on the spontaneous rate of JH biosynthesis, and that HMG‐CoA synthase does not appear to be a target for the allatostimulating factors acting on the CA through the NCA‐I.
Differential alaserpin expression during development of the antennae in the tobacco hawkmoth, Manduca sexta
Authors: Hanneman Eric H. , Kanost Michael R.
Published: February 2005

The expression of alaserpin, a serine protease inhibitor, was studied in the developing adult olfactory system of Manduca sexta. Alaserpin mRNA in the antenna was identical to alaserpin from larval fat body, by restriction mapping and partial sequencing of clones isolated from a male pupal stage 6 antennal cDNA library. Northern and primer extension analysis showed that alaserpin transcripts were the same length at all stages and were present at stages 3 and 6, declined at stages 9–15, reappeared at stage 18, and persisted in adults. Western blotting revealed that alaserpin was present at stages 3–12, declined at stage 15, and disappeared at stage 18 and in adults. Immunocytochemistry at stage 6 revealed labeled cells closely associated with neurite bundles and scattered throughout the receptor cell layer. An occasional cell was seen in the antennal lumen. Labeled cells were segmentally distributed along the length of the antenna. The results suggest that alaserpin may have a role in antennal morphogenesis and olfactory neuron axon outgrowth.
Purification and characterization of pyruvate carboxylase from the honeybee and some properties of related biotin‐containing proteins in other insects
Authors: Tu Zhijian , Hagedorn Henry H.
Published: February 2005

Our discovery of a Mr 128 k biotin‐containing protein in an extract of the white‐fly was an unexpected outcome of the use of an avidin‐biotin‐peroxidase method to visualize a western blot. This major biotin‐containing protein was shown to be present in several tissues of 10 different species of insects by doing a western blot and staining it with streptavidin‐linked peroxidase. The amount of this protein in the thorax of the mosquito, Aedes aegypti, increased during development. The non‐flying grasshopper, Barytettix psolus, had reduced amounts of this protein in their thoraces compared to a flying grass‐hopper, Schistocerca americana. The major biotin‐containing protein was purified from the thoraces of honeybee, Apis mellifera, using an avidin Sepharose affinity column. The purified biotin‐containing protein was shown to be pyruvate carboxylase, an enzyme that catalyzes the specific transfer of a carboxyl group to pyruvate, yielding oxaloacetate. The purified honeybee pyruvate carboxylase was characterized enzymatically and structurally. This protein had a single subunit of Mr 128 k and formed a native molecule of about Mr 500 k consisting of four of these subunits. The amino acid composition of the protein was also obtained. The enzymatic activity of this protein required acetyl‐CoA, ATP, and Mg2+. The Kms of the enzyme for bicarbonate and pyruvate were similar to pyruvate carboxylase from other oganisms. The biotin‐containing protein was also partially purified from mosquito thoraces using the same methods and was shown to be pyruvate carboxylase. The comparison between insect pyruvate carboxylase and that of other organisms is provided and the possible physiological role of the pyruvate carboxylase in the thoracic muscles of insects is discussed.
The oviduct musculature of the stable fly Stomoxys calcitrans: Properties of its spontaneous motility and neural regulation
Authors: Cook Benjamin J.
Published: February 2005

The muscles of the stable fly oviduct were striated and in the common oviduct both circular and longitudinal fiber layers were present. Although two muscle layers were evident in the lateral oviducts, both consisted of an irregular lattice of longitudinal fibers with extensive branching. This branching between fibers seemed to provide a pathway for the spread of excitation because semi‐isolated preparations of the oviduct severed from their connections to the central nervous system continued to contract in a spontaneous and rhythmic manner. Compression or shortening of the longitudinal muscles of the oviduct was the predominant mode of activity. Both phasic contractions (1–2 s in duration) and tonic events (15–30 s in duration) were observed. Innervation of the oviduct arises from the four major branches of the median abdominal nerve trunk that enter the ovipositor. Five to seven peripheral nerve cells were found along these four branches. Electrical stimulation of the severed median abdominal nerve trunk caused the oviduct to respond to a single pulse. However, these individual responses approached a fusion at 6 pulses per second (pps). Two of five preparations showed an inhibition of spontaneous phasic concentrations and a noticeable drop in baseline tonus during and after a 10–20 s interval of stimulation at a frequency of 0.5–2 pps. Endogeneous electrical activity was recorded from branch nerves in the ovipositor after connections to the central nervous system were cut. Potentials of two distinct durations were evident in this recorded activity. The significance of this and other findings are discussed.
Partial characterization of digestive tract proteinases from western corn rootworm larvae, Diabrotica virgifera
Authors: Gillikin Jeffrey W. , Bevilacqua Steven , Graham John S.
Published: February 2005

