The fate and possible role of arylphorin during the metamorphosis of Mamestra brassicae.

1. Arylphorin, one of the storage proteins has been isolated from the hemolymph of Mamestra brassicae. 2. It has been established that Mamestra arylphorin is the most similar to manducin from among the known storage proteins of other species. 3. A rabbit polyclonal antibody has been developed against arylphorin, and its concentration changes have been determined quantitatively by ELISA in the hemolymph and fat body from the 1st day of the last larval instar to the 3rd day of the imago stage. 4. Histological sections were made on each day during the investigated period and it was shown by immunohistochemical methods that the main quantity of arylphorin was accumulated in the storage protein granules of the fat body and it could be detected even in the imaginal fat body. 5. The uptake of arylphorin by the fat body is induced by 20-hydroxyecdysone. 6. During differentiation of the imaginal cuticle arylphorin is incorporated first in the epidermal cells and it is built in the endocuticular layer of the integument thereafter.     

Possible involvement of lumichrome in the binding of storage protein to its receptor in Sarcophaga peregrina

Previously, a receptor molecule on the surface of fat body cells of Sarcophaga peregrina larvae that is involved in the uptake of storage protein from the hemolymph was shown to be a 120-kDa protein (Ueno, K., and Natori, S. (1984) J. Biol. Chem. 259, 12107-12111). This paper reports evidence that lumichrome (7,8-dimethylalloxazine) may be present in the binding site of the receptor and mediate the binding of storage protein and receptor. A stoichiometric amount of lumichrome was shown to bind to the storage protein under conditions in which the latter bound to its receptor.     

Changes of acid phosphatase content and activity in the fat body and the hemolymph of the flesh fly Neobellieria (sarcophaga) bullata during metamorphosis

Changes in the specific and total activity of the lysosomal marker enzyme acid phosphatase (Acph) and in the amount of enzyme protein were examined in the fat body and the hemolymph from the last larval molt to the larval-pupal apolysis. The specific activity showed minor changes during the last larval period. In contrast, the total activity of the enzyme was low during the feeding period and higher during the wandering stage and strikingly increased at the time of puparium formation. We purified a protein having para-nitrophenyl phosphate phosphatase (Acph) activity and raised antisera against it. The amount of Acph protein in the fat body and hemolymph was examined using an ELISA. The specific Acph content showed little variation, but the total amount of the enzyme protein showed a stepwise increase in both organs during last larval stage and was markedly elevated in the pupal stage in the fat body. In contrast, a considerable decrease in the amount of Acph protein was observed in the hemolymph during this period. These data were in agreement with immunohistochemical observations showing an accumulation of the enzyme protein in fat body cells during the prepupal stage with a concomitant disappearance of the enzyme from the hemolymph. Inhibition of ecdysteroid secretion by water stress prevented the changes both in total enzyme activity and in the amount of Acph protein. However, Acph protein content and enzyme activity could be restored when the water stress was followed by a 20-hydroxyecdysone (20-HE) treatment. Taken together, our data show that Acph is secreted by fat body cells into the hemolymph during the larval stage, where it is stored in an inactive form. Increase in the 20-HE titer at the end of last larval stage reverses this process, and the enzyme is taken up by the fat body cells, where it becomes activated and appears in auto- and heterophagic vacuoles. Arch. Insect Biochem. Physiol. 34:369–390, 1997. © 1997 Wiley-Liss, Inc.     

Induction of selective phosphorylation of a fat body protein of Sarcophaga peregrina larvae by 20-hydroxyecdysone.

20-Hydroxyecdysone was shown to induce selective phosphorylation of a fat body protein of Sarcophaga peregrina larvae with a molecular mass of 30 kDa. This phosphorylation was not associated with synthesis of new protein. Fractionation of 32P-labelled fat body by differential centrifugation showed that this protein was mainly present in the membrane-rich fraction, although we could not specify the membrane. Thus, 20-hydroxyecdysone may modify the function of the fat body by inducing phosphorylation of a specific membrane protein.     

