In vitro synthesis, release and uptake of storage proteins by the fat body of Manduca sexta: putative hormonal control

1. Two major proteins (P1 and P2) are synthesized by the fifth instar larval fat body of Manduca sexta and then released into the hemolymph. 2. These proteins are later sequestered by the pre-pupal fat body. 20-Hydroxyecdysone does not appear to affect the synthesis of either protein. 3. When day 2 fifth instar larvae are neck-ligated there is an excessive synthesis (supersynthesis) of P2 (arylphorin). 4. Juvenile hormone I (JH I) applications to ligated animals had no effect, but brain homogenate injections resulted in the inhibition of P2 synthesis. 5. Neck ligations of larvae between days 5 and 6 revealed a head critical period between day 5 + 12 hr and day 5 + 18 hr, after which the head is unnecessary for the sequestration of either protein by the fat body. 6. JH I and JH III applications to ligated larvae before the head critical period do not restore the ability of the fat body to sequester the storage proteins. 7. P1 and P2 appear to be synthesized differentially and P2 is sequestered by the fat body to a much lesser extent than P1. 8. P2 is the hemolymph storage protein of both larval and pupal stages, whereas P1 appears to be the storage protein of the pupal fat body. 9. The data indicate that the synthesis of arylphorin and the resorption of both proteins are controlled by a putative head factor(s).     

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。     

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.     

Site of synthesis,tissue distribution,and lipophorin transport of hydrocarbons in Blattella germanica (L.) nymphs

The site of hydrocarbon (HC) synthesis and the amount of HC in various tissues were investigated in relation to developmental stage in the last larval stadium of the German cockroach, Blattella germanica. Abdominal integument linearly incorporated [1-(14)C]propionate into HC for at least 6h in vitro, whereas other body parts synthesized little or no HC. The third through sixth abdominal sternites and tergites were the principal sites of synthesis. High rates of HC synthesis resulted in a fivefold increase in internal HC during the last stadium. We examined the distribution of HC in the hemolymph, fat body, and the developing imaginal cuticle. Hemolymph HC titer was relatively constant at approximately 8&mgr;g/&mgr;l. However, as hemolymph volume increased from 5 to 11&mgr;l in the first 4days of the last stadium, HC content increased and then remained stable the remainder of the stadium. Lipophorin, immunoprecipitated with adult lipophorin polyclonal antibodies, was the only HC carrier protein in nymphal hemolymph and its HC profile was identical to that of hemolymph and similar to that of the epicuticle. The concentration and total amount of hemolymph lipophorin increased until 3days before adult eclosion and declined immediately after ecdysis. The HC content of non-biosynthetic integument (legs, pronotum) doubled during formation of the imaginal cuticle, as did the HC content of sternites, which synthesize HC. HC content of fat body, however, increased threefold during the same period, suggesting that the fat body serves as a storage site for HC during cuticle formation. We conclude that in the last stadium HC is synthesized by abdominal oenocytes, loaded onto hemolymph lipophorin, and transported to fat body and both nymphal and imaginal cuticle. Hydrocarbons associate with the imaginal integument several days before eclosion.     

Structural determination of the N-glycans of a lepidopteran arylphorin reveals the presence of a monoglucosylated oligosaccharide in the storage protein

The structures of the oligosaccharides attached to arylphorin from Chinese oak silkworm, Antheraea pernyi, have been determined. Arylphorin, a storage protein present in fifth larval hemolymph, contained 4.8% (w/w) of carbohydrate that was composed of Fuc:GlcNAc:Glc:Man=0.2:4.0:1.4:13.6 moles per mole protein. Four moles of GlcNAc in oligomannose-type oligosaccharides strongly suggest that the protein contains two N-glycosylation sites. Normal-phase HPLC and mass spectrometry oligosaccharide profiles confirmed that arylphorin contained mainly oligomannose-type glycans as well as truncated mannose-type structures with or without fucosylation. Interestingly, the most abundant oligosaccharide was monoglucosylated Man9-GlcNAc2, which was characterized by normal-phase HPLC, mass spectrometry, Aspergillus saitoi alpha-mannosidase digestion, and 1H 600 MHz NMR spectrometry. This glycan structure is not normally present in secreted mammalian glycoproteins; however, it has been identified in avian species. The Glc1Man9GlcNAc2 structure was present only in arylphorin, whereas other hemolymph proteins contained only oligomannose and truncated oligosaccharides. The oligosaccharide was also detected in the arylphorin of another silkworm, Bombyx mori, suggesting a specific function for the Glc1Man9GlcNAc2 glycan. There were no processed glucosylated oligosaccharides such as Glc1Man5-8GlcNAc2. Furthermore, Glc1Man9GlcNAc2 was not released from arylophorin by PNGase F under nondenaturing conditions, suggesting that the N-glycosidic linkage to Asn is protected by the protein. Glc1Man9GlcNAc2 may play a role in the folding of arylphorin or in the assembly of hexamers.     