We have partially characterized the proteolytic activity within luminal contents of the digestive tracts of larvae of the western corn rootworm, Diabrotic virgifera LeConte. At least 15 proteinases were detected based on chromatographic behavior on ion exchange high‐performance liquid chromatography and their mobility on sodium dodecyl sulfate gels containing gelatin. Inhibitors of proteolytic activity indicated that these enzymes are primarily sulfhydryl proteinases. Native polycrylamide gel electrophoresis revealed that a single proteinaceous inhibitor, egg white cystatin, was capable of abolishing a substantial part of the proteolytic acitivity. Our data suggest, accordingly, that gene transfer experiments utilizing the cystatin gene may generate lines of maize which have increased resistance to western corn rootworm larvae. © 1992 Wiley‐Liss, Inc.
Regulation of ooplasmic sodium and potassium activities in developing locust eggs
Authors: Hawkins Erika , O'Donnell Michael J.
Published: February 2005

Ooplasmic activities of potassium and sodium were measured with ion sensitive microelectrodes before and during the period of maximal water uptake which occurs 3–5 days after oviposition for eggs incubated at 37°C. Potassium activity increased from 84 mM in eggs before fertilization at 118 mM in eggs 1 day after fertilization (d1). Sodium activity increased from 8 mM to 29 mM over the same period. These changes exceeded those predicted from the decrease in water content (8%) during the first day after oviposition. Between d1 and d3, potassium and sodium activities decreased to values predicted on the basis of the 88% increase in egg water content. Although water content increased an additional 46% between d3 and d5, ooplasmic sodium activity remained constant at 11 mM and potassium activity increased from 64 mM to 74 mM during this time. Declines in concentrations of sodium and potassium measured in whole eggs by atomic absorption spectrometry mirrored the increase in egg water content. The results suggest that regulation of ooplasmic sodium and potassium activities is accomplished by release of these ions from internal stores, possibly the york spheres. © 1992 Wiley‐Liss, Inc.
Stimulation of sex pheromone production by proteinaceous extracts of the bursa copulatrix in the redbanded leafroller moth
Authors: Fabriás Gemma , Jurenka Russell A. , Roelofs Wendell L.
Published: February 2005

Pheromone biosynthesis in the redbanded leafroller moth, Argyrotaenia velutinana, was stimulated by homogenates of the bursa copulatrix. Although pheromonotropic activity was also extractable from the ovary, the activity of pheromone biosynthesis activating neuropeptide (PBAN) or bursa extracts was not impaired in isolated abdomens by removal of the ovary. Response to the bursa extracts was dependent on the dose administered and the time of incubation. Amounts of pheromone present in adult females of different ages appeared to be correlated with the extractable amount of pheromonotropic activity from their bursa copulatrix. Decapitation did not result in the suppression of burse factor production. Homogenates of the bursa elicited similar effects in both isolated gland and isolated abdomen incubations, but the brain neuropeptide, PBAN, was less active in the former than in the latter. Bursa extracts stimulated pheromone production in isolated abdomen incubations deprived of the bursa copulatrix, but PBAN did not. Loss of activity of bursa homogenates after treatment with either pronase E or carboxypeptidase Y indicated that the pheromonotropic factor is a proteinaceous substance. The mechanism through which pheromone production is regulated in redbanded leafroller moths is discussed. © 1992 Wiley‐Liss, Inc.
Juvenile hormone analog and injection effects on locust hemolymph protein synthesis
Authors: Wyatt Gerard R. , Kanost Michael R. , Chin Beth C. , Cook Kathryn E. , Kawasoe Barbara M. , Zhang Jianzhong
Published: February 2005