Synthesis of two storage proteins during larval development of the tobacco hornworm, Manduca sexta

Studies of synthesis and accumulation of the two storage proteins arylphorin and female-specific protein (FSP) during the final two larval instars of the tobacco hornworm showed both stage and temporal specificity. Arylphorin was present in both stages, but its synthesis ceased during the molt, during starvation, and at the wandering stage, and then resumed about 24 hr after the onset of feeding. During the larval molt about 25% of injected iodinated arylphorin was incorporated into the newly forming fifth instar cuticle. The cessation of arylphorin synthesis was mimicked by exposure of the fat body to 1 microgram/ml 20-hydroxyecdysone (20HE) in complete Grace's medium or to dilutions of Grace's medium greater than 50%. Lower concentrations of 20HE were ineffective, indicating that the cessation of synthesis in vivo was likely due to a combination of lack of excess nutrients and the hormonal milieu. The female-specific protein was not synthesized until the final larval instar, appearing first in females on Day 2 and later in males at the time of wandering, with synthesis continuing throughout the prepupal period. In vitro studies showed that this protein was synthesized as a 620-kDa protein, and then during secretion a 730-kDa immunoreactive form also appeared. Synthesis of FSP was inhibited by exposure of Day 2 fat body to 1 microgram/ml 20HE for 24 hr. Ligation followed by 20HE infusion showed that the disappearance of FSP from the hemolymph during the prepupal period was controlled by the rising ecdysteroid titer.     

Characterization of juvenile hormone-binding proteins of hemolymph of Locusta migratoria

The binding of juvenile hormone (JH) by components from hemolymph of adult female Locusta migratoria was characterized to establish whether hemolymph JH-binding proteins could be distinguished from a protein of fat body (BP-1) that may be a JH receptor. Hemolymph was analyzed by the hydroxyapatite assay, gel separation chromatography, polyacrylamide gel electrophoresis, and density gradient centrifugation. Three fractions that bound JH were separated from whole hemolymph by DEAE cellulose column chromatography, and these differed from all three cytosol-binding components. The major hemolymph component (H-A) showed relatively stable binding of JH, a slight loss of binding capacity after delipidation, and a K_d for JH-I of 16 nM. The K_ds for JH-l and JH-lll with unfractionated hemolymph were 26 and 42 nM respectively. The order of effectiveness of competitors for binding of [³H]JH-l was JH-lll > JH-l ? methoprene > hydroprene ? acids of methoprene and hydroprene. The data indicated that unlabeled JH-lll was bound more effectively than its radioactive counterpart. The sedimentation values determined by sucrose density gradient ultracentrifugation were 13-14 S for hemolymph, and the sedimentation value was not altered by the inclusion of 0.4 M KCl throughout the gradient. The data indicated that H-A resembled the specific JH carriers and differed from the putative receptor of fat body cytosol by several criteria.     

Site of hemolymph lectin production and its activation in vitro by 20-hydroxyecdysone

To identify the tissues which produce hemolymph lectin in larvae of Bombyx mori, ovary, testis, fat body, and hemocytes from 5th-instar larvae were cultured in vitro and the culture medium was partially purified and assayed for hemagglutinating activity. Among the tissues tested, hemocytes appeared to be a major source of the hemolymph lectins. Ovary produced lectins to about one-tenth of the amount observed for the hemocytes, whereas testis and fat body were not productive. To study the hormonal control of hemolymph lectin production by hemocytes, hemocytes from 4th-instar larvae were cultured in vitro. Hemagglutinating activity in the hemolymph of 4th-instar larvae was immunostainable with the monoclonal antibody raised against 350,000 dalton lectin found in the 5th-instar hemolymph, but their molecular sizes were larger than the 5th-instar hemolymph lectins. When 20-hydroxyecdysone was added into the medium, production of the lectin by the hemocytes was remarkably enhanced, depending upon the hormone concentration.     

Endocrine aspects of ovary growth and gestation in the Argentinian cockroach,Blaptica dubia (Dictyoptera,Blaberidae)

Titers of juvenile hormone (JH) III and free ecdysteroids were studied in the hemolymph of the ovoviviparous Argentinian cockroach, Blaptica dubia, related to fat body depletion and reproduction. Adult females were analyzed during the first (days 5–25) and second vitellogenic cycle (days 80–100) and during the periods of gestation. Body weight changes of adult females were closely related to ovarian growth, ootheca formation, ootheca deposition, and hatching of the nymphs. Biochemical analysis of the fat body revealed lipids as the main storage compounds, followed by glycogen, proteins, and free carbohydrates. Changes in the fat body weight and in the chemical constituents of the fat body correlated with the processes of vitellogenesis and gestation. Concentrations of JH and free ecdysteroids in the hemolymph were measured by high pressure liquid chromatography-mass spectrometry. JH III was the only JH homolog found. JH III titers were high during vitellogenesis as well as toward the end of the gestation period. Changes in the concentrations of ecdysone and 20-hydroxyecdysone were less clear. The results reveal JH III as the major gonadotropic hormone in adult females of B. dubia.     

