Properties,synthesis, and accumulation of storage proteins in Pieris rapae L.

Two kinds of storage proteins (SP-1, SP-2) were confirmed in hemolymph and fat body of Pieris rapae during metamorphosis. Both proteins were present in high concentrations in the hemolymph during the last larval instar. Hemolymph concentrations of SP-1 and SP-2 dropped after pupation as the proteins were being deposited in fat bodies. SP-2 is present in a larger amount than SP-1. Detailed studies on storage proteins determined their properties, mode of synthesis, and accumulation in the fat body. SP-1 has a molecular weight of 500,000 and consists of one type of subunit (M_r 77,000), while SP-2 has a molecular weight of 460,000 and is composed of two types of subunits (M_r 80,000 and 69,000). The pl values of SP-1 and SP-2 were determined to be 6.97 and 7.06, respectively. Fat body cells from 1-day-old fifth instar larvae synthesized storage proteins in large amounts, whereas those from late prepupae exhibited high protein sequestration. Proteins taken up in fat body accumulated in dense granules during the pupal stage but sharply decreased at the adult stage. Morphological changes in the fat body tissues were observed during the larval-pupal transformation; the nuclei of fat body cells became irregularly shaped, and the boundaries between cells seemed to be obscure. Synthesis, storage, or degradation of storage proteins in fat body during development is closely associated with morphological changes in the tissues.     

Fat body protein granules and storage proteins in the silkmoth, Hyalophora cecropia

Fat body cells of silkmoth pupae (Hyalophora cecropia ) contain granules, showing a less dense outer zone and a denser, often crystalline, inner portion appear after cocoon spinning and increase until the larval-pupal ecdysis; more granules are formed in females than in males. Urate granules, appearing fibrous in internal structure, first form about the same time, but their accumulation is more gradual, and continues in the pupa. Both types have been isolated by centrifugation. Protein granules dissolve in buffers to yield proteins 1 and 2, with distinct electrophoretic and antigenic properties. These proteins have been isolated individually from pupal fat body extracts by using their different thermal stabilities in phosphate buffer containing MgCl2 and (NH4)2SO4, respectively, and purification was completed by gel chromatography. Protein 1 has a molecular weight of 480,000 and a subunit of 85,000 daltons, while protein 2 gives values of 530,000 and 89,000, respectively. Their amino acid compositions are similar but distinct. Proteins 1 and 2 accumulate in the hemolymph, beginning 3 days before spinning, reach maximal levels at spinning, and then decline in the hemolymph while granules are formed in the fat body, although the total hemolymph protein concentration does not decline at this time. It is concluded that the fat body of the late, feeding larva synthesizes two related "storage proteins" and secretes them in partially crystalline granules as protein reserves for metamorphosis.     

Developmental and sex-dependent regulation of storage protein synthesis in the silkworm,Bombyx mori

The mechanism of sex-dependent expression of a major plasma protein, referred to as storage protein 1 (SP-1) was studied during development of the silkworm, Bombyx mori. SP-1 occurred in the hemolymph of the female as well as in the male larvae until the end of the fourth larval instar. In the last instar larvae, the amount of SP-1 in the hemolymph greatly increased in females, but markedly declined in males. The level of fat body mRNA for SP-1 reflected the developmental and sex-dependent changes in the hemolymph concentration of SP-1. The developmental patterns of hemolymph proteins in the third and the fourth instar larvae of sex-mosaic individuals were quite analogous to those observed in normal larvae at the same developmental stages. The hemolymph concentration of SP-1 at the last larval instar of the sex mosaics varied among individuals irrespective of the gonad compositions. In vitro culture of the fat body cells dissected from several locations of a sex-mosaic larva provided evidence that each fat body cell in a common hemolymph milieu synthesizes a high (female type) or a low (male type) level of SP-1 depending on the sex chromosome composition. The amount of vitellogenin in the hemolymph of the sex-mosaic pupae was in proportion to that of SP-1 at the last larval instar. From these results, it is suggested that the sex-dependent expression of SP-1 and vitellogenin in B. mori is genetically determined and developmentally regulated without participation of the reproductive organs or any sex-specific humoral factors.     