A function for pericardial cells in an insect

The pericardial cells (PCs) of fifth instar Calpodes ethlius larvae are functionally adapted for filtering hemolymph and sequestering and digesting proteins. They also have a structure appropriate for the synthesis of proteins for secretion. PC secretion has been investigated by labelling the cells with [³⁵S]methionine ti vitro with detection of newly synthesized polypeptides appearing in the medium by electrophoresis and fluorography. Sources possibly contributing to the appearance of newly synthesized polypeptides in the medium, such as cell breakdown and fat body contamination have been ruled out. The post-incubation medium of PCs contains at least six newly synthesized polypeptides. Three of these polypeptides, having relative molecular masses of 82, 57 and 43 kDa, react with antibodies to hemolymph. At least one additional polypeptide is similar by two-dimensional analysis to that naturally present in hemolymph. PCs incubated together with the heart to which they are normally attached, secrete additional polypeptides that are presumed to come from the heart. The 82 kDa polypeptide secreted by the PCs is similar to the subunits of arylphorin secreted by fat body and other tissues. We conclude that PCs secrete proteins into the hemolymph although the amount may be small relative to that of the fat body.     

Adsorptive endocytosis of vitellogenin,lipophorin, and microvitellogenin during yolk formation in Hyalophora

Yolk in Hyalophora cecropia is a mixture of proteins that are derived from the extracellular medium. We have measured for five of these proteins the number of moles deposited in each egg, the molarity of their precursors in the hemolymph at a midpoint in vitellogenesis (day 18 of adult development), and the degree to which they are concentrated by the oocyte, relative to inulin. The proteins were isolated by gel permeation and ion exchange chromatography and used to generate antibodies in rabbits. Preliminary studies established that yolk proteins are essentially quantitatively extractable in media suitable for measuring antigen concentrations by precipitation with antibodies and that yolk and hemolymph forms of the five proteins have, effectively, the same antibody-binding specificities as the isolated standards. Content per egg was about 900 pmol for vitellogenin, 600 pmol for microvitellogenin, and 300 pmol for lipophorin. By contrast, two hemolymph storage hexamers, arylphorin and a flavoprotein, occurred at less than 3 pmol per egg. In principle, yolk precursors are taken in both as solutes in the fluid phase of the endocytotic vesicles and as ligands adsorbed to vesicle membranes. Measurements of inulin uptake indicated that fluid phase endocytosis could account for only 4% of vitellogenin, 1% of microvitellogenin, and 15% of lipophorin in the yolk, when hemolymph precursors are at their day 18 concentrations. By the same comparison, arylphorin and flavoprotein appear to be excluded from the yolk, relative to inulin.     

Growth and mitogenic effects of arylphorin in vivo and in vitro

In insects, developmental responses are organ- and tissue-specific. In previous studies of insect midgut cells in primary tissue cultures, growth-promoting and differentiation factors were identified from the growth media, hemolymph, and fat body. Recently, it was determined that the mitogenic effect of a Manduca sexta fat body extract on midgut stem cells of Heliothis virescens was due to the presence of monomeric alpha-arylphorin. Here we report that in primary midgut cell cultures, this same arylphorin stimulates stem cell proliferation in the lepidopterans M. sexta and Spodoptera littoralis, and in the beetle Leptinotarsa decemlineata. Studies using S. littoralis cells confirm that the mitogenic effect is due to free alpha-arylphorin subunits. In addition, feeding artificial diets containing arylphorin increased the growth rates of several insect species. When tested against continuous cell lines, including some with midgut and fat body origins, arylphorin had no effect; however, a cell line derived from Lymantria dispar fat body grew more rapidly in medium containing a chymotryptic digest of arylphorin.     

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.     

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.     