In adult female Locusta migratoria, at about day 8 after eclosion, when vitellogenin (Vg) is first produced as a result of induction by juvenile hormone (JH), the intensity of hemolymph protein electrophoretic bands at about 75 kDa and 20 kDa increases sharply, suggesting that JH may induce additional proteins. A major component of the elevated protein is persistent storage protein (PSP; subunit 74 kDa). Administration of the JH analog, methoprene, to precocene‐treated adult locusts was followed by a rise in hemolymph levels of PSP but not in apolipophorin III (19 kDa), identified immunochemically and electrophoretically. The synthesis of PSP in adult fat body was confirmed by incorporation of [3H]leucine. At 48 h after treatment with methoprene, Vg synthesis was induced in females (as previously observed) and synthesis of PSP in both sexes was elevated above controls, while synthesis of apolipophorin III was not stimulated. We conclude that in adult locust fat body the synthesis of several proteins responds in different ways to the JH analog: Vg (and a 21 kDa protein described elsewhere) is induced de novo solely in females; PSP (and a 19 kDa protein described elsewhere) is stimulated in both sexes but is not fully JH‐dependent; apolipophorin III is not stimulated. In these experiments, methoprene was administered both by injection in mineral oil and topically in acetone. After injection of mineral oil as a vector control, incorporation into secreted proteins was stimulated at 24 h, presumably due to a wound effect; topical application of acetone avoids this effect and is a preferred route for administration of JH analog. © 1992 Wiley‐Liss, Inc.
Effects of diet and temperature on Morimus funereus larval hemolymph cation concentrations
Authors: Janović‐Hladni Miroslava , Chen Andrew C. , Ivanović Jelisaveta , Djordjević Suzana , Stanić Vlasta , Perić Vesna , Frusić Marina
Published: February 2005

The effects of diet and different constant temperatures on hemolymph cation concentrations (Na+, K+, Mg2+, Ca2+) have been studied in Morimus funereus larvae collected from natural habitat, fed natural (oak or beech bark) or artificial diet, as well as in larvae reared from hatching on an artificial diet. In the hemolymph of larvae maintained under natural conditions Mg2+ was dominant, whereas Na+ concentration was very low. In their natural diets concentrations of Na+ and K+ were very low, while those of Ca2+ and Mg2+ were high. In larvae continuously reared on an artificial diet, hemolymph Mg2+ concentration was significantly decreased and Na+ concentration increased more than fourfold compared to the results obtained in oak‐fed larvae. Na+ and K+ are the dominant cations in the artificial diet. The concentrations of K+ and Ca2+ in the hemolymph of larvae fed natural or artificial diet are nearly identical, suggesting the existence of an internal regulatory mechanism in this insect for these cations. The hemolymph cation concentrations of M. funereus larvae are predominantly dependent upon the diet consumed, much less upon the environmental temperatures. The most stable concentrations of cations were observed in larvae continuously fed an artificial diet and exposed to different constant temperatures. There was much less stability in the hemolymph cation concentration in oak larvae fed either natural or artificial food after their transfer to constant temperatures. With respect to the response to the external factors studied, the most sensitive are the Na+ concentrations, the most stable seems to be K+. © 1992 Wiley‐Liss, Inc.
Comparative studies on the mode of action of proctolin and phorbol‐12, 13‐dibutyrate in their ability to contract the locust mandibular closer muscle
Authors: Baines Richard A. , Downer Roger G. H.
Published: February 2005

The role of proctolin has been further investigated in the locust (Locusta migratoria) mandibular closer muscles. Radioactive calcium uptake measurements were made using protease‐dissociated muscle cells. Both the phorbol ester, phorbol‐12, 13‐dibutyrate, and proctolin produce tonic contractions which are associated with the influx of extracellular calcium. The thresholds for proctolin and the phorbol ester to contract the muscle were 1‐10 nM and 10‐100nM, respectively, while their respective thresholds for evoking measurable calcium influx into the muscle cells were 0.1‐1 nM for proctolin, and 0.1‐1 pM for phorbol‐12, 13‐dibutyrate. The effect of phorbol‐12, 13‐dibutyrate is blocked by a number of protein kinase inhibitors (at a concentration of 0.1 mM), suggesting that an activation of protein kinase can lead to calcium influx. These inhibitors, however, do not block the effect of proctolin, indicating that these two compounds work through different pathways, possibly coverging on the same final target. In light of this finding, a number of other compounds have been tested to try to ascertain how proctolin mediates an increased calcium influx. © 1992 Wiley‐Liss, Inc.
Separation of insect hemolymph proteins by cascade‐mode multiaffinity chromatography
Authors: Samaraweera Preminda , Law John H. , Porath Jerker
Published: February 2005

