Crystal structure of Bombyx mori arylphorins reveals a 3:3 heterohexamer with multiple papain cleavage sites

In holometabolous insects, the accumulation and utilization of storage proteins (SPs), including arylphorins and methionine‐rich proteins, are critical for the insect metamorphosis. SPs function as amino acids reserves, which are synthesized in fat body, secreted into the larval hemolymph and taken up by fat body shortly before pupation. However, the detailed molecular mechanisms of digestion and utilization of SPs during development are largely unknown. Here, we report the crystal structure of Bombyx mori arylphorins at 2.8 Å, which displays a heterohexameric structural arrangement formed by trimerization of dimers comprising two structural similar arylphorins. Our limited proteolysis assay and microarray data strongly suggest that papain‐like proteases are the major players for B. mori arylphorins digestion in vitro and in vivo. Consistent with the biochemical data, dozens of papain cleavage sites are mapped on the surface of the heterohexameric structure of B. mori arylphorins. Hence, our results provide the insightful information to understand the metamorphosis of holometabolous insects at molecular level.     

Location of the LSP-1 Genes in Drosophila Species by IN SITU Hybridization

The locations of the larval serum protein one (LSP-1) α, β and γ genes were determined in Drosophila melanogaster and in 14 other species of Drosophila by in situ hybridization to polytene chromosomes. The LSP-1 α gene mapped to bands 11B on the X chromosome, the LSP-1 β gene mapped to bands 21D-E on chromosome 2L, and the LSP-1 γ gene mapped to band 61A in all the melanogaster subgroup species. In eight other species, both the LSP-1 α and β genes mapped to one site on Muller's element E which corresponds to chromosome 3R of D. melanogaster. No hybridization of LSP-1 γ was detected in these eight species. Restriction enzyme digestion and analysis of genomic DNA by filter transfer hybridization confirmed the presence of LSP-1 α-like and β-like genes in seven of these species. These results are discussed with respect to conservation of the chromosomal elements in the genus Drosophila.     

Histochemical analysis of the ecdysterone-regulated expression of the Drosophila genes P1 and LSP-2

3H-labeled DNA probes for the ecdysterone-inducible Drosophila genes P1 and LSP-2 were hybridized in situ to RNA in sections of embryos and larvae. Intense hybridization was detected specifically in fat body cells of third-instar larvae and not in other cells of third-instar larvae nor in any cells at earlier stages. These results confirm the stringent tissue- and stage-specificity of P1 and LSP-2 expression. Hybridization of both probes occurred to virtually all the cells in the fat bodies, indicating that both genes are expressed in the same cells. Since P1 expression begins several hours later than LSP-2 expression, and appears to be induced directly by ecdysterone, this finding implies that one or more of the fat body components mediating the response to ecdysterone is gene-specific.     

Detection of haemocyte proteins in the integument of the developing Mediterranean fruit flyCeratitis capitata

Summary Studies of the synthesis of integumental proteins during the feeding and non-feeding stages ofCeratitis capitata demonstrated stage specificity. The synthetic profile changed dramatically, showing a maximum of protein synthesis just before the larval wandering stage, followed by an abrupt decline. The comparison between synthetic and accumulation profiles indicated that some polypeptides must be internalized into the integument from the haemolymph. The major haemolymph proteins or arylphorins have already been documented to be incorporated into the integument. In the present work, we demonstrated the interalization of some haemocyte proteins into the integument. For that purpose, polyclonal antibodies were raised against total haemocyte proteins. Immunoblot analysis of haemocyte salt extractable proteins revealed that the protein bands at 36, 54, 58, 84, 110 and 130 kDa were immunoreactive with the total haemocyte antibodies. Cell-free protein synthesis, organ culture experiments and immunoblot analysis indicated that the 36-, 54- and 58-kDa polypeptides were synthesized only in the haemocytes and were probably internalized into the integument from the serum. The 36-kDa polypeptide was also demonstrated to be internalized into the fat body of white puparia. The immunofluorescence experiments suggested that the internalization of haemocyte proteins first occurs into the epidermal cells and then into the cuticle. The presence of haemocyte proteins in the integument was also demonstrated by immunofluorescence experiments in twoC. capitata mutants. These mutations affect the darkening and stiffening of the cuticle. The demonstration of 36-, 54- and 58-kDa haemocyte polypeptides in the integument reveals a hitherto unknown function of this cell type. Moreover, the demonstration of tyrosine binding to the 54- and 58-kDa polypeptides points to their potential involvement in the sclerotization process in the cuticle.     