Evidences for a stage-specific juvenile hormone binding protein in the hemolymph of the silkworm,Bombyx mori L.: Identification and characterization by photoaffinity labeling and immunological analyses

Two molecular forms of juvenile hormone binding proteins were identified in the larval hemolymph of Bombyx mori by photoaffinity labeling. One form having an Mr of 33 kDa was present constantly in the hemolymph of the third to the fifth instar larvae while the other form having an Mr of 35 kDa was detected in the hemolymph until in the early fifth instar larvae but not in the prewandering larvae and prepupae. A 33 kDa binding protein was purified by hydrophobic interaction chromatography, gel filtration, and native PAGE. Antiserum against 33 kDa binding protein cross-reacted with 35 kDa binding protein on Western blots, suggesting that these binding proteins shared the same epitopes. From the results of saturation binding assays, it was inferred that 33 and 35 kDa binding proteins had a similar binding affinity for JH 1. It was revealed that one of these binding proteins, 35 kDa binding protein, was produced in the fat body in a stage-specific manner: fat body of the early fifth instar larvae synthesized both 33 and 35 kDa binding proteins while that of prewandering larvae synthesized only 33 kDa binding protein. © 1996 Wiley-Liss, Inc.     

丽蝇蛹集金小蜂寄生对寄主蛹可溶性蛋白与芳基蛋白组成与含量的影响

为了探讨蛹期寄生蜂对寄主蛋白代谢的寄生生理效应,利用Bradford蛋白含量测定法、Western免疫印迹法及酶联免疫吸附检测法研究了棕尾别麻蝇Boettcherisca peregrina蛹被丽蝇蛹集金小蜂Nasonia vitripennis寄生后其脂肪体和血淋巴中可溶性蛋白及芳基蛋白组成与含量的变化。结果表明：寄生蛹脂肪体和血淋巴中可溶性蛋白的组成与未寄生相比基本无明显差异; 不论寄生与否寄主蛹脂肪体和血淋巴中芳基蛋白亚基分子量均为80 kDa,该亚基在脂肪体中未出现降解现象,而在血淋巴中仅于寄生后12 h的寄主蛹中呈现2条分子量相近的Western免疫印迹带,说明其降解可能先于未寄生对照。就含量而言,寄生蛹脂肪体中可溶性蛋白含量除寄生后24 h外均显著低于未寄生对照,芳基蛋白含量除寄生后48 h外也均显著低于未寄生对照,其中寄生后12 h的含量仅为未寄生的32.0%。寄生蛹血淋巴中可溶性蛋白含量多低于未寄生蛹,且寄生后2,12,24 h的差异达显著水平;芳基蛋白的含量均有低于未寄生的趋势,其中寄生后12 h的含量为未寄生的17.0%。综合认为,丽蝇蛹集金小蜂的寄生可导致寄主脂肪体和血淋巴中可溶性蛋白及芳基蛋白含量下降。     

Selective transport of the mulberry leaf urease from the midgut into the larval hemolymph of the silkworm,Bombyx mori

Just before spinning, larvae of the silkworm, Bombyx mori, absorb intact urease of the host plant (mulberry leaf) from the midgut lumen into the hemolymph. In order to investigate whether the transport of the mulberry leaf urease is selective, crude proteins extracted from the mulberry leaves were labeled with biotin and orally administered to the fifth instar larvae. The biotinylated proteins transported into the hemolymph were detected by ligand blotting using streptavidin. When the biotinylated proteins were administered to 5-day-old fifth instar larvae, a strong signal of a biotinylated protein was detected in the hemolymph 2 days after the administration. In contrast, when the biotinylated mulberry leaf proteins were administered to 3-day-old fifth instar larvae, no signal derived from the biotinylated proteins was detected in the hemolymph. The signal weakened when the biotinylated proteins had been immunoprecipitated before administering to the larvae, indicating that the signal came from the mulberry leaf urease. These results show that the transport of the mulberry leaf urease from the midgut into the hemolymph is selective and larval-stage specific. Subsequently, binding assays were carried out to test the binding ability of the mulberry leaf urease to the brush border membrane in the epithelial cells of larval midgut. The urease was not bound to the brush border membrane vesicles (BBMV) from the midgut of 3-day-old fifth instar larvae, while more than 60% of the total amount of incubated urease was bound to the BBMV from the midgut of 6-day-old fifth instar larvae. The urease binding ability of BBMV correlated with the uptake of the mulberry leaf urease. This suggests that a urease binding molecule(s) exists in the BBM of the midgut epithelium, which is involved in the uptake of the mulberry leaf urease. In addition, the uptake of the mulberry leaf urease into the hemolymph was induced by 20-hydroxyecdysone.     