The hemolymph of the adult female Manduca sexta was fractionated by cascade‐mode multiaffinity chromatography (CASMAC) on a main‐line tandem column chain containing Zn2+‐TED, T‐gel, Ni2+‐DPA, and phenylsepharose and a side‐line column containing Zn2+‐DPA. The technique separated some of the previously described major hemolymph proteins, and yielded a number of fractions with simple composition. Some of these fractions contained only less abundant proteins of Manduca hemolymph. Thus, it appears that CASMAC would be a very useful fractionation technique for purification and characterization of the minor proteins of insect hemolymph. © 1992 Wiley‐Liss, Inc.
Storage proteins of the larval root weevil Diaprepes abbreviatus (Coleoptera: Curculionidae): Riboflavin binding and subunit isolation
Authors: Shapiro Jeffrey P. , Niedz Randall P. , Silhacek Donald L.
Published: February 2005

Proteins present at high concentrations in hemolymph of the larval weevil Diaprepes abbreviatus were previously shown to bind a synthetic coumarin, 7‐amino‐3‐phenyl coumarin (coumarin‐10). One of the two native proteins previously identified (protein I) is now shown to separate into two distinct bands (proteins la and lb) using native gradient pore‐limiting electrophoresis. The high concentration of proteins la, lb, and ll in larval hemolymph, their disppearance from hemolymph upon pupation, and an apparent hexameric structure shown by chemical crosslinking identify them as hexameric storage proteins (hexamerins). At least one chromatographic form of lb isolated by anion exchange HPLC is now shown to bind riboflavin (Rb). Binding was also demonstrated by quenching of Rb fluorescence by a partially isolated mixture of the storage proteins. Lipophorin did not quench Rb fluorescence. Rb was heat‐extracted from whole hemolymph and identified by its fluorescence spectra and by reverse phase HPLC with fluorescence detection. The two subunits shared by the three holoproteins have been isolated by sequential density gradient ultracentrifugation, gel permeation HPLC, and reverse phase HPLC. All three holoproteins shared the α subunit (M, 75,000), while the β subunit (M, 71,000) was lacking from one of the three. Repeated passage through an anion exchange column yielded two of the three proteins (lb and ll) in homogeneous form. Chemical crosslinking with dimethylsuberimidate indicated a hexameric structure for the holoproteins. All subunits and holoproteins stained as high mannose glycoproteins when probed with biotinylated concanavalin A on PVDF membranes. The α subunit was high in Met, His, and Thr, and the β subunit was high in Lys. Both were high in Pro and had approximately 16% Phe + Tyr. Sequences of the 20 N‐terminal amino acid residues of each subunit showed 45–60% homology between subunits. These coleopteran proteins also showed some sequential homology but no immunological cross‐reactivity with storage proteins from the lepidopterans Galleria mellonella and Heliothis virescens. © Wiley‐Liss, Inc.
Regulation of fat body glycogen phosphorylase in larval tobacco hornworm, Manduca sexta
Authors: Siegert Karl J.
Published: February 2005

Activation and inactivation of fat body glycogen phosphorylase was investigated in ligated abdomens of larval Manduca sexta and in vitro. After maximal activation through Manduca adipokinetic hormone (AKH) or chilling, inactivation of glycogen phosphorylase commenced as soon as the stimulus for the activation was removed indicating that the enzyme system in the fat body is fine‐tuned to low phosphorylase activities which is necessary to allow glycogen synthesis. In intact ligated abdomens phosphorylase can be activated repeatedly by either stimulus showing that the fat body system does not lose its responsiveness. It was impossible to achieve complete conversion of the inactive form of phosphorylase into the active form even after administration of AKH and simultaneous chilling. © 1992 Wiley‐Liss, Inc.
Colocalization and differential expression of PBAN‐ and FMRFamide‐like immunoreactivity in the subesophageal ganglion of Helicoverpa zea (Lepidoptera: Noctuidae) during development
Authors: Blackburn Michael B. , Kingan Timothy G. , Raina Ashok K. , Ma Michael C.
Published: February 2005

Pheromone biosynthesis activating neuropeptide (PBAN) stimulates sex pheromone production in several species of moths. We have used an antiserum to PBAN to map the distribution of PBAN‐like immunoreactivity (PLI) in the subesophageal ganglion (SEG) of the larval and pupal stages of the corn earworm, Helicoverpa zea, and compared this distribution with that of the adult. The distribution of PLI within the SEG of larvae and pupae was quite similar to that found in adults, suggesting a function for PBAN other than the stimulation of pheromone biosynthesis. In all developmental stages studied, three groups of nerve cells in the SEG, and their projections, contained PLI. In adults and larvae, most of the cells containing PLI also had FMRFamide‐like immunoreactivity (FLI). In pupae, however, a number of cells which appeared to have PLI and FLI in both larvae and adults, did not contain andy detectable FLI, while the PLI appeared unchanged. The FLI returns to these cells during adult development. These immunocytochemical observations were corroborated by quantifying PLI and FLI by competitive ELISA; the level of extractable FLI in pupae was about 25% of that found in larvae, while PLI levels were unchanged. © 1992 Wiley‐Liss, Inc.
Characterization of fall armyworm mitochondrial DNA (Lepidoptera: Noctuidae)
Authors: Ke Li Dao , Pashley Dorothy P.
Published: February 2005

