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.     

Clathrin‐dependent endocytosis predominantly mediates protein absorption by fat body from the hemolymph in Bombyx mori

During insect larval–pupal metamorphosis, proteins in the hemolymph are absorbed by the fat body for the maintenance of intracellular homeostasis; however, the type of proteins and how these proteins are internalized into the fat body are unclear. In Bombyx mori, the developmental profiles of total proteins in the hemolymph and fat body showed that hemolymph‐decreased protein bands (55–100 kDa) were in accordance with those protein bands that increased in the fat body. Inhibition of clathrin‐dependent endocytosis predominantly blocked the transportation of 55–100 kDa proteins from the hemolymph into the fat body, which was further verified by RNA interference treatment of Bmclathrin. Six hexamerins were shown to comprise ～90% of the total identified proteins in both the hemolymph and fat body by mass spectrum (MS) analysis. In addition, hemolymph‐specific proteins were mainly involved in material transportation, while fat body‐specific proteins particularly participated in metabolism. In this paper, four hexamerins were found for the first time, and potential proteins absorbed by the fat body from the hemolymph through clathrin‐dependent endocytosis were identified. This study sheds light on the protein absorption mechanism during insect metamorphosis.     

Tyrosine and phenylalanine concentrations in haemolymph and tissues of the American cockroach, Periplaneta americana, during metamorphosis

Phenylalanine and tyrosine concentrations were measured in the haemolymph, fat body, and abdominal integument of the American cockroach, Periplaneta americana, during the pre- and post-ecdysial periods of cuticle formation and sclerotization.Gas-liquid chromatography of trimethylsilyl derivatives of phenylalanine, tyrosine, and their metabolites provided a very sensitive and rapid method for determining those amino acids in small haemolymph and tissue samples.Haemolymph tyrosine increased in two stages: initially near apolysis and 16 to 25 hr pre-ecdysis, reaching its highest concentration at ecdysis (3·5 μg tyrosine/mg haemolymph). During that time, total haemolymph tyrosine increased by approximately 700 μg/insect. Fat body and abdominal integument began to accumulate tyrosine near apolysis. Fat body tyrosine peaked between ecdysis and 3·3 hr post-ecdysis whereas abdominal integument tyrosine peaked at ecdysis. Maximum concentrations were 6·0 μg and 4·1 μg tyrosine/mg wet wt. of tissue, respectively. Between ecdysis and 24 hr post-ecdysis, the period of maximum sclerotization, total tyrosine in haemolymph and fat body decreased by approximately 600 μg and 420 μg/insect, respectively. Phenylalanine concentrations did not change significantly in the haemolymph, fat body, or abdominal integument during the pre- and post-ecdysial periods.The cockroach apparently does not store free phenylalanine or tyrosine in the fat body during larval development as compared to tyrosine storage in some Diptera. The rapid increase of haemolymph, fat body, and integument tyrosine just prior to ecdysis suggests another form of storage for this important amino acid.     

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.     

Carbohydrate metabolism in starved fifth instar larvae of Manduca sexta

The influence of starvation on carbohydrate metabolism in fifth instar larvae of Manduca sexta was studied. The percentage of active fat body glycogen phosphorylase increased from 10% to approximately 50% within 3 h of starvation; afterward the enzyme was slowly inactivated. The increase of phosphorylase activity might have been caused by a peptide(s) from the CC. The amount of fat body glycogen in starved animals decreased over 24 h by approximately 20 mg. The released glucose molecules seem to be converted mainly to trehalose because the hemolymph trehalose concentration in starved animals was always slightly higher than in the fed controls, and the glucose concentration decreased even when phosphorylase was activated. The chitosan content in starved larvae increased during the first 9 h of treatment to the same extent as in fed controls. It is suggested that fat body glycogen phosphorylase was activated during starvation to provide substrates for chitin synthesis and energy metabolism.     

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.     

