Isolation of the diapause hormone from the silkworm,Bombyx mori

The diapause hormone (DH) secreted from the suboesophageal ganglion is responsible for the embryonic diapause in the silkworm, Bombyx mori. It was extracted from two million mated male adult heads. At least two species of DH were separated from the extracts (a complex lipid fraction) by gel permeation chromatography with Sephadex LH-20 by organic eluants. One of them was further purified by repeating gel permeation column chromatography with Merckogel^® Type OR 6000 in the cold and dark to give finally a single peak with high DH activity. This preparation seems to be chemically pure, and its molecular weight is chromatographically estimated to be between 2000 and 4000. Other characteristics of DH are also presented. Biological activity of the final DH preparation is discussed with reference to other insect hormones such as juvenile hormone and α-ecdysone.     

Neuroendocrine control of diapause hormone secretion in the silkworm,Bombyx mori

To clarify the control mechanism of diapause hormone (DH) secretion in the silkworm Bombyx mori a series of anatomical and pharmacological experiments were carried out. The arrangement of 'diapause' and 'non-diapause' eggs in the ovarioles of the moths was determined by the coloration method to estimate the accumulation of 3-hydroxykynurenine in the eggs. The females destined to lay non-diapause eggs (non-diapause producers) had diapause eggs in their ovaries if their subesophageal ganglions (Sg) had been surgically removed at 2days after larval-pupal ecdysis or later. In contrast when the surgical extirpation extended to the brain and the corpora cardiaca (CC)-corpora allata (CA) complex in addition to the Sg, the non-diapause producers had no diapause eggs. When the Sg was removed from the females destined to lay diapause eggs (diapause-producers), diapause eggs appeared in response to the treatment at 2days after larval-pupal ecdysis, but the appearance of diapause eggs was delayed by 2days when the brain-CC-CA complex was included among the organs removed. These observations suggested that DH is produced in Sg and transferred to the CC-CA complex, and that the secretion of DH from the complex is suppressed in non-diapause producers. The pattern of diapause and non-diapause eggs induced by the transection of the subesophageal connective in diapause and non-diapause producers suggested a regenerative and secretory capacity of the neurosecretory cells after the operation. The appearance of diapause eggs in non-diapause producers with transected protocerebrum of the brain confirmed that there was an inhibitory center in the protocerebrum. Changes in parts of the ovarioles containing diapause and non-diapause eggs with time of injection of gamma-aminobutyric acid (GABA) and picrotoxin suggested that a GABAergic inhibitory mechanism in DH secretion may be active in non-diapause producers but inactive in diapause producers throughout the pupal stage.     

Cleavage of Rabbit Myelin Basic Protein by Plasmin: Isolation and Identification of the Major Products

Rabbit myelin basic protein (BP) was subjected to partial cleavage with plasmin, and 15 cleavage products were isolated by a combination of gel filtration and ion-exchange chromatography. Their identification was achieved by amino acid analysis and tryptic peptide mapping, supplemented in some instances by carboxy-terminal analyses with carboxypeptidases A, B, and Y and amino-terminal analyses with dipeptidyl aminopeptidase I. The results showed that major plasmic cleavage sites included the Lys89-Asn90, Lys133-Ser134, and Lys153-Leu154 bonds. Cleavages also occurred at the Arg31-His32, Lys53-Arg54, and Arg25-His26 bonds, but these appeared to be less extensive. A large number of additional peptides were produced in relatively low yield. The smaller of these were isolated from heterogeneous fractions by high-voltage electrophoresis-TLC. Amino acid analysis of these peptides showed that minor cleavage sites included the Arg9-His10, Lys13-Tyr14, Lys103-Gly104, Lys137-Gly138, Lys140-Gly141, and Arg160-Ser161 bonds. In spite of a lower selectivity toward peptide bonds in BP as compared with pepsin, cathepsin D, and thrombin, plasmin has the advantage over the former proteinases in that it does not cleave at or near the Phe44-Phe45 bond. Instead it cleaves at the Arg31-His32 and Lys53-Arg54 bonds, thus preserving the entire hydrophobic sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe as well as short sequences to either side.     