Yolk polypeptide synthesis in the fat body of Sarcophaga bullata: Localization,hormonal induction and cell-free translation

The fat body of adult Sarcophaga bullata consists of different cell-types. The yolk polypeptides (YPs) are localized in secretory granules in the cytoplasm of female trophocyte fat body cells while the oenocytes and larval fat body cells are immunonegative. An antiserum against the larval serum protein 1 of Drosophila crossreacts on immunoblotting with several polypeptide bands in the haemolymph with mol. wt ∼80 kD. This antiserum specifically reacts with some storage granules of the persisting larval fat body cells and not with the other fat body cell types. The trophocyte fat body cells of male Sarcophaga treated with 20-OH-ecdysone, display a similar granular type of immunoreaction with an anti-YP antiserum as in vitellogenic females. Moreover, 20-OH-ecdysone induced in the fat body of males, in contrast to methoprene, synthesis of mRNA coding for YPs to a level as high as that in vitellogenic females, as shown in the reticulocyte lysate cell-free system.     

Some aspects of regulation of fat body lipolytic activity in Galleria mellonella

The degradation of ¹⁴C-trioleate by the homogenate of fat body of normally developing specimens of both sexes of Galleria mellonella at different days after the larval-pupal ecdysis, and of specimens subjected to different experiments was studied. It has been confirmed that the lipolytic activity of female fat body being low after the larval-pupal ecdysis, rises distinctly by about 6 days later. In contrast to this, the lipolytic activity of this tissue in males is high and does not appear to undergo changes.Ovariotectomy causes a significant fall of lipolytic activity.Preincubation of fat body of ovariotectomized females in the medium containing ovaries of pharate adults 6 days after the larval-pupal ecdysis brings about a rise of lipolytic activity of this tissue.     

Storage proteins of the fall webworm,Hyphantria cunea drury

Two storage proteins, storage protein-1 (SP1) and storage protein-2 (SP2), were found in hemolymph and fat body during the development of Hyphantria cunea, the fall webworm. Both storage proteins show similiar quantitative changes during development in males and females; however, SP1 is more abundant. The hemolymph of last instar larvae contains high concentrations of the storage proteins. However, following pupation, the storage proteins accumulate in fat bodies. SP1 peaks in the hemolymph of males and females late in last instar larvae (8-day-old 7th instar larvae). SP1 has a native molecular weight of 460,000 and consists of six identical subunits (Mr = 76,700), while SP2 has a molecular weight of 450,000 and is composed of two different subunits (Mr = 74,100 and 72,400). Both SP1 and SP2 are hexamers and are phosphorylated glycolipoproteins. The pl values of SP1 and SP2 were determined to be 5.70 and 5.50, respectively. Antibodies raised against SP1 react positively with vitellogenin and ovary extract, as well as with proteins in the hemolymph from last instar larvae and proteins in pupal fat bodies. Storage protein synthesis starts in fat bodies of a 4-day-old 7th instar larvae and in female peaks at 6–8 days of the 7th instar.     

Hormonal control of storage protein synthesis and uptake by the fat body in the silkworm, Bombyx mori

The female silkworm, Bombyx mori, rapidly accumulates two storage proteins, that are synthesized by the fat body, in the haemolymph during the feeding stage of the last-larval instar, and then sequesters them from the haemolymph into fat body during the larval-pupal transformation.The rapid synthesis and uptake of storage proteins by the fat body are shown to be induced by allatectomy in the early-penultimate larval instar. A juvenile hormone analogue, methoprene, is highly effective in inhibiting the allatectomy-induced synthesis, and, in a higher dosage, further blocks the uptake. Allatectomy in the late-penultimate larval instar shortly before moulting does not enhance the storage protein synthesis, but causes the uptake to occur two days earlier in the last-larval instar. Injection of 20-hydroxyecdysone is not stimulatory for synthesis of the proteins, but is effective to induce their uptake. Starvation during the early last-larval instar completely blocks the synthesis.From these results, it is suggested that storage protein synthesis is induced in the absence of juvenile hormone by some supplementary stimulus, possibly the supply of nutrient after feeding, and uptake is induced by ecdysteroids after a decline in the juvenile hormone level.     