Ecdysone 20-hydroxylation in imaginal wing discs of Pieris brassicae (lepidoptera): Correlations with ecdysone and 20-hydroxyecdysone titers in pupae

Ecdysone 20-hydroxylase activity has been detected in pupal wing discs of Pieris brassicae. This activity is due to an enzyme system located in microsomal fractions. Its apparent Km is 58 nM for ecdysone. The enzyme is inhibited by the reaction product 20-hydroxyecdysone with an apparent Ki of 2.6 μM. Its activity varied during pupal-adult development with a maximum on day 4, when ecdysone levels are the highest in the animal. Although low, the peak activity is sufficient to assure 25% of the conversion of endogenous ecdysone into 20-hydroxyecdysone in pupae. Ecdysone and 20-hydroxyecdysone levels were measured in hemolymph and whole animals; ecdysone appears to be mainly located in hemolymph, whereas 20-hydroxyecdysone seems to be equally distributed between hemolymph and tissues. All these findings are discussed in relation to the roles of ecdysone and 20-hydroxyecdysone during pupal-adult development.     

Storage protein uptake in helicoverpa zea: Arylphorin and VHDL share a single receptor

The major lepidopteran storage protein arylphorin is selectively taken up into perivisceral fat body of prepupae by receptor mediated endocytosis. The arylphorin receptor was identified by ligand blotting and in vitro binding studies. Fat body membranes contain a glycosylated receptor protein with an apparent molecular weight of 80,000 that binds arylphorin with a K_D of 9.02 × 10^?8 M. Competitive binding experiments revealed that the arylphorin receptor is identical with the previously identified VHDL receptor. Apparently a single receptor mediates the uptake of structurally distinct storage proteins. This storage protein receptor is present only in perivisceral fat body and only during the period around pupation when both storage proteins are sequestered. © 1994 Wiley-Liss, Inc.     

家蝇的卵黄发生及其激素调节

用5—15%SDS-PAGE分析表明,家蝇Musce domestica viaina卵黄蛋白由三个亚基组成,其亚基分子量分别为58KD、50KD、48KD.火箭免疫电泳的结果表明,脂肪体、血淋巴和卵巢内卵黄原蛋白的变化具有密切的相关性,卵黄原蛋白在体内最早出现在羽化后30小时左右,然后迅速增加,在羽化后48小时,脂肪体和血淋巴中卵黄原蛋白含量达到最大值,卵巢开始沉积卵黄蛋白在羽化后30小时,到产卵前达到最大值,脂肪体在离体培养条件下,通过测定³H-亮氨酸掺入卵黄原蛋白的量,对不同发育时期家蝇脂肪体合成卵黄原蛋白的能力及激素的调节作用进行了研究,结果表明,羽化12小时后,合成能力迅速上升,48小时时形成高峰,60小时后迅速下跌直至产卵,其合成能力一直维持在低水平,产卵后合成能力又迅速回升,激素处理结果表明,保幼激素可以促进卵黄发生前期和后期家蝇脂肪体的卵黄原蛋白合成,20-羟基蜕皮酮可以大幅度促进卵黄发生期家蝇脂肪体的卵黄原蛋白合成.当二种激素共同处理时,对卵黄发生前期和卵黄发生期的家蝇脂肪体有协同促进作用,而对卵黄发生后期的脂肪体没有这种作用.本文还对家蝇卵黄发生过程中脂肪体、血淋巴和卵巢三者之间的关系及家蝇卵黄发生的激素调节进行了讨论.     

Identification, characterization, and developmental regulation of two storage proteins in the bamboo borer Omphisa fuscidentalis

Two insect storage proteins, OfSP1 (75 kDa) and OfSP2 (72 kDa), were purified using three different chromatographies from the hemolymph of Omphisa fuscidentalis larvae during diapause, and their genes were cloned. OfSP1 and OfSP2 concentrations in the hemolymph were high during diapause. During pupation, OfSP1 levels decreased in the male hemolymph and disappeared from the female hemolymph. OfSP1 and OfSP2 mRNA levels in the fat bodies were low during the third instar, but increased greatly during the fourth and fifth larval instars. During diapause, mRNA expression continued at a lower level than during the feeding period. The injection of 20-hydroxyecdysone (20E) into diapausing larvae caused an increase in OfSP1 and OfSP2 mRNA levels 2-3 days post-injection, followed by a decrease in expression until pupation, which occurred 2-4 days thereafter. When larvae were treated with juvenile-hormone analog (JHA), OfSP1 and OfSP2 mRNA levels gradually decreased until the onset of pupation. In Omphisa, OfSP1 and OfSP2 proteins are produced and released by the larval fat bodies in the fourth and fifth-instar larvae, and the proteins accumulate in the hemolymph until the insects enter diapause. OfSP1 may be reabsorbed by the fat bodies at the end of diapause for subsequent re-use during pupation.     