Mitochondrial DNA from the fall armyworm, Spodoptera frugiperda (J.E. Smith), was cloned and characterized using restriction enzyme mapping. Genome size is approximately 16.3 kilobase (Kb), consistent with that of most animals. Three fragments, 8.1 Kb, 6.2 Kb, and 2.0 Kb, were produced by digestion with restriction enzyme Xbal and cloned into a pUC19 vector. Twenty‐nine restriction enzymes were used to generate a detailed physical restriction enzyme map of the three cloned fragments. These data represent the first detailed characterization of a lepidopteran mitochondrial genome. © 1992 Wiley‐Liss, Inc.
Certain hemocyte proteins of the medfly, Ceratitis capitata, are responsible for nonself recognition and immobilization of Escherichia coli in vitro
Authors: Marmaras Vassilis J. , Charalambidis Nektarios
Published: February 2005

The results indicate that certain hemocyte proteins of the medfly, Ceratitis capitata, are responsible for the recognition of foreignness, since they are able to bind to the surface of Escherichia coli in vitro. Furthermore, when the E. Coli‐hemocyte protein complex was incubated in the presence of tyrosine and phenoloxidase, the bacteria were immobilized, forming large aggregates. The formation of aggregates seems to be due to reactive tyrosine intermediate (quinone) generated by the action of phenoloxidase. © 1992 Wiley‐Liss, Inc.
Acetylcholine receptors of thoracic dorsal midline neurones in the cockroach, Periplaneta americana
Authors: Bai Donglin , Erdbrugger Heinrich , Breer Heinz , Sattelle David B.
Published: February 2005

The actions of acetylcholine and cholinergic ligands have been studied using dorsal midline neurones from the metathoracic ganglion of the cockroach Periplaneta americana. Both nicotine and oxotremorine depolarized dorsal midline neuronal cell bodies. Dose‐response curves for nicotine and oxotremorine saturated at different levels. Nicotine‐induced depolarizations were completely or partially blocked by mecamylamine, d‐tubocurarine, strychnine, and bicuculline, but were insensitive to α‐bungarotoxin (100 nM), atropine (100 μM), scopolamine (10 μM), and pirenzepine (50 μM). Following pretreatment with collagenase, the dorsal midline neurones were sensitive to high doses of α‐bungarotoxin (3 μM). Oxotremorine‐induced depolarizations were blocked by scopolamine (10 μM), atropine (100 μM), and pirenzepine (50 μM) and were insentive to mecamylamine (10 μM) and d‐tubocurarine (100 μM). The results indicate the coexistence of at least two distinct acetylcholine receptors on dorsal midline neuronal cell bodies in the cockroach metathoracic ganglion. © 1992 Wiley‐Liss, Inc.
Interference of the insect growth regulator methoprene in the process of larval‐pupal differentiation
Authors: Shaaya Eli
Published: February 2005

Studies with epidermal cells of a number of insects showed that a sequence of physiological events takes place prior to and during the process of commitment of the epidermal cells from the larval to the pupal stage. At the onset of metamorphosis RNA synthesis, including that of RNA, was suppressed for about 30 h just prior to the wandering stage. At the completion of the wandering stage, pupally committed epidermal cells synthesize a new type of RNA termed hetero‐disperse nuclear RNA (hnRNA). It is suggested that this RNA species could play a role in the permanent shutting‐off of larval‐specific genes. Methoprene, a juvenile hormone analogue (JHA), delayed the larval‐pupal transformation and prevented these changes in RNA synthesis when applied prior to the appearance of the hnRNA, but had no effect when applied after this period. Treatment with the JHA in the early last larval instar prevented not only the normal rise in ecdysteroid titer, but also the increase in the activity of chitinolytic enzymes. Treatment of young pupae had no effect on either the ecdysteroids titer or on enzyme activity. A causal relationship between molting hormone titer and the activity of chitinolytic enzymes is proposed. © 1993 Wiley‐Liss, Inc.