Carbohydrate metabolism during starvation in the silkworm Bombyx mori

The effect of starvation on carbohydrate metabolism in the last instar larvae of the silkworm Bombyx mori was examined. Trehalose concentration in the hemolymph increased slightly during the first 6 h of starvation and decreased thereafter, whereas glucose concentration decreased rapidly immediately after diet deprivation. Starvation-induced hypertrehalosemia was completely inhibited by neck ligation, suggesting that starvation stimulates the release of a hypertrehalosemic factor(s) from the head. The percentage of active glycogen phosphorylase in the fat body increased within 3 h of starvation and its glycogen content decreased gradually. These observations suggest that production of trehalose from glycogen is enhanced in starved larvae. However, hypertrehalosemia during starvation cannot be explained by the increased supply of trehalose into hemolymph alone, as similar changes in phosphorylase activity and glycogen content in the fat body were observed in neck-ligated larvae, in which hemolymph trehalose concentration did not increase but decreased gradually. When injected into larvae, trehalose disappeared from hemolymph at a rate about 40% lower in starved larvae than neck-ligated larvae. The hemolymph lipid concentration increased during starvation, suggesting that an increased supply of lipids to tissues suppresses the consumption of hemolymph trehalose and this is an important factor in hypertrehalosemia.     

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.     

Absence of female-specific protein in the hemolymph of stable fly Stomoxys calcitrans (L.) (Diptera: Muscidae)

Changes, during the reproductive cycle, in fat body, hemolymph, and ovarian proteins of the stable fly Stomoxys calcitrans were characterized quantitatively and qualitatively using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein content of all three tissues increased after blood feeding. Fat body protein increased first, followed by hemolymph and ovarian proteins. SDS-PAGE failed to identify vitellogenin in both female hemolymph and fat body samples. No single protein or group of proteins predominated at any stage of the reproductive cycle. Comparisons between male and female stable fly hemolymph and fat body proteins failed to detect female-specific proteins. Female-specific proteins, however, were detected in the hemolymph of four other species of Diptera.     

Transport of lipids in insects

Many insect species are almost completely dependent on lipids for their metabolic needs, although this is usually a function of developmental stage. The primary storage organ is the fat body, which can constitute 50% of the fresh weight of the insect and also acts as the major metabolic center (analogous to the vertebrate adipose tissue and liver). Bathing the fat body (and all other tissues and organs) is the hemolymph, the main functions of which are to transport nutrient substrates to utilization sites and to deliver metabolic wastes to the excretory system. Although neutral lipids are stored as triglycerides, in times of need they appear to be endergonically released into the hemolymph as diglycerides in the majority of insects thus far studied (particularly silkmoths and locusts). Indeed, diglycerides constitute the largest neutral lipid fraction in the hemolymph of silkmoths, locusts, cockroaches, bugs, etc. In the hemolymph the diglyceride is found as a constituent of specific lipoproteins, and one specific lipoprotein class (lipoprotein I; high density lipoprotein) appears to be necessary for the transport of diglyceride from the fat body cell into the hemolymph. This particular lipoprotein is also involved in the transport of cholesterol from the gut into the hemolymph. Thus, lipoprotein I appears to be the major neutral lipid and sterol transport agent in the insects studied and, in addition, plays a regulatory role in the release of both diglycerides and sterols. Hemolymph lipoprotein II (very high density lipoprotein) may be important in providing protein and lipid to the insect ovary during oogenesis. Ecdysone, the polyhydroxy steroidal insect molting hormone, is probably carried "free" in the hemolymph, although reports exist of specific hemolymph-binding proteins in some species. The other major insect growth hormone, juvenile hormone, is transported by hemolymph lipoproteins in silkmoths and locusts and by a lower molecular weight hemolymph protein in the tobacco hornworm.     

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.     

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.     