粘虫不同发育期体内储存蛋白的检测及苦皮藤素Ⅴ对其含量的影响

采用PAGE和SDS-PAGE以及Western blot 的方法,分析了粘虫Mythimna separata幼虫、蛹及成虫体内的储存蛋白。结果表明,粘虫体内存在两种储存蛋白,其中一种为SP-1,即幼虫特异性储存蛋白,从6龄粘虫幼虫的2日龄开始出现在血淋巴中,到末日龄时达到峰值,停止取食后从血淋巴中消失;另一种为SP-3,在化蛹时开始出现在脂肪体中,一直到成虫期仍可持续表达,因此属于持续性储存蛋白。SP-1为分子量约94 kD和100 kD的2种亚基组成的蛋白质,而SP-3为分子量约94 kD的1种亚基组成的蛋白质。SP-1含8.16%的芳香类氨基酸,3.06%的甲硫氨酸。经苦皮藤素Ⅴ亚致死剂量处理5龄粘虫幼虫后的6龄2、3、4日龄粘虫幼虫体内储存蛋白的含量明显低于对照组,对5日龄后粘虫处理组和对照组体内储存蛋白的含量及雌性成虫产卵量没有明显影响。     

Studies on the cytophysiology of the fat body of the American silkmoth

Summary A developmental study at the electron microscopic level was conducted of the fat body cells of Hyalophora cecropia (L.). During the last larval instar the fat body increases in volume and the cells exhibit a well developed rough endoplasmic reticulum and protein bodies of diverse sizes. In the pupal fat body, the protein bodies appear to be enclosed by a double membrane and contain glycogen granules, ribosomes and mitochondrion-like structures. In addition, there are large lipid globules, cytolysomes and rough endoplasmic reticulum. The ultrastructure of the protein bodies suggests the development of large bodies by fusion of smaller protein bodies. Changes in fat body cell ultrastructure were followed during adult development and cytological evidence was obtained for the depletion of protein, glycogen and lipid in the female during this period. The female adult fat body cell contains free ribosomes, protein bodies, many mitochondria, a few lipid globules and glycogen granules. The male moth fat body cells have many mitochondria, a few glycogen granules, essentially no protein bodies, but an abundance of large lipid globules.Studies on the influence of egg maturation on the morphology of the fat body of Hyalophora gloveri (L.) revealed that ovariectomy of pupae yielded adults having more fat body than normal females, and that the fat body cells of the ovariectomized animals contained more glycogen, lipid and protein. Male pupae receiving ovarian implants developed into adults containing eggs and possessed more fat body than normal females but less than normal males. Very few glycogen granules were found in the fat body cells of normal males or males with implanted ovaries.Supported by grant AM-02818 from the National Institutes of Health.We thank Dr. James Oschman for his helpful suggestions and constructive criticisms.     

The fate of the larval storage protein calliphorin during adult development of Calliphora vicina

Purified calliphorin labelled with [¹⁴C]phenylalanine by in vivo synthesis was injected into mature Calliphora vicina larvae to examine the distribution of label during metamorphosis and in young adults. About 2.5% of the administered radioactivity was expired during adult development, and 10% was incorporated in the puparium. At the middle of adult development calliphorin was the only detectable radioactive soluble protein in contrast to the situation in the teneral adult where most soluble proteins were labelled. All organs and/or tissues of 4-day flies were radioactive; nearly half the phenylalanine of calliphorin was incorporated into flight muscle, actin and myosin being strongly labelled. Earlier hypotheses that calliphorin is a larval storage protein are now experimentally verified.     

粘虫不同发育期体内储存蛋白的检测及苦皮藤素Ⅴ对其含量的影响

采用PAGE和SDS-PAGE以及Western blot 的方法,分析了粘虫Mythimna separata幼虫、蛹及成虫体内的储存蛋白。结果表明,粘虫体内存在两种储存蛋白,其中一种为SP-1,即幼虫特异性储存蛋白,从6龄粘虫幼虫的2日龄开始出现在血淋巴中,到末日龄时达到峰值,停止取食后从血淋巴中消失;另一种为SP-3,在化蛹时开始出现在脂肪体中,一直到成虫期仍可持续表达,因此属于持续性储存蛋白。SP-1为分子量约94 kD和100 kD的2种亚基组成的蛋白质,而SP-3为分子量约94 kD的1种亚基组成的蛋白质。SP-1含8.16%的芳香类氨基酸,3.06%的甲硫氨酸。经苦皮藤素Ⅴ亚致死剂量处理5龄粘虫幼虫后的6龄2、3、4日龄粘虫幼虫体内储存蛋白的含量明显低于对照组,对5日龄后粘虫处理组和对照组体内储存蛋白的含量及雌性成虫产卵量没有明显影响。     