Characterization in rat pancreatic juice of a protein homologous to the human pancreatic stone protein

1. The pancreatic stone protein (PSP, Mr 15,000) which has been discovered in human calculi derives from the native glycosylated forms of the protein (Mrs 17,500-22,000) which are present in human pancreatic juice through tryptic cleavage of the Arg 11-Ile 12 bond. 2. In the present study, a homologous native form of the protein (Mr 17,000) was purified from rat pancreatic juice. 3. Its N-terminal amino acid sequence was found to display a high degree of homology with that of the human native protein forms, apart from the fact that it was not glycosylated. 4. In rat as in human, tryptic cleavage of the Arg 11-Ile 12 bond transforms a soluble protein into one which is practically insoluble at neutral pH.     

Chronic low-frequency stimulation of rabbit fast-twitch muscle induces partial inactivation of the sarcoplasmic reticulum Ca2(+)-ATPase and changes in its tryptic cleavage

Persistently increased contractile activity as induced by low-frequency stimulation in fast-twitch rabbit muscle elicits a partial inactivation of the sarcoplasmic reticulum Ca2(+)-ATPase function with regard to Ca2+ transport and ATP hydrolysis. Electron microscopy showed no differences in the frequency and structure of the two-dimensional Ca2(+)-ATPase crystals between microsomal fractions from normal and stimulated muscles. However, differences existed between the tryptic digestion of the Ca2(+)-ATPase in both the membrane-bound and solubilized enzyme at the first tryptic cleavage site, named T1 (Arg505). This followed from a delayed appearance of the A and B fragments of the Ca2(+)-ATPase in the electrostimulated muscle. No differences existed with regard to the second tryptic cleavage site, named T2 (Arg198). Confirming previous results, fluorescein isothiocyanate (FITC) binding to the enzyme of the chronically stimulated muscle was markedly reduced. The FITC-labeled fraction of the enzyme from both the normal and the stimulated muscle followed similar time courses of tryptic cleavage. The fraction of Ca2(+)-ATPase that did not bind TITC was identified by immunoblot analysis as the trypsin-resistant form. In view of the vicinity of T1, the FITC- and the ATP-binding sties, these results point to a modification of the enzyme in that region leading to an inactivation of about 50% of the sarcoplasmic reticulum Ca2(+)-ATPase molecules.     

Amino acid sequence of the Pseudomonas putida cytochrome P-450. I. Sequences of tryptic and clostripain peptides

In order to elucidate the complete amino acid sequence of Pseudomonas putida cytochrome P-450, tryptic digestion was performed on the S-carboxymethylated enzyme. Although cleavage did not occur at every lysyl and arginyl bond, 31 tryptic peptides ranging in size from 1 to 55 residues were isolated. These were sequenced by manual Edman degradation and carboxypeptidase digestion. Overlaps of some od these tryptic peptides were obtained by data obtained from partial Edman degradation and amino acid composition of the clostripain cleavage products. These results, together with data from the cyanogen bromide and acid cleavage peptides reported in the accompanying paper, established the complete amino acid sequence of P. putida cytochrome P-450.     

Activation of pro-phenoloxidase by the activating enzyme of the silkworm,Bombyx mori

The activation reaction of prophenoloxidase was analyzed using a homogeneous pro-enzyme preparation and highly purified pro-phenoloxidase activating enzyme (abbreviated as PPAE) obtained from silkworm larvae.The activation of pro-enzyme by PPAE results in release of a peptide having a molecular weight of about 5000 calculated from its amino acid composition and the difference between the molecular weights of the pro-enzyme and phenoloxidase. PPAE seems to be a highly specific enzyme capable of limited proteolysis, since no further degradation of phenoloxidase or release of any other peptide could be detected.Phenoloxidases having different kinetic properties and substrate specificities were obtained depending on the pH at which activation took place, although the molecular weights of phenoloxidase determined by sodium dodecylsulphate-electrophoresis were the same irrespective of the operating pH. This suggests that a process subsequent to peptide bond cleavage in the activation reaction is influenced by pH, resulting in phenoloxidases with different properties.Phenoloxidase seems to be present as progressively higher molecular weight aggregates. The enzyme is bound strongly to foreign surfaces such as glass, DEAE-cellulose and hydroxyapatite, from which much of the enzyme activity cannot be eluted by high NaCl or phosphate concentration. This may be important in relation to the intracellular binding and occurrence of multiple forms of phenoloxidase.     