造血器官机能障碍后家蚕血淋巴中蛋白质成分的变化

为了研究家蚕Bombyx mori造血器官机能障碍后其血淋巴中蛋白质成分的变化,利用重离子射线局部照射家蚕幼虫的造血器官,检测了照射后家蚕血淋巴中的蛋白质成分及注射大肠杆菌后在体内诱导出现的应急蛋白量的变化。结果表明,照射蚕血淋巴中的蛋白质含量与对照蚕之间没有明显的差异。但在成分分析时发现,5龄起蚕血淋巴中70 kD附近的3条蛋白质谱带比对照蚕的浓度要高,随着个体的发育两者的浓度都上升;5龄后期则相反,对照蚕的浓度比照射蚕高;脂肪体中贮藏蛋白质的含量具有相似的变化趋势。用家蚕贮藏蛋白质SP-1及SP-2的抗血清进行免疫印迹反应的结果显示：70 kD附近的3条蛋白质谱带的最上面的一条为贮藏蛋白质SP-1,下面的二条为贮藏蛋白质SP-2;同时照射蚕血淋巴中分子量约为24 kD的蛋白质成分也发生变化,5龄前期的浓度比对照蚕低,5龄第3天几乎检测不到;全体照射与造血器官局部照射蚕之间的结果相似。照射蚕注射大肠杆菌后在体内诱导出现的应急蛋白量明显比对照蚕要少。由此认为家蚕幼虫造血器官与血淋巴中的蛋白质成分有关,造血器官的机能障碍、血球的数量减少可影响脂肪体中蛋白质的合成,从而使存在于血淋巴中的蛋白质成分发生变化。     

Hormonal regulation of phase polymorphism and storage-protein fluctuation in the common cutworm, Spodoptera litura

Three storage proteins are synthesised by Spodoptera litura last-instar larvae as detected by an antiserum against pupal fat body proteins. The putative pupal storage proteins 1 and 2, appear in the haemolymph of the last-instar larvae 36 h after ecdysis under crowded rearing conditions: they appear 1 day later in isolated conditions. The appearance of these proteins in the haemolymph is prevented by juvenile hormone treatment and enhanced by allatectomy. Injection of 20-hydroxyecdysone into ligatured larvae does not induce appearance of these 2 proteins. Accumulation of protein 3 that reacts with Bombyx mori arylphorin antiserum is not blocked by juvenile hormone and is similar in both phases. It also accumulates to a small extent in the haemolymph during the moult to the final-larval instar and then disappears at ecdysis. One-hundred ng/ml ecdysteroid caused the sequestration of these proteins by the fat body, but a higher concentration of ecdysteroid (200 ng/ml) produced pupal cuticle in the isolated abdomens, suggesting that different ecdysteroid concentrations are necessary for these two events.     

Isolation of an imaginal disc growth factor homologue from Pieris rapae and its expression following parasitization by Cotesia rubecula

Endoparasitoid insects introduce maternal factors into the body of their host at oviposition to suppress cellular defences for the protection of the developing parasitoid. We have shown that transient expression of polydnavirus genes from a hymenopteran parasitoid Cotesia rubecula (CrPDV) is responsible for the inactivation of hemocytes from the lepidopteran host Pieris rapae. Since the observed downregulation of CrPDV genes in infected host tissues is not due to cis-regulatory elements at the CrV1 gene locus, we speculated that the termination of CrPDV gene expression may be due to cellular inactivation caused by the CrV1-mediated immune suppression of infected tissues. To test this assumption, we isolated an imaginal disc growth factor (IDGF) that is expressed in fat body and hemocytes, the target of viral infection and expression of CrPDV genes. Time-course experiments showed that the level of P. rapae IDGF is not affected by parasitization and polydnavirus infection. However, the amount of highly expressed genes, such as storage proteins, arylphorin and lipophorin, are significantly reduced following parasitization.     

Developmental changes of storage proteins and biliverdin-binding proteins in the haemolymph and fat body of the common cutworm,Spodoptera litura

The concentrations of three storage proteins (SL-1,SL-2 and SL-3, hexamers of 70-80kDa subunits) and two biliverdin-binding proteins (BP-A and BP-B, dimers of 165kDa) in the haemolymph and fat body during larval and pupal development of Spodoptera litura were determined by immunodiffusion tests using polyclonal antisera. SL-1 and SL-2 (methionine-rich) first appeared in the haemolymph of one-day-old sixth (final) instar larvae, prominently increased in the haemolymph during the later feeding period and were almost totally sequestered by the fat body after gut purge. SL-3 (arylphorin) was first detected in the haemolymph during the molting period to the final larval ecdysis, increased in concentration throughout the entire feeding period of the final larval instar and was partly sequestered by the fat body several hours later than the other storage proteins. BP-A showed nearly the same pattern in the haemolymph as SL-3: BP-B increased during feeding period and decreased during molting period and attained a maximum level during the penultimate larval instar, however its concentration decreased considerably and remained low in the final larval instar. BP-A was partly and BP-B was almost totally sequestered by the fat body 8 h after sequestration of SL-1 and SL-2, rendering the fat body blue in colour. These facts suggest an additional function of biliverdin-binding proteins as amino acid storage proteins and the results show a differential uptake mechanism for these proteins by the fat body.     