Inhibition of the formation of protein storage granules by glutaurine in the larval fat body cells of Mamestra brassicae (Insecta, Lepidoptera)

Correlative changes in the protein contents of haemolymph and fat body and the accumulation of protein storage granules in the fat body cells of Mamestra brassicae were investigated during the last larval stage in normally developing larvae and following administration of glutaurine (1 X 10(-4) mg/g body weight). The protein content of the haemolymph of untreated larvae increased up to the 4th day of the stage, declined during days 5 and 6, and increased again before pupation. In the glutaurine-treated larvae the amount of proteins in the haemolymph was as high as in the controls during the first four days but continued to rise up to the end of the stage. The protein content of the fat body started to increase from the 3rd day and heavy accumulation of protein storage granules in the cells of fat body was observed on the 5th and following days. The protein content of the fat body of glutaurine-treated larvae remained at a low level and the protein storage granules were absent in the cells. The inhibition of the selective uptake of haemolymphatic storage proteins by fat body following glutaurine treatment is suggested.     

PROTEIN UPTAKE INTO MULTIVESICULAR BODIES AND STORAGE GRANULES IN THE FAT BODY OF AN INSECT

The fat body in Calpodes ethlius (Lepidoptera, Hesperiidae) takes up protein from the blood throughout the larval stage before pupation. Depending upon the phase of development, the protein appears in multivesicular bodies, in large storage granules, and in structures of intermediate form. There are three phases in the 8 days of the last larval stage; the first devoted to growth (molting to 66 hr), the second to synthesis for storage or export (M + 66 to M + 156 hr), and the third to preparation for pupation (M + 156 to pupation at M + 192 hr). From M + O to M + 156 and from M + 180 to M + 188 hr, protein is taken up into multivesicular bodies. Larger MVB's form a continuous series with the protein granules formed from M + 162 to M + 180 hr. Blood proteins increase in concentration and amount from M + 66 to M + 156 hr at the same time as the fat body cells have a high rate of incorporation of amino acids and a structure appropriate for protein synthesis. During granule formation, both amino acid incorporation and blood protein concentration decrease. Since foreign proteins injected into the blood appear in the granules, they are probably made mainly from sequestered blood. Protein uptake involves two stages: concentration between the cells, and ingestion in pinocytotic vesicles. The vesicles fuse to become MVB's or storage granules, depending upon their rates of growth and the addition of lytic enzymes. Since MVB's do not accumulate in the fat body and since many of them contain acid phosphatase and appear empty, they are presumed to be concerned in protein turnover.     

An electrophoretic study of proteins of the ovary,fat body,and haemolymph in the migratory grasshopper, Melanoplus sanguinipes

Electrophoretic separation of saline extracts from the ovary revealed 14 proteins. Twelve proteins were detected in the fat body, of which seven had electrophoretic mobilities identical to those in the ovary. Similarly, eight of 16 proteins in the haemolymph of vitellogenic females ahad electrophoretically identical counterparts in the ovary. As these proteins accumulate in the haemolymph of ovariectomized females, the findings suggest that most yolk proteins are synthesized in the fat body. Although most female haemolymph proteins are present in males, two of the predominant yolk protiens are absent and represent female-specific proteins.Although certain proteins accumulate in the haemolymph of allatectomized females, the major ovarian proteins are absent or present in low concentrations. However, 48 hr after allatectomized females are treated with a juvenile hormone analogue, the haemolymph protein pattern resembles that of a normal female. This suggests that the corpora allata stimulate the synthesis of female-specific and other vitellogenic proteins. The median neurosecretory cells (mNSC) are also necessary for synthesis of female-specific proteins. Furthermore, proteins which are present in allatectomized females are absent in mNSC-cauterized insects suggesting that the mNSC stimulate general protein synthesis.     