Structural and functional studies of the interaction of the eukaryotic elongation factor EF-2 with GTP and ribosomes

The structure of the guanosine nucleotide binding site of EF-2 was studied by affinity labelling with the GTP analogue, oxidized GTP (oGTP), and by amino acid sequencing of polypeptides generated after partial degradation with trypsin and N-chlorosuccinimide. Native EF-2 contains two exposed trypsin-sensitive cleavage sites. One site is at Arg66 with a second site at Lys571/Lys572. oGTP was covalently bound to the factor between Arg66 and Lys571. After further cleavage of this fragment with the tryptophan-specific cleavage reagent N-chlorosuccinimide, oGTP was found associated with a polypeptide fragment originating from a cleavage at Trp261 and Trp343. The covalent oGTP . EF-2 complex was capable of forming a high-affinity complex with ribosomes, indicating that oGTP, in this respect, induced a conformation in EF-2 indistinguishable from that produced by GTP. Although GTP could be substituted by non-covalently linked oGTP in the factor and ribosome-dependent GTPase reaction, the factor was unable to utilize the covalently bound oGTP as a substrate. This indicates that the conformational flexibility in EF-2 required for the ribosomal activation of the GTPase was inhibited by the covalent attachment of the nucleotide to the factor. EF-2 cleaved at Arg66 were unable to form the high-affinity complex with ribosomes while retaining the ability to form the low-affinity complex and to hydrolyse GTP. The second cleavage at Lys571/Lys572 was accompanied by a total loss of both the low-affinity binding and the GTPase activity.     

Hydrolysis of intact and Cys-Phe-cleaved human atrial natriuretic peptide in vitro by human tissue kallikrein.

Atrial natriuretic peptide (ANP) is a 28-amino-acid hormone involved in the regulation of fluid balance. In circulation, the proteolytic inactivation of ANP has been demonstrated to involve both membrane metalloendopeptidase and an aprotonin-sensitive activity, probably corresponding to kallikrein [Vanneste, Y., Pauwels, S., Lambotte, L., Michel, A., Dimaline, R. & Deschodt-Lanckman, M. (1990) Biochem. J. 269, 801-806]. In the present study, we focused on the aprotinin-sensitive pathway of ANP metabolism. In order to identify the cleavage sites recognized by kallikrein within the sequence of the hormone, tissue kallikrein was purified to homogeneity from human urine and the degradation of human ANP by the enzyme preparation was studied. Our results demonstrate that both intact and Cys7-Phe8-cleaved ANP, the initial metabolite produced in circulation by the metallo-endopeptidase, are substrates in vitro for purified tissue kallikrein. However, the Cys-Phe-cleaved peptide was degraded approximately fourfold faster than the intact hormone by the purified enzyme. The first degradation step of ANP by tissue kallikrein involves two cleavages occurring at the bonds Arg3-Arg4 and Gly16-Ala17, generating an inactive, open-ring metabolite. Incubation of ANP for a longer period with the enzyme led to the generation of several additional degradation fragments. Ten peaks were separated by HPLC and characterized by amino acid analysis. The results allowed the identification of a total of eight peptide bonds susceptible to hydrolysis by tissue kallikrein in the sequence of ANP: Arg3-Arg4, Ser5-Ser6, Cys7-Phe8, Arg11-Met12, Gly16-Ala17, Gly20-Leu21, Ser25-Phe26 and Arg27-Tyr28. These results indicate that the aprotinin-sensitive activity involved in the metabolism of ANP in circulation could correspond to tissue kallikrein. However, clear identification of ANP as a novel physiological substrate of the enzyme will need further investigation.     

Effect of actin on the tryptic digestion of myosin subfragment 1 in the weakly attached state.

The structure of myosin subfragment 1 (S1) in the weakly attached complex with actin was studied at three specific sites, at the 50-kDa/20-kDa and 27-kDa/50-kDa junctions, and at the N-terminal region, using tryptic digestion as a structure-exploring tool. The structure of S1 at the vicinity of the 50-kDa/20-kDa junction is pH dependent in the weakly attached state because the tryptic cleavage at this site was fully protected by actin at pH 6.2, but the protection was only partial at pH 8.0. Since the actin protection is complete in rigor at both pH values, the results indicate that the structure of S1 at the 50-kDa/20-kDa junction differs in the two states at pH 8.0, but not at pH 6.2. Actin restores the ADP-suppressed tryptic cleavage after Lys213 at the 27-kDa/50-kDa junction in the strongly attached state, but not in the weakly attached state, which indicates structural difference between the two states at this site. ATP and ADP open a new site for tryptic cleavage in the N-terminal region of the S1 heavy chain between Arg23 and Ile24. Actin was found to suppress this cleavage in both weakly and strongly attached states, which shows that, in the vicinity of this site, the structure of S1 is similar in both states. The results indicate that the binding of S1 to actin induces localized changes in the S1 structure, and the extent of these changes is different in the various actin-S1 complexes.     

