Regulation of expression of arylphorin and female-specific protein mRNAs in the tobacco hornworm, Manduca sexta

Two non-cross-hybridizing cDNA clones were isolated from a lambda gt11 cDNA library prepared from Day 2 fifth instar female fat body of Manduca sexta and shown by hybrid selection to code respectively for the two storage proteins arylphorin and female-specific protein (FSP). Analysis of the developmental expression of arylphorin showed its presence during the feeding phases of the penultimate (fourth) and final (fifth) larval instars and its absence during the molt. Abdominal ligation of larvae followed by infusion of Grace's medium showed that this amino acid-rich medium was able to maintain arylphorin expression in fourth instar larvae, but not continued high expression in fifth instar larvae. This nutrient medium however was sufficient to allow initiation of expression in newly ecdysed fifth larval abdomens. Infusion of 5 micrograms 20-hydroxyecdysone (20HE) caused a significant reduction of arylphorin RNA in ligated fourth larval abdomens, whereas 50 micrograms was required in Day 2 fifth larval abdomens to suppress this RNA. Thus, both the lack of incoming nutrients and the rising titer of ecdysteroid contribute to the loss of arylphorin mRNA at the molts and at wandering. By contrast, FSP mRNA was first detected in females on Day 2 of the fifth instar, but not in males until wandering, and then was present throughout the prepupal period. In females allatectomy caused the precocious appearance of FSP mRNA which was prevented by application of 10 micrograms methoprene, a juvenile hormone analog. Expression of FSP mRNA in males however appeared to be independent of hormonal milieu.     

Developmental profiles of the mRNAs for Manduca arylphorin and two other storage proteins during the final larval instar of Manduca sexta

When fat body mRNA from the tobacco hornworm larva, Manduca sexta, was translated in a rabbit reticulocyte lysate system, three major polypeptides were found, each having a different developmental profile. One mRNA coded for a 74 kilodalton (K) polypeptide doublet precipitated by an antibody to the arylphorin (manducin). This mRNA was present only during the intermolt feeding phase of the penultimate and the final larval instars. Its appearance 16–24 hr after larval ecdysis was dependent upon the incoming nutrient supply and independent of the juvenile hormone (JH) level. Immunoblots of proteins of the fat body, epidermis, and cuticle revealed the presence of arylphorin in all three tissues. Additionally, several small polypeptides that cross-reacted with the arylphorin antibody were found in the fat body during and up to 24 hr after the last larval molt and in the tanning pupal cuticle. The larval epidermis was also found to contain a small amount of arylphorin mRNA. At the time of the JH decline prior to the onset of metamorphosis, a female-specific mRNA coding for a 79 K translation product appeared. In allatectomized larvae this mRNA was detectable earlier, and its appearance in intact larvae was prevented by application of methoprene, indicating that JH regulates its appearance. At wandering a new mRNA that also codes for a 79 K polypeptide appeared in both sexes and was the major messenger present during the prepupal stage. Neither it nor the female-specific mRNA were translatable after pupal ecdysis.     

Stage and segment specificity of the secretory cell of the dermal glands of the tobacco hornworm, Manduca sexta

The pair of epidermally derived Verson's glands on each segment of the tobacco hornworm, Manduca sexta, secretes at ecdysis proteinaceous products which coat the epicuticle. These proteins are produced by a single secretory cell which displays both stage- and segment-specificity during development. Three major 12-kDa polypeptides are synthesized at the larval molts, while higher molecular weight (14-93 kDa) polypeptides are produced at the pupal molt. In the pupa, but not in the larva, there are three segment-specific protein patterns, each involving both qualitative and quantitative differences: (1) thoracic (T) segments 1 and 2; (2) T3 and abdominal (A) segment 1; (3) A2-A8. Larval-specific proteins were found to be synthesized in low amounts throughout the penultimate fourth instar, with enhanced synthesis occurring during the molt, coincident with the molting surge of ecdysteroids. Synthesis of the major pupal products commenced about the time of wandering, with enhanced synthesis occurring throughout prepupal development, coincident with the prepupal surge in ecdysteroids. The onset of synthesis of the major pupal products differed, both within and between segments. Culture of fifth instar Day 2 glands in vitro showed that this synthesis depended on 20-hydroxyecdysone. The differential regulation within and between segments observed in vivo was also seen in vitro.     

Changes in the chemical composition of fat body during the last larval instar of Manduca sexta.

The fat body increases in weight throughout the last larval instar in spite of the loss in total body weight during the wandering and prepupal stages. The protein content of fat body increases dramatically and is greatly responsible for the increase in fat body weight in the wandering and prepupal stages. Lipids do not contribute significantly to the fat body weight gain except during the feeding stage. The amount of total fat body RNA increases until wandering then drops abruptly in the prepupal stage. The total amount of fat body DNA peaks before the onset of wandering and prepupal stages.     