Subcellular localization of uric acid storage in the fat body of Manduca sexta during the larval-pupal transformation

The fat body of the tobacco hornworm, Manduca sexta, serves as the major site for uric acid storage during metamorphosis. Light and electron microscopic examinations of fat body stained with reduced silver to show the location of stored uric acid have revealed that most, if not all, fat body cells store uric acid. The extent of specific staining is proportional to the increase in uric acid concentration in fat body during the initial stages of metamorphosis. Storage is associated with discrete membrane-bound structures, designated as uric acid storage vacuoles. In larval fat body, the structures are round or elliptical-shaped vacuoles with electron-dense fibrous interiors and are about the size of observed mitocondria (0.5–1.0 μm). During the larval-pupal transformation, the storage vacuoles double in size and appear as fibrous cores with spaces between the cores and the surrounding membranes. Before pupal ecdysis, the storage vacuoles are concentrated around the nucleus of each cell but after that event they are more uniformly distributed within fat body cells.     

Distribution and accumulation of storage protein-1 in ovary of Hyphantria cunea Drury

Storage protein-1 (SP-1) is a major storage protein found in the hemolymph and fat body of Hyphantria cunea. In this study, the uptake and accumulation of SP-1 into the ovary of H. cunea was investigated using biochemical and immunocytochemical methods. SP-1 in H. cunea has a high methionine content (4.6%) but is not female-specific, like other high methionine storage proteins. In the 6-day-old pupal ovary, SP-1 was detectable in trace amounts but accumulated to significant levels toward the end of the pupal stage. After adult emergence, SP-1 rapidly decreased in the ovarian follicles and remained low in the egg. This suggest that SP-1 is either extensively modified or degraded, causing a loss of its antigenic property in the ovary after adult emergence. During vitellogenesis, SP-1 is present in the hemolymph and penetrates through the tunica propria to reach the perioocytic space. From there, SP-1 is incorporated into yolk bodies. These results clearly show that SP-1 is taken up by the developing oocyte. Its disappearance suggests that SP-1 might be an amino acid reservoir for providing precursors for egg formation, in contrast to yolk proteins, which are utilized during postembryonic development. Arch. Insect Biochem. Physiol. 37:115–128, 1998. © 1998 Wiley-Liss, Inc.     

Metabolic shift from lipogenesis to glycogenesis in the last instar larval fat body of the silkworm,Bombyx mori

When [¹⁴C]glucose was injected into the last instar larvae of the silkworm, Bombyx mori, the label was incorporated into various tissues at varying degrees depending on the developmental stages. Fat body exhibited high incorporation rates throughout the feeding periods. Silk glands became active in incorporation but midgut decreased toward larval maturation. The pulse labeling experiment clearly demonstrated that the metabolic shift from lipogenesis to glycogenesis occurred in fat body at the middle of the last instar; a predominant incorporation was found in lipids when [¹⁴C]glucose was injected at the early stage, while at the late stage glycogen synthesis became most active. Incorporation into fat body proteins was not a major factor throughout the instar. Extirpation of silk glands enhanced incorporation into glycogen and proteins at the late stage but did not affect lipid synthesis. Long-term chase showed that fat body lipids and proteins synthesized at the early stage were totally carried over into the pupal fat body, while much glycogen produced at the late stage was used during the larval-pupal transformation with the remainder carried over into the pupa.From these results the metabolic shift from lipogenesis to glycogenesis in fat body is discussed in relation to the storage function of the fat body for pupal metamorphosis.     

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.     

Stage- and tissue-specific expression of the genes encoding calliphorin,the major larval serum protein of Calliphora vicina

Summary The stage- and tissue-specific biosynthesis of calliphorin was analysed during the development of the blowfly, Calliphora vicina. Western blot analyses show that the protein is not present in eggs, whereas it can be detected in fat body, brain, imaginai disk, salivary gland and epidermis throughout all postembryonic stages, including the adult one. By Northern analysis a unique 2.6 kb mol.wt. mRNA coding for calliphorin is identified exclusively in the fat body tissue of larvae, pupae and adults. Hybridization experiments of in vivo labelled poly(A)⁺ RNA with filter-bound calliphorin genes indicate that the genes are transcribed until pupariation. However, the translation of the calliphorin mRNA stops at the end of the feeding stage, as shown by [³⁵S]-methionine incorporation.