Regulation of synthesis of the larval serum proteins of Drosophila melanogaster

We have determined the relative amounts of subunits of larval serum proteins (LSPs) 1 and 2 during larval development in Drosophila melanogaster. These results indicate that synthesis of polypeptide subunits of LSP-1 and LSP-2 is coordinate: the proteins are first detected at the same time; they accumulate in a coordinate fashion; their RNAs are first detected at the same time; the RNAs also accumulate in similar relative amounts. Analyses of fat body polypeptides and fat body RNA indicate that synthesis of LSP-1 declines at a time when there are still substantial quantities of LSP-1 RNA in the cytoplasm. Cessation of LSP-1 subunit synthesis occurs before cessation of LSP-2 synthesis, indicating that at late times the genes (or mRNAs) for these two proteins are subject to different "switch-off" controls.     

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.     

Quantitative analysis of the amount of larval serum protein-1 (LSP-1) synthesized by flies with different doses of the LSP-1 coding sequences

Larval serum protein-1 (LSP-1) and LSP-2 are the major proteins of Drosophila larval serum. The amount of LSP-1 synthesized is strictly proportional to the number of LSP-1 genes present within the range 1-10. The normal number in female flies is 6. Flies with extreme amounts of LSP-1 were, by our criteria, as fit as the wild type. The ratio of LSP-2:LSP-1 was analyzed in 169 different stocks and was constant in 164 of these. The significantly different ratios in five stocks were all due to the lack of one of the LSP-1 gene products.     

Protein phosphorylation in the larval epidermis of Locusta migratoria: Role of cyclic AMP and 20-hydroxyecdysone

Cyclic nucleotide-dependent and cyclic nucleotide-independent protein kinase (PK) activities were quantified in the wing-pad epidermis of Locusta migratoria during the fifth larval instar. A low level of cyclic nucleotide-independent PK activity was detected in this tissue, whereas no PK-C (Ca²⁺- and phospholipid-dependent enzyme) activity was found. The main cyclic nucleotide-PK activity was cAMP-PK and large changes were observed during the intermoult. Concomitant increases in cAMP-PK activity and cAMP-dependent phosphorylation of several endogenous polypeptide substrates occurred during the last part of the intermoult cycle. The most marked ³²P-incorporation was observed in a 43 kDa polypeptide. These changes could be correlated with the peak of 20-OH-ecdysone in both hemolymph and integument.In vitro incubation of wing-pads with 20-OH-ecdysone (48 h) did not change cAMP-PK activity, but there were major changes in the pattern of endogenous phosphoproteins in particular the appearance of 43 and 45 kDa phosphoproteins. Two-dimensional analysis revealed several groups of phosphorylated spots. The results indicate a close relationship between certain cAMP-dependent phosphorylated polypeptides and the action of 20-OH-ecdysone.     

Identification and molecular analysis of a multigene family encoding calliphorin, the major larval serum protein of Calliphora vicina

A library of Calliphora vicina genomic DNA was constructed in the λEMBL3 vector and screened for recombinant phages containing chromosomal segments encoding calliphorin, the major larval serum protein (LSP) of Calliphora. A large series of recombinants hybridizing with in vitro labelled poly(A)⁺ RNA from Calliphora larval fat bodies and with specific probes derived from the LSP-1 genes of Drosophila melanogaster was isolated. Five of these phages, chosen at random, were shown by hybrid selection to retain calliphorin mRNA specifically. Eleven calliphorin mRNA-homologous regions were located on restriction maps of these phages by hybridization with 5' end-labelled poly(A)⁺ RNA from Calliphora larval fat bodies. Each phage contains at least two calliphorin genes arranged in direct repeat orientation and seperated by 3.5–5 kb intergenic regions. The genes display similar but not identical restriction patterns. Filter hybridization and heteroduplex analysis indicate that they share a detectable homology with the LSP-1β gene of D. melanogaster. Whole genome Southern analysis showed that these genes belong to a large family of closely related calliphorin genes which were found by in situ hybridization to polytene chromosomes of trichogen cells to be clustered in region 4a of chromosome 2 of Calliphora vicina.     