The effects of actinomycin D,cycloheximide and puromycin on the 20-hydroxyecdysone induced autophagocytosis in larval fat body cells of Pieris brassicae

The effects of 20-hydroxyecdysone (5 μg/g body weight), cycloheximide (5 μg/g body weight), puromycin (30 μg/g body weight) and actinomycin D (30 μg/g body weight) were investigated on the larval fat body cells of 48–70-hours-old last instar larvae of Pieris brassicae.20-Hydroxyecdysone was found to induce the formation of autophagic vacuoles 3 hr after its administration, but this was, however, prevented by simultaneous cycloheximide treatment in a parallel experiment. On the contrary, puromycin proved to induce autophagocytosis. These diverse effects of the two translational inhibitors on hormone-induced autophagocytosis may be explained by differences in their modes of action.Actinomycin D, when administered 21 hr prior to the hormone, exerted a preventive effect on induced autophagocytosis, but was ineffective when applied 3 hr before the injection of 20-hydroxyecdysone.It was concluded that the presence of RNA synthesized several hours prior to the hormone treatment was a prerequisite for induction of autophagocytosis by 20-hydroxyecdysone.     

A steroid hormone affects sodium channel expression in Manduca central neurons

Neuronal differentiation is characterized by stereotypical sequences of membrane channel and receptor acquisition. This is regulated by the coordinated interactions of a variety of developmental mechanisms, one of which is the control by steroid hormones. We have used the metamorphosis of the holometabolous insect, Manduca sexta, as a model to study effects of 20-hydroxyecdysone on the maturation of thoracic neuron membrane channel expression. To test for direct hormone action, neurons were dissociated into primary cell culture on the first day of pupal life. In situ hybridization demonstrated that the amount of expression of the acetylcholine receptor alpha subunit, MARA1, was not affected by 20-hydroxyecdysone. Immunocytochemistry with an antibody directed against the SP19 segment of voltage-gated sodium channels revealed no effect of 20-hydroxyecdysone treatment during the first 6 days in culture. SP19 sodium channel protein was evenly distributed along all neurites. In contrast, after 8 days in culture, 20-hydroxyecdysone increased the amount of SP19 protein expression and strongly affected its distribution in differentiating neurons. In the presence of 20-hydroxyecdysone, patches of high densities of SP19 sodium channel protein were found in growth cones close to the base of filopodia. This is a further step toward unraveling the blend of membrane proteins under the control of steroids during the development of the central nervous system of postembryonic Manduca. Our results, taken together with previous studies, indicate that 20-hydroxyecdysone does not affect the expression of potassium membrane current or of the nicotinic acetylcholine receptor but instead regulates the amplitude of the calcium membrane current and the amount and distribution of SP19 sodium channel protein.     

Changes in the titer of the female-predominant storage protein (81K) during larval and pupal development of the waxmoth,galleria mellonella

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

Dynamics of lipid accumulation by the fat body of Rhodnius prolixus: the involvement of lipophorin binding sites

In insects, lipids are stored in the fat body, mainly as triacylglycerol (TAG). In Rhodnius prolixus, a hematophagous hemipteran, lipids are accumulated after blood meal to be used later on. In adult females, at the second day after feeding, the amount of TAG was 57+/-17 microg/fat body, it increased almost five times and at fourth day it was 244+/-35 microg/fat body. TAG content remained constant until day 13, but it then decreased and, at day 20th it was very low (31+/-4.9 microg/fat body). Radiolabeled free fatty acid was used to follow lipid accumulation by the fat body, as it was previously shown that, in R. prolixus, injected free fatty acids associate with lipophorin, a major hemolymphatic lipoprotein. (3)H-palmitic acid was injected into the hemocoel of R. prolixus females. It disappeared from the hemolymph very rapidly, and radioactivity was incorporated by the fat body. Sixty minutes after injection, radioactivity in the fat body was found mainly in TAGs. The capacity of the fat body to incorporate fatty acids from the hemolymph varied according to the days after blood meal, and it was maximal around the fourth day. Lipophorin binding to specific sites in fat body membrane preparations also showed variation at different days. When membranes obtained from insects at the second, fifth and tenth days were compared, binding was highest at fifth day after feeding.