The temporal pattern of vitellogenin synthesis in Drosophila grimshawi

The temporal pattern of protein production and, in particular, vitellogenin protein synthesis during the sexual maturation of Drosophila grimshawi females has been studied in vivo by briefly feeding the flies with 35S-methionine and 3H-amino acids. The overall level of incorporation was very low in young flies; it then progressively increased to reach a maximum with the onset of sexual maturity at 13-15 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses revealed three classes of proteins: those synthesized throughout the age spectrum, which constitute the majority of protein species; proteins synthesized primarily or only in young flies; and proteins synthesized only by the older flies. In this Drosophila species, the three vitellogenins (V1, V2, and V3) appeared to be synthesized in a two-phase pattern. In the first phase, small quantities of V1 and V2 were detected immunologically in the fat body and hemolymph of newly emerged and 1 day-old flies. These proteins did not accumulate in the hemolymph or the ovaries, apparently being unstable proteins. The second phase commenced in early vitellogenesis (7-9 days of age) with synthesis in the fat body of small quantities of V1 and V2, followed by V3 proteins. These proteins were secreted and accumulated in the hemolymph and 24 h later were found in the ovaries. Their quantities increased rapidly and a steady state of synthesis, release into the hemolymph, and uptake by the ovaries was reached by days 13-15. We have estimated that during the steady state of vitellogenin synthesis, a fly can synthesize in 24 h at least 152 micrograms of vitellogenins, which is more than 2% of its body weight, at an average rate of about 6.3 micrograms vitellogenins/h. About 2 micrograms of this are synthesized in the fat body, and about 4 micrograms in the ovaries. These findings are discussed in terms of their physiological implications and contrasted with the available data on Drosophila melanogaster.     

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。     

Effects of long-term starvation on body weight and body composition of juvenile channel catfish, Ictalurus punctatus, with special emphasis on amino acid and fatty acid changes

Triplicate groups of 30 channel catfish Ictalurus punctatus (initial weight: 76.13 ± 0.78 g) were stocked in indoor flow‐through fiberglass tanks and starved for 80 days. Body weight, morphometric parameters, body composition, amino acid and fatty acid changes in muscle and liver tissues were investigated to determine the effect of long‐term starvation on body weight and body composition of juvenile channel catfish. During the starvation period, body weight, condition factor (CF), viscerosomatic index (VSI), hepatosomatic index (HSI) and intraperitoneal fat ratio (IPR) declined (P < 0.05). In the whole body, both protein and lipid decreased while changes in the amount of fat were relatively rapid. Hepatic lipid and carbohydrate contents declined as starvation progressed, but crude protein and moisture contents increased (P < 0.05). In contrast, muscle crude protein showed a greater decline than did muscle lipids, and muscle glycogen remained relatively constant. During the 80‐day starvation period the ratio of total essential amino acids (EAA) to total non‐essential amino acids (NEAA) in muscle and liver (P < 0.05) increased. In muscle tissue, total mono‐unsaturated fatty acids (MUFA) and n‐6 fatty acids decreased, but total saturated fatty acids (SFA) and n‐3 fatty acids, as well as the ratio of n‐3 to n‐6 fatty acids increased (P < 0.05). However, in the liver, starvation resulted in the relative increase of total MUFA and reduction in n‐3 fatty acid contents as well as the ratio of n‐3 to n‐6 fatty acids (P < 0.05). Based on these observations, lipids and glycogen can be considered as more important sources of catabolizable energy in liver, whereas protein might be preferentially mobilized in muscle; lipids played a more important role as energy reserves on a relative basis in the whole body. Channel catfish preferentially utilized NEAA to EAA as an energy substrate and preferentially reserved EAA during starvation. Mobilization of fatty acids showed more variation in the muscle and liver during starvation.     