Characterization of the lac repressor species produced by limited tryptic cleavage

Tryptic cleavage of native lac repressor under very mild conditions has been found to yield preparations suitable for detailed physical and chemical analysis. Sephadex G-200 chromatography of the digest produces one main protein peak followed by small peptides. The protein from the main peak was analyzed by automated Edman degradation and revealed two unique cleavage sites, one at residue 51 and the other at 59. The tryptic core protein under native conditions is tetrameric and exhibits a circular dichroism spectrum similar to that of native lac repressor.     

Localization of the sites of ADP-ribosylation and GTP binding in the eukaryotic elongation factor EF-2

Tryptic cleavage of EF-2, molecular mass 93 kDa, produced an 82-kDa polypeptide and a 10-kDa fragment, which was further degraded. By a slower reaction the 82-kDa polypeptide was gradually split into a 48-kDa and a 34-kDa fragment. Similarly, treatment with chymotrypsin resulted in the formation of an 82-kDa polypeptide and a small fragment. In contrast to the tryptic 82-kDa polypeptide the corresponding chymotryptic cleavage product was relatively resistant to further attack. The degradation of the 82-kDa polypeptide with either trypsin or chymotrypsin was facilitated by the presence of guanosine nucleotides, indicating a conformational shift in native EF-2 upon nucleotide binding. No effect was observed in the presence of ATP, indicating that the effect was specific for guanosine nucleotides. After affinity labelling of native EF-2 with oxidized [3H]GTP and subsequent trypsin treatment the radioactivity was recovered in the 48-kDa polypeptide showing that the GTP-binding site was located within this part of the factor. Correspondingly, tryptic degradation of EF-2 labelled with [14C]NAD+ in the presence of diphtheria toxin showed that the site of ADP-ribosylation was within the 34-kDa polypeptide. By cleavage with the tryptophan-specific reagent N-chlorosuccinimide the site of ADP-ribosylation could be located at a distance of 40-60 kDa from the GTP-binding site and about 4-11 kDa from the nearest terminus.     

The primary structure of hemoglobins from the domestic cat (Felis catus, Felidae)

The complete primary structure of the two hemoglobin components of the domestic cat (Felis catus) is presented. The major component (A) accounts for 60-70% whereas the minor component (B) constitutes 30-40% of the total hemoglobin. Separation of the polypeptides was carried out in buffers containing 8M urea on CM-Cellulose. The sequence was studied by Edman degradation of tryptic and cyanogen bromide cleavage products in a liquid phase sequencer. The sequence is compared for homology with human hemoglobin. The beta-chain of the minor components (beta B) has a blocked N-terminal residue identified as acetylserine whereas that of the major component (beta A) is free glycine. The two hemoglobins have identical alpha-chains and differ with respect to their beta-chains at the following positions (beta B/beta A): beta NA1 Ac-Ser/Gly, beta A1 Ser/Thr, beta H17 Ser/Asn and beta HC1 Arg/Lys. The structural and functional aspects of these exchanges are discussed.     

Establishment of a sandwich ELISA system to detect diapause hormone, and developmental profile of hormone levels in egg and subesophageal ganglion of the silkworm, Bombyx mori