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.     

Fat body stimulation of ecdysone synthesis in Heliothis zea

Prothoracic glands of Heliothis zea pupae require both a humoral factor and prothoracicotropic hormone (PTTH) to synthesize ecdysone. The humoral factor is absent when pupae are maintained at diapause-sustaining temperatures. Thus, pupae remain in diapause despite the release of PTTH at or before larval-pupal ecdysis.Tissue implantation experiments revealed that a diapause-terminating factor is present in the fat body of non-diapausing pupae. Other tissue implantation experiments showed that, when diapausing pupae were transferred from 19 to 27°C, diapause-terminating activity appeared first in the fat body and then the fat body into the haemolymph. HPLC separation of the haemolymph and fat body fractions followed by bioassay demonstrated that fractions containing diapause-terminating activity eluted from both tissues within 28–30 min. These results suggest that the factors found in the fat body and haemolymph may be the same compound.Evidence from ecdysone radioimmunoassay experiments ruled out the possibility that the diapauseterminating activity was due to either free or conjugated ecdysteroids. Corresponding in vitro experiments in which the prothoracic glands were cultured with brain extracts versus fat body and haemolymph fractions also indicated that the haemolymph/fat body factor was not PTTH.     

Interactions between beta-ecdysone and fat body during wing disk development in vitro

We have investigated the actions of beta-ecdysone and fat body on wing disks of Plodia interpunctella in a series of sequential incubations in vitro. These experiments revealed that extended treatment times of beta-ecdysone at concentrations of 0·5 μg/ml or greater inhibited development of disks, and confirm that the presence of a fat body factor in the culture medium prevents this inhibition.     

The vitellogenin of Leucophaea maderae: Synthesis as a large phosphorylated precursor

Phosphorylation of vitellogenin (yolk protein precursor) and vitellin (major yolk protein) polypeptides of Leucophaea maderae was studied by [³²P]ortho phosphate labeling and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography. The vitellogenin molecule was isolated from the hemolymph and fat body by antibody precipitation and high-performance liquid chromatography (HPLC), and shown to consist of at least five polypeptides (“subunits”) which had apparent molecular masses of 155, 112, 95, 92 and 54 kD. Labeling studies with ³²P showed that the covalently attached phosphorus was distributed in an uneven fashion among the five polypeptides. The two heavily-phosphorylated polypeptides, 112 and 54 kD, corresponded to the large and small, mature vitellin subunits. Quantitative SDS-PAGE analysis of long-term ³²P-labeled vitellin showed that these large and small “subunits” contained 55 and 30%, respectively, of the total radioactivity.When fat body was pulse-labeled with ³²P we found a heavily-phosphorylated intracellular 215 kD polypeptide which was precipitable with anti-vitellogenin. The synthesis of this intracellular precursorform of vitellogenin (pre-Vg) was under absolute juvenile hormone control. In vitro³²P pulse-chase experiments showed that pre-Vg was proteolytically processed within the fat body into some (or possibly all) of the mature vitellogenin subnits. Furthermore, peptide mapping confirmed that all of the phosphorylated vitellogenin subunits were derived from pre-Vg. Since previous studies have shown that phosphoserine residues account for essentially all of the covalently-attached phosphorus of the native vitellogenin molecule, we speculate that the asymmetric pattern of vitellogenin and vitellin subunit-phosphorylation is due to an uneven distribution of phosphoserine residues along the initial pre-Vg polypeptide chain. Finally, we conclude that phosphorylation of vitellogenin occurred post-translationally in the fat body endoplasmic reticulum because we could identify ³²P-labeled pre-Vg in purified microsomal vesicles but not in nascent vitellogenin polypeptide chains attached to vitellogenin polyribosomes.     