N-β-Alanyldopamine levels and synthesis in integument and other tissues of Manduca sexta (L.) during the larval-pupal transformation

N-β-Alanyldopamine (NBAD) and other diphenols in tissues of the fifth larval instar of the tobacco hornworm, Manduca sexta (L.), were analyzed by HPLC with electrochemical detection. NBAD accumulated in the integument during the intermolt feeding period, with maximal levels in the wandering stage (6 mmol/g). It then declined to a low level during apolysis and endocuticle digestion, while hemolymph NBAD increased during the same interval to a peak concentration (3 mM) shortly before pupal ecdysis. Trachea and foregut contained lesser amounts of NBAD (0.5 mmol/g), perhaps associated with cuticle, whereas fat body, muscle, midgut and hindgut had 0.1 mmol/g or less. Dopamine (DA), N-acetyldopamine and 3,4-dihydroxyphenylalanine (DOPA) were at least 10-fold less abundant than NBAD in the integument. NBAD synthase, which catalyzes the formation of NBAD from DA and β-alanine, was assayed in both integument and fat body. Highest activity was detected in the integument, where two peaks were observed, one at day 3 near the end of larval feeding and the other at day 9 as pupal cuticle tanning was initiated. Fat body enzyme was substantially less and was detected only in the pharate pupa. Maximal NBAD synthesis by integument cultured in vitro was dependent upon DA supplementation of at least 1.4 mM. 20-Hydroxyecdysone did not alter NBAD synthesis in vitro in either the integument or the fat body, even though injection of this hormone into isolated larval abdomens induced synthesis and/or transport of integumental NBAD back into the hemolymph. The rate-limiting steps in the NBAD biosynthetic pathway appear to be the production of DOPA and DA, because β-alanine occurs in the hemolymph at relatively high levels throughout larval-pupal development.     

Preferential loss of body fat during starvation in dietary obese rats.

This study was undertaken to examine whether diet-induced obesity alters the amount and/or composition of weight lost during starvation. The amount and composition of weight lost during a 4-day period of starvation was determined before and at 17, 30 and 42 weeks after rats (350 g of body weight) were given a high fat diet (HFD). To control for effects of aging, a second group of rats, fed standard laboratory chow, was also subjected to similar periods of starvation. Although total weight loss during starvation was never greater for HFD rats than for chow-fed rats, the former group showed a clear patter of increasing loss of body fat and total energy and conservation of fat-free tissues with periods of starvation later in life. In addition, chow-fed rats showed substantial energy conservation during each period of starvation (i.e. they lost less energy each day than their pre-starvation energy requirements). In contrast, HFD rats demonstrated substantial energy conservation only at 17 weeks and not at 30 or 42 weeks; during the last period of starvation, their average daily loss of carcass energy exceeded their pre-starvation energy requirements. This suggests the increased fat mass of these rats may have led to increased fuel availability and to an increased metabolic rate during starvation. If these results are applicable to humans, the more obese subjects are likely to show greater total loss of energy than lean subjects, but show a lesser loss of lean body mass, at least initially. If protein requirements are reflected by the ability to mobilize protein during food restriction, protein requirements would be substantially lower in the dietary obese rats than in controls. In summary, diet-induced obesity leads to preferential loss of body fat and conservation of lean mass during starvation.     

Regulation of synthesis of larval serum proteins after transplantation of larval fat body into adult Drosophila melanogaster

The larval serum proteins, LSP1 and LSP2, of Drosophila melanogaster are synthesized by the fat body during the third instar. We examined the potential for LSP synthesis by fat body implants in adult flies. Fat body from third instar donors will continue to synthesize LSPs in both males and females. Implants from late second instar larvae will start synthesizing LSP1 and LSP2 in females but only LSP1 in males, suggesting that regulation of these proteins is not the same and that the physiological milieu in the two sexes differs. The newly synthesized LSPs are secreted into the hemolymph for approximately 48 hr when secretion stops but synthesis continues. This sequence follows the pattern for LSP secretion in situ. Fat body from mid second instar larvae is variable in its ability to synthesize LSPs. LSPs are not detected in implants from first instar larvae despite there being a high level of protein synthesis in the implant and considerable growth of the fat body cells. We conclude that there is a critical stage of differentiation during the latter half of the second instar when the fat body becomes independent of the larval milieu and can synthesize LSPs in the adult.