In the silkworm Bombyx mori, diapause hormone (DH) is produced in the female subesophageal ganglion (SG) and induces embryonic diapause by targeting developing ovaries. DH is processed from a precursor protein consisting of DH, pheromone biosynthesis activating neuropeptide (PBAN) and three other neuropeptides (SGNPs). Because these five neuropeptides share a common sequence, FXPRLamide, at the C-terminus, a direct and specific assay for DH itself is required in order to understand the profile of concentration changes. In this study, we produced a mouse monoclonal antibody (anti-DH[N] mAb) against the N-terminal region of DH and developed a sandwich enzyme-linked immunosorbent assay using the anti-DH[N] mAb and a rabbit polyclonal antibody against the C-terminus of DH. This procedure enabled us to specifically quantify the DH molecule at femtomolar levels (equivalent to 1/10 of SG). We then plotted DH levels in eggs and SGs during embryonic and post-embryonic development. DH was present in late-stage embryos that had been destined for the production of both diapause and nondiapause eggs. DH levels in SG gradually increased in both types during larval development and peaked at the early pupal stage. At the middle pupal stage, DH levels in SG and SG-brain complex decreased markedly in the diapause-egg producing type, thus indicating active release of DH into the hemolymph. From 5th instar larva to adult, no sexual differences in DH levels were observed in SGs or SG-brain complexes from diapause and nondiapause egg-producing types.     

Binding of substrate in two conformations to human prothrombinase drives consecutive cleavage at two sites in prothrombin

Thrombin formation results from cleavage of prothrombin following Arg(271) and Arg(320). Both bonds are accessible for cleavage, yet the sequential action of prothrombinase on Arg(320) followed by Arg(271) is implied by the intermediate observed during prothrombin activation. We have studied the individual cleavage reactions catalyzed by prothrombinase by using a series of recombinant derivatives: wild type prothrombin (II(WT)) contained both cleavage sites; II(Q271) contained a single cleavable site at Arg(320); II(Q320) and II(A320) contained a single cleavable site at Arg(271); and II(QQ) was resistant to cleavage. Cleavage at Arg(320) in II(Q271) could account for the initial cleavage reaction leading to the consumption of either plasma prothrombin or II(WT), whereas cleavage at Arg(271) in either II(Q320) or II(A320) was found to be approximately 30-fold slower. Equivalent kinetic constants were obtained for three of the four possible half-reactions. Slow cleavage at Arg(271) in intact prothrombin resulted from an approximately 30-fold reduction in V(max). Thus, the observed pathway of bond cleavage by prothrombinase can be explained by the kinetic constants for the four possible individual cleavage reactions. II(Q320) was a competitive inhibitor of II(Q271) cleavage, and II(QQ) was a competitive inhibitor for each reaction with K(i) approximately K(m). The data are inconsistent with previous proposals and suggest a model in which substrates for each of the four possible half-reactions bind in a mutually exclusive manner and with equal affinity to prothrombinase in a cleavage site-independent way. Despite equivalent exosite binding interactions between all four possible substrates and the enzyme, we propose that ordered bond cleavage results from the constraints associated with the binding of substrates in one of two conformations to a single form of prothrombinase.     

Altered inactivation pathway of factor Va by activated protein C in the presence of heparin.

Inactivation of factor Va (FVa) by activated protein C (APC) is a predominant mechanism in the down-regulation of thrombin generation. In normal FVa, APC-mediated inactivation occurs after cleavage at Arg306 (with corresponding rate constant k'306) or after cleavage at Arg506 (k506) and subsequent cleavage at Arg306 (k306). We have studied the influence of heparin on APC-catalyzed FVa inactivation by kinetic analysis of the time courses of inactivation. Peptide bond cleavage was identified by Western blotting using FV-specific antibodies. In normal FVa, unfractionated heparin (UFH) was found to inhibit cleavage at Arg506 in a dose-dependent manner. Maximal inhibition of k506 by UFH was 12-fold, with the secondary cleavage at Arg306 (k306) being virtually unaffected. In contrast, UFH stimulated the initial cleavage at Arg306 (k'306) two- to threefold. Low molecular weight heparin (Fragmin) had the same effects on the rate constants of FVa inactivation as UFH, but pentasaccharide did not inhibit FVa inactivation. Analysis of these data in the context of the 3D structures of APC and FVa and of simulated APC-heparin and FVa-APC complexes suggests that the heparin-binding loops 37 and 70 in APC complement electronegative areas surrounding the Arg506 site, with additional contributions from APC loop 148. Fewer contacts are observed between APC and the region around the Arg306 site in FVa. The modeling and experimental data suggest that heparin, when bound to APC, prevents optimal docking of APC at Arg506 and promotes association between FVa and APC at position Arg306.     