Molecular characterization of the sans fille gene in Antheraea pernyi: A highly conserved gene during the evolution of animals

Sans-fille (SNF) is the Drosophila homologue of mammalian general splicing factors U1A and U2B″, and plays an important role in sex determination in Drosophila melanogaster. In this study, the snf gene from Antheraea pernyi (Lepidoptera: Saturniidae), an economically important insect, was isolated and characterized. The obtained 925 bp cDNA sequence contains an open reading frame of 669 bp encoding a polypeptide of 222 amino acids, showing 78% sequence identity to that from D. melanogaster. A database search revealed that SNF protein homologs are present in many animals, including invertebrates and vertebrates, with more than 70% amino acid sequence identities, suggesting that they were highly conserved during the evolution of animals. Phylogenetic analysis revealed that A. pernyi SNF was closely related to Bombyx mori SNF. Quantitative real-time PCR (qRT-PCR) analysis showed that the A. pernyi snf gene was transcribed during five larval developmental stages, and in six tested tissues (ovaries, testes, silk glands, fat body, integument, and hemolymph), with the most abundance determined in the gonads (ovaries or testes). Investigation of expression changes throughout embryonic development indicated that A. pernyi snf mRNA was expressed at a low level from days 0 to 4, and reached a maximum level at day 10, but decreased to a low level before hatching. These results suggest that the product of the snf gene may play important roles in the development of A. pernyi.     

The developmental profiles of two major haemolymph proteins from Drosophila melanogaster.

There are four major protein species in the haemolymph of the late 3rd instar of Drosophila. Two of these, LSP-1 and LSP-2, have been studied in detail. Larvae, pupae, and flies of different ages were measured for wet weight, total extractable protein and using an immunoassay, the amounts of LSP-1 and LSP-2. The synthesis of both proteins begins after the 2nd larval ecdysis and at puparium formation they represent 9% and 1.5% of total extractable protein. This value remains constant during the first part of metamorphosis, then falls rapidly. The function of these proteins and their suitability as systems for the study of gene control and protein synthesis in Drosophila are discussed.     

Time studies of ecdysone-action on in vitro apolysis of Chilo suppressalis integument

The relationship between induction of in vitro apolysis and the duration of hormone treatment, and the effects of metabolic inhibitors on the ecdysone-induced apolysis were investigated in the cultured integument taken from the rice stem borer larva, Chilo suppressalis. When fragments of integument were subjected to 0.3 μg/ml β-ecdysone for more than 5 hr and then transferred to hormone free medium, they were induced to apolyse one day after treatment. If the fragments of integument were treated with hormone for 1 to 4 hr at first and then treated with hormone for 2 to 5 hr again after a 5 day interval in hormone free medium, almost all the fragments were induced to apolyse one day after treatment. This result suggests that the action of β-ecdysone on the cultured integument is accumulative. If the fragments of integument were cultured in the medium containing actinomycin-D and then transferred to medium containing β-ecdysone, a strong inhibitory effect on the apolysis of the integument was observed. Similarly, an inhibitory effect appeared when fragments of integument were treated first with hormone and then with puromycin. These results show that the m-RNA synthesis necessary for apolysis was completed within 6 hr after hormone treatment. However, the protein synthesis required for apolysis was not. The relationship of the results obtained from these in vitro experiments to the mode of action of ecdysone is discussed.     

Physiological control of the chromatophores of Austrolestes annulosus (Odonata)

The chromatophore-containing integument of Austrolestes annulosus always changes from a dark to a blue colour phase in vitro and therefore functions independently of a central control mechanism. In isolated abdominal segments, the reverse colour change occurs independently of the presence of central nervous system. The amount of change decreases with the size of the piece of integument; it varies over different parts of the body of the same intact insect, and it varies both in vitro and in vivo with time of day.A variety of experiments, involving ligation of abdominal segments and ablation of their ganglia, show that the terminal ganglion is primarily responsible for the release of a darkening factor which migrates in an anterior direction. Other ventral ganglia have a similar function but are of less importance.Transplants of terminal ganglia and injection of their extracts fail to restore capacity for colour change in ligature-isolated segments which have had their own ganglia removed. These findings, supported by histological observations, suggest that the darkening factor is a neurosecretion.