Sho Sakurai

Hemolymph ecdysteroid titer and ecdysteroid-dependent developmental events in the last-larval stadium of the silkworm, Bombyx mori: role of low ecdysteroid titer in larval–pupal metamorphosis and a reappraisal of the head critical period

The endocrine regulation of larval-pupal metamorphosis was studied in the silkworm, Bombyx mori, by measuring the following changes: hemolymph ecdysteroid titer, the secretory activity of prothoracic glands and the responsiveness of larvae to ecdysteroids and prothoracicotropic hormone (PTTH), with regard to developmental events such as the occurrence of spinneret pigmentation, initiation of cocoon spinning and onset of wandering stage as indicated by gut purge. These measurements were concentrated especially on the time before and after the head critical period (HCP) which falls 3-4 days before the gut purge ([Sakurai, 1984]). A small increase in the hemolymph ecdysteroid titer was first found during the HCP, and then the titer increased with daily fluctuations. Small but significant titer peaks were found prior to the occurrence of both spinneret pigmentation and gut purge, indicating that an individual titer peak could possess a specific role in development. Responsiveness of larvae to exogenous 20-hydroxyecdysone (20E) after the HCP was markedly higher than that before the HCP. The sensitivity of the prothoracic gland to PTTH also changed during the HCP. The results thus showed that the HCP is not the period after which an additional PTTH release is not required for the developmental events occurring on schedule, but rather it is the period during which complex events occur not only in the endocrine glands but also in the peripheral tissues. In addition, various developmental phenomena before gut purge are brought about by the hemolymph ecdysteroid whose concentration gradually increased with daily fluctuations, and these precise changes in the titer appeared to be important for the sequential occurrence of developmental events in the larval-pupal metamorphosis.

Development changes in juvenile hormone and juvenile hormone acid titers in the hemolymph and in-vitro juvenile hormone synthesis by corpora allata of the silkworm, Bombyx mori.

A simple method was developed to quantify hemolymph juvenile hormone (JH) and JH acid in hemolymph extracts from Bombyx mori with an established radioimmunoassay (RIA) for JH I. When various organic solvent extracts of hemolymph were assayed by RIA, levels of non-specific binding of the labeled ligand in the assay were determined to be greater than 50% of the maximum amount of the label bound by the antiserum. When hemolymph was diluted with methanol:water:8.4N ammonium hydroxide (10:9:1) and extracted with isooctane, non-specific binding was only 50% higher than control levels obtained with the assay buffer alone. The organic phase contained only JH and aqueous phase, JH acid. Consequently, this extraction method was used to prepare samples for RIA and enabled the separate measurement of JH and JH acid in hemolymph. With this method, changes in the hemolymph titers of JH and JH acid were determined from the third instar through early pupal stage of Bombyx mori. Changes in the in vitro secretory activity of corpora allata and brain-corpora cardiaca-corpora allata complexes from fifth instar larvae were also determined by using JH I RIA of the incubation medium.

Programmed cell death triggered by insect steroid hormone, 20-hydroxyecdysone, in the anterior silk gland of the silkworm, Bombyx mori

Abstract Silk gland is a larval specific tissue of lepidopteran insects and begins to degenerate shortly before pupation. Programmed cell death (PCD) of the anterior silk gland of Bombyx mori last instar larvae was studied in vivo and in vitro, focusing on the effects of 20- hydroxyecdysone (20E). The glands began to exhibit signs of PCD in vivo 2 days after gut purge and comp-leted PCD by 48 h. In vitro, 20E prematurely induced PCD, and its completion took 144 h (6 days). An oligo-nucleosomal ladder pattern was observed in DNA extracted at the end of PCD. Caspase 3 inhibitor inhibited attainment of full PCD, but it did not block chromatin condensation as revealed by acridine orange staining. α-Amanitin inhibited the PCD induced by 20E in vitro if added to the culture in the first 8 h. Similarly, cycloheximide and emetine completely blocked PCD when applied in the first 18 h of culture with 20E. These results indicate that 20E-stimulated transcription and protein synthesis for PCD are completed in 8 h and 18 h, respectively. Nevertheless, withdrawal of 20E from the medium at different times showed that 20E must be present in vitro for 42 h to elicit full PCD. Current results indicate that the effects of 20E on the progression of PCD are mediated by two distinct processes – one through nuclear hormone receptors, and the other independent from de novo gene expression.

Juvenile hormone inhibits ecdysone secretion and responsiveness to prothoracicotropic hormone in prothoracic glands of Bombyx mori

The role of juvenile hormone (JH) in the regulation of prothoracic gland activity was investigated during the early days of the last (fifth) larval instar of Bombyx mori. Allatectomy on the day of larval ecdysis into the fifth instar or 1 day before ecdysis shortened the time between larval ecdysis and gut purge. Prothoracic glands of the freshly ecdysed fifth instar larvae were inactive and did not respond to the prothoracicotropic hormone (PTTH), whereas those larvae that were allatectomized 1 day before ecdysis exhibited secretory activity in vitro and were capable of responding to PTTH. When corpora allata were removed from freshly ecdysed fifth instar larvae, the prothoracic glands became competent to respond to PTTH in 6 hr and exhibited secretory activity in vitro 9 hr after the allatectomy. Treatment of allatectomized larvae with a JH analog resulted in the recovery of the normal inactive state of the glands. These data suggest that JH acts during the early stages of the instar to suppress both the secretory activity of prothoracic glands and also the acquisition of competence to respond to PTTH.

Coordinate responses of transcription factors to ecdysone during programmed cell death in the anterior silk gland of the silkworm, Bombyx mori

Programmed cell death (PCD) in Bombyx mori anterior silk glands (ASGs) is triggered by 20‐hydroxyecdysone (20E). We examined the expression profiles and effects of 20E on 11 transcription factor genes in the fifth instar to determine whether they demonstrate the hierarchical control seen in Drosophila PCD. Results indicate that EcR‐A and usp‐2, but not EcR‐B1 or usp‐1, may be components of the ecdysone receptor complex. Up‐regulation of E75A, BHR3, and three BR‐C isoforms, but not E75B, appeared to be associated with the induction of PCD. βFTZ‐F1 was not expressed during PCD execution. Thus, gene control in B. mori ASGs differs from that in Drosophila salivary glands, despite both tissues undergoing PCD in response to 20E at pupal metamorphosis.

Regulation of soluble and membrane-bound trehalase activity and expression of the enzyme in the larval midgut of the bamboo borer Omphisa fuscidentalis

Diapausing larvae of Omphisa fuscidentalis contain soluble and membrane-bound trehalase in the midgut. Soluble trehalase activity accounts for three-fourths of the total trehalase activity in midgut homogenates. The exposure of diapausing larvae to juvenile hormone analog (JHA) induced pupation, accompanied by an increase in soluble trehalase activity at the beginning of the prepupal period. Injection of 20-hydroxyecdysone (20E) increased the level of soluble trehalase activity 5 days postinjection in a dose-dependent manner. In contrast, no increase in membrane-bound trehalase activity was observed under the same conditions. We cloned the cDNAs that encode the soluble and membrane-bound forms of trehalase in O. fuscidentalis trehalase-1 (OfTreh-1) and trehalase-2 (OfTreh-2), respectively. Treh-1 encodes a 581-aa protein while Treh-2 encodes a 648-aa protein with one putative transmembrane domain near the C-terminus. The mRNA expression level of Treh-1 was 27-fold higher than that of Treh-2 in diapausing larval midgut. Following the exposure of diapausing larvae to JHA, Treh-1 mRNA expression increased gradually until the prepupal period whereupon it increased dramatically; in