The degradation of bovine parathyroid hormone by leukocytes

Human peripheral leukocytes catalyse an elastase-like cleavage of bovine parathyroid hormone, with an identical specificity to that previously observed with a neutral proteinase (EC 3.4.21-) isolated from the outer surface of plasma membranes from human leukocytes. Parathyroid hormone is not hydrolysed by human erythrocytes, and polymorphonuclear leukocytes are much more effective in hormone degradation than lymphocytes. Despite the fact that purified human leukocyte granular elastase (EC 3.4.21.37) catalyses an identical cleavage reaction, the hydrolysis observed with intact cells is clearly not due to proteinases secreted by the cells. The reaction is inhibited by human serum and by purified human alpha-1-antitrypsin, but not by antibodies to human leukocyte granular elastase. The possible significance of these phenomena to the in vivo metabolism of parathyroid hormone are discussed.     

Structural comparison of bovine erythrocyte, brain, and liver NADH-cytochrome b5 reductase by HPLC mapping

NADH-cytochrome b5 reductases purified from bovine erythrocytes and from bovine brain and liver microsomes solubilized with lysosomal protease were subjected to structural analysis by using HPLC mapping, amino acid analysis of the resulting peptides, and NH2-terminal sequence analysis of apoproteins. HPLC maps of the tryptic peptides derived from these enzymes were very similar to each other, and amino acid analysis of the HPLC-separated peptides indicated that the structures of these enzymes are identical except for the NH2-terminal region. The NH2-terminal sequence of the brain enzyme determined by automated Edman degradation was as follows: NH2-Phe-Gln-Arg-Ser-Thr-Pro-Ala-Ile-Thr-Leu-Glu-Asn-Pro-Asp- Ile-Lys-Tyr-Pro-Leu-Arg-Leu-Ile-Asp-Lys-Glu-Val-Ile- This sequence is identical to that of liver enzyme except that the liver enzyme started at the 3rd Arg or 4th Ser. The NH2-terminal amino acid residue of the soluble erythrocyte enzyme was not detected by automated Edman degradation. The sequence analysis of a tryptic peptide from the erythrocyte enzyme indicated that Leu is present before the NH2-terminal Phe of the brain enzyme. The recently reported sequence of the apparently identical protein (Ozols et al. (1985) J. Biol. Chem. 260, 11953-11961) differs in two amino acid assignments from our sequence.     

Reaction of human chorionic somatomammotropin and human pituitary growth hormone with tetranitromethane at 0 degrees C

The comparative reactivity of the eight tyrosine residues which occur at homologous positions in human chorionic somatomammotropin and human pituitary growth hormone has been investigated by their reaction with tetranitromethane at 0 °C. The derivatives were characterized by circular dichroism spectra, spectrophotometric titrations, rate of tryptic digestion, and immunodiffusion. Pigeon crop-sac stimulating activities were fully retained in these derivatives. The extent of modification for human chorionic somatomammotropin and human pituitary growth hormone was 2.5 and 4.2 out of 8 residues, respectively. The location of each modified tyrosine residue in the derivatives was determined by amino acid analysis of isolated nitrated peptides after cyanogen bromide cleavage and enzymatic digestion. It was found that tyrosine-143 was highly reactive in the pituitary hormone but unreactive in the placental hormone.     

Physiological differentiation of DH-PBAN-producing neurosecretory cells in the silkworm embryo

Embryonic diapause of the silkworm, Bombyx mori, is induced by a neuropeptide hormone, the diapause hormone (DH), which is secreted from a limited number of neurosecretory cells in the subesophageal ganglion (SG) at the maternal generation. We examined the developmental fate of the hormone-producing cell (DH-pheromone biosynthesis activating neuropeptide [PBAN]-producing cell) in the embryonic stage at the level of gene expression and cell biology. The DH-PBAN gene expression started at the histogenesis stage and gradually increased toward hatching. DH is an amidated peptide belonging to FXPRLamide family. The immunoreactive somata against anti FXPRLamide antiserum were found in the SG from blastokinesis. Immunoreactive neural processes with varicosites were also found on the corpus cardiacum and the corpus allatum. The implantation of a part of a developing embryo including the SG into the pupae with the SG removed induced diapause eggs in the progeny. These results were obtained from eggs incubated under diapause-averting conditions as well as diapause-inducing conditions. Thus, a neurosecretory system responsible for biosynthesis of FXPRLamide neuropeptides is established as early as histogenesis, although the system to regulate the secretion of neuropeptide hormones has not been fully formed by that time.