Selectivity in storage hexamerin clearing demonstrated with hemolymph transfusions between Hyalophora cecropia and Actias luna.

When Hyalophora cecropia hemolymph was injected into wandering Actias luna larvae, a methionine-rich hexamerin was selectively transferred to the host's fat body, and completely cleared from the hemolymph by the time of pupal eclosion. Donor arylphorin was 30-40% removed from the hemolymph, and riboflavin-binding hexamerin was even less completely cleared. During the pupal-adult molt, these rates were reversed: methionine-rich hexamerin disappeared no faster than bovine serum albumin, while riboflavin-binding hexamerin was rapidly and completely cleared from the hemolymph, even though A. luna hemolymph lacks a homologue of this protein; arylphorin, again, was cleared at an intermediate rate. Selective clearing of the three hexamerins occurred at similar stages in H. cecropia, their species of origin. Developmentally programmed clearing, with selectivity at least partially conserved between genera, was also demonstrated with transfused vitellogenin: in A. luna females that were forming yolk, H. cecropia vitellogenin was cleared more rapidly than bovine serum albumin; but in younger females, and in males at all stages of metamorphosis, this Mr 510,000 molecule was instead an indicator of nonselective, large protein clearing. Nonselective clearing was more complete during adult development than during pupation. It also showed signs of being more effective for small than for large proteins, insensitive to carbohydrate conjugates, and unsaturated at the protein levels used.     

Alteration of hemolymph polypeptides in Manduca sexta larvae parasitized by Cotesia congregata: A two-dimensional electrophoretic analysis and comparison with major bacteria-induced proteins

Analyses using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) previously demonstrated that parasitization by the braconid wasp Cotesia congregata significantly alters the normal hemolymph polypeptide profile of host Manduca sexta larvae. In the present study two-dimensional gel analyses corroborated our earlier findings and provided additional evidence that multiple parasitism-specific polypeptides were induced, which varied according to the stage of development of the wasps. Parasitization additionally elicited changes in the total protein concentration detected in the blood. Initially an elevation was observed, with newly parasitized larvae exhibiting a twofold elevation in hemolymph protein concentration by 12–24 h postoviposition. In contrast, terminal-stage hosts with second instar parasites had significantly less protein in the hemolymph, likely due to reduced growth and inhibition of arylphorin synthesis by the fat body during the final stages of parasitism. Comparison of the array of hemolymph polypeptides produced in unparasitized larvae injected with 10⁶cells of the gram-negative bacterium Enterobacter cloacae with those proteins induced by parasitization indicated the two classes are different. Our findings confirm that the hostresponse to parasitism is a specific one, and not mimicked by bacterial challenge. Duringshort-term in vitro culture of wasp larvae dissected from the host hemocoel, several proteins were detected in the medium using SDS - PAGE, with their appearance in vitro suggestive of secretion by the wasps in vivo. Moreover, hemolymph from the parasites had significant amounts of putative host proteins, including an arylphorin - like polypeptide and a protein with a mobility similar to that of insecticyanin. Thus, a dynamic interchange of proteins may occur, with the parasites accumulating host proteins while simultaneously secreting a variety of factors into the host hemocoel.     

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.     

Arylphorin from Manduca sexta: carbohydrate structure and immunological studies

The major hemolymph protein in the last larval stage of Manduca sexta is a hexameric glycoprotein, arylphorin (Mr = 450,000). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified arylphorin reveals the presence of two subunits, A1 and A2. Both subunits are glycosylated and have apparent Mr = 77,000 and 72,000, respectively. Pronase digestion of arylphorin yielded a single major glycopeptide. 250 MHz NMR spectroscopy of arylphorin glycopeptide revealed a Man9GlcNAc2 oligosaccharide structure similar to that observed in mammalian glycoproteins. Endoglycosidase-H treatment of arylphorin was employed to remove covalently linked carbohydrate residues. The carbohydrate removal lowered the apparent Mr of subunits A1 and A2 to 72,000 and 69,000, respectively, indicating that the difference in arylphorin subunit size is not due to levels of glycosylation. Immunoblotting experiments with anti-arylphorin antiserum and Bombyx mori storage proteins indicated cross reactivity with the corresponding arylphorin of this insect. Preparation of subunit A2 monospecific antibodies, followed by immunoblotting of arylphorin showed a close immunological relationship between subunits A1 and A2.     

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.