Developmental expression of three genes for larval cuticular proteins of the tobacco hornworm, Manduca sexta

Three cDNA clones coding for the 12.8, 13.3, and 14.6 kDa larval cuticular proteins of the tobacco hornworm, Manduca sexta, were isolated and characterized. Hybridization to abdominal epidermal RNA from different stages showed that the genes for the 12.8 and 13.3 kDa proteins were expressed only during larval life. By contrast, the gene for the 14.6 kDa protein was expressed throughout the segment during the feeding, growing larval stages, then only in the flexible intersegmental regions during the deposition of endocuticle in the pharate pupa and adult. Quantitative RNA dot blot hybridizations showed that the RNA for each protein disappeared during the larval molt when the ecdysteroid titer was high, then reappeared during the preecdysial deposition of endocuticle. All disappeared when the epidermis became pupally committed at the onset of wandering. Exposure of the fourth instar epidermis to 20-hydroxyecdysone (20HE) in vitro under conditions that lead to the formation of a new larval cuticle by 48 hr caused the disappearance of these RNAs by 18 hr. Exposure of Day 2 fifth instar epidermis to 20HE in vitro caused a depression of these RNAs which in the case of the RNAs coding for the 12.8 and 13.3 kDa proteins was partially prevented by simultaneous exposure to methoprene, a juvenile hormone (JH) mimic. By contrast, the RNA for the 14.6 kDa protein was suppressed by exposure to methoprene alone. Thus, each of these larval cuticular genes is turned off by high ecdysteroid; the presence or absence of JH determines whether or not this suppression is permanent in some or all cells.     

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).     

The role of juvenile hormone in endocrine control of pigmentation in Manduca sexta

Spatial and temporal distribution of insecticyanin was studied in the fourth and fifth larval instars of Manduca sexta. The protein was distributed between the epidermis (62%), the hemolymph (37%) and the pericardial cells (0.5%). Hemolymph insecticyanin (HINS) was highest (0.6 mg/ml) in the very early fourth instar, gradually declining to 0.3 mg/ml. Levels in the fifth instar decreased after ecdysis (0.15 mg/ml), began to rise at wandering, and nearly doubled by the time of pupation. Titers of epidermal insecticyanin (EINS) followed the general growth patterns during the fourth and early fifth instar. At 76 hr after fifth instar ecdysis, titers of EINS dropped precipitously and then rose again to peak just after the wandering stage. Levels of EINS again rapidly declined and could not be detected after 180 hr. Ecdysteroids appear to shut off synthesis of EINS but this response is quantitatively modified in the presence of JH. Endocrine manipulation of the last larval-larval molt indicated that juvenile hormone (JH) acts quantitatively on EINS to induce a dose-dependent increase. The JH-induced increase can be as much as 4-fold, depending upon the body region.     

20-Hydroxyecdysone mediated activation of larval haemolymph protein uptake by fat body cells of Corcyra cephalonica (Insecta)

Evidence is presented here to show that 20-hydroxyecdysone is essential for the activation of the larval fat body for differential uptake of larval haemolymph proteins (LHPs). By using radiolabelled LHPs it is shown that the fat body cells of Corcyra cephalonica selectively incorporate LHPs during late-larval and prepupal development. Fluorographic analysis of the labelled fat body proteins from prepupal stage separated on sodium dodecyl-sulphate polyacrylamide gels suggests that the LHPs are sequestered without any degradation. Although, during the last larval instar the uptake of all the three LHPs (LHP 1, LHP 2 and LHP 3) by the fat body cells is very low, 20-hydroxyecdysone treatment of early, mid or late-last instars causes a significant increase in uptake of all the three LHPs. However, the response to hormone treatment was more pronounced in late-last instar when compared to early and mid-last instar.     

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.     

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.     

Regulation of dopa decarboxylase gene expression in the larval epidermis of the tobacco hornworm by 20-hydroxyecdysone and juvenile hormone

Dopa decarboxylase (DDC) which converts dopa to dopamine is important for cuticular melanization and sclerotization in insects. An antibody to Drosophila DDC was found to precipitate both DDC activity and a 49-kDa polypeptide synthesized by the epidermis of molting Manduca larvae. Using the Drosophila DDC gene, we isolated the Manduca DDC gene which on hybrid selection produced a 49-kDa translation product precipitable by the Drosophila DDC antibody. The 3.1-kb DDC mRNA appeared 12 hr after head capsule slippage (HCS) and reached maximal levels 7 hr later. Peak expression was twofold higher in melanizing allatectomized larvae and could be depressed to normal levels by application of 0.1 micrograms juvenile hormone I at HCS. Infusion of 1 microgram/hr 20-hydroxyecdysone (20-HE) for 18 hr beginning 2 hr after HCS or addition of 1 microgram/ml 20-HE to the culture medium for 24 hr prevented the normal increase in DDC mRNA. When Day 2 fourth instar epidermis was explanted before the molting ecdysteroid rise and cultured with 1-3 micrograms/ml 20-HE for 17 hr and then for 24 hr in hormone-free medium, DDC expression was three- to fourfold higher than that in epidermis cultured in the absence of hormone. Twelve or more hours of incubation with 20-HE was required for an increase in DDC mRNA, but continuous exposure to 20-HE prevented the increase. In all cultures an initial rapid increase in DDC mRNA was observed which decayed with time in vitro and apparently was associated with the wound response. Thus, ecdysteroid during a larval molt is necessary to program the later expression of DDC, but the subsequent decline of the ecdysteroid is required for this expression to occur.     

Biochemical and ontogenetic aspects of glycoprotein synthesis in Drosophila virilis salivary glands

After SDS-polyacrylamide gel electrophoresis two glycosylated glue proteins are found in the salivary glands of Drosophila virilis late third instar larvae. Synthesis of larval glue protein 1 occurs in three successive steps: at first a precursor protein with a molecular weight of about 138,000 daltons is formed. This is modified by two subsequent steps of glycosylation, the first one involving hexosamine, the second one hexoses. Studies with tunicamycin and β-hydroxynorvaline suggest that glycosylation occurs at threonine residues. Larval glue protein 2 has a molecular weight of approximately 15,000 daltons and is weakly glycosylated. The synthesis of glue proteins is stage specific. It starts at about 120 hr after oviposition and attains its maximal rate about 20 hr later. At this time the larvae leave the food. Between ecdysone release and puparium formation (146–151 hr) larval glue protein synthesis is terminated. Throughout the prepupal stage a different set of glycoproteins is synthesized. Thus, the larval-prepupal transition is accompanied by the reprogramming of glycoprotein synthesis in salivary glands. The secretion products formed during the two developmental stages seem to possess different biological functions.     

Different pathways of DNA synthesis are operative in the fat body of Spodoptera litura (insecta) during larval and pupal development

In Spodoptera litura, the DNA content of the fat body increases during the last larval instar. Radiolabelling studies reveal high degree of H3-thymidine incorporation in the fat cell DNA, during last larval stage of development. At prepupal stage the DNA synthesised during larval development is degraded, at least partially and the resultant nucleotides are most likely used as building blocks for the fat cell DNA synthesis during pupal development and pupal-adult metamorphosis, because very little H3 -thymidine injected into pupae is incorporated in DNA, though in fact, significant amount of DNA is synthesised during this period.     

DNA replication events during larval silk gland development in the silkworm, Bombyx mori

The silk gland is an important organ in silkworm as it synthesizes silk proteins and is critical to spinning. The genomic DNA content of silk gland cells dramatically increases 200-400 thousand times for the larval life span through the process of endomitosis. Using in vitro culture, DNA synthesis was measured using BrdU labeling during the larval molt and intermolt periods. We found that the cell cycle of endomitosis was activated during the intermolt and was inhibited during the molt phase. The anterior silk gland, middle silk gland, and posterior silk gland cells asynchronously exit the endomitotic cycle after day 6 in 5th instar larvae, which correlated with the reduced expression of the cell cycle-related cdt1, pcna, cyclin E, cdk2 and cdk1 mRNAs in the wandering phase. Additional starvation had no effect on the initiation of silk gland DNA synthesis of the freshly ecdysed larvae.     

Protein degradation in silkworm peritracheal athrocytes and its physiological role in metamorphosis

The major functions of silkworm peritracheal athrocytes (nephrocytes) include endocytosis. Although athrocytes are also believed to function in protein degradation, there is limited experimental evidence for this. In this study, we detected the uptake and degradation of foreign proteins in peritracheal athrocytes by immunohistochemical, Western blot, and ex vivo analyses. IgG-FITC was detected in the athrocytes of silkworm larvae following injection, and LysoTracker analysis showed endosomal and lysosomal colocalizations. Athrocytes from larvae injected with IgG were incubated in Grace's medium for 2 days before being analyzed for the degradation of IgG by Western blotting. The level of incorporated IgG decreased and degradation products appeared following ex vivo culture. The highest level of IgG incorporation and degradation in the athrocytes was observed at the early pupal stage. The athrocytes also incorporated arylphorin, a major larval haemolymph protein and storage protein in silkworms. At the early pupal stage, arylphorin was actively degraded in the athrocytes. These results indicate that, in cooperation with the fat body, peritracheal athrocytes may function in the digestion of arylphorin during silkworm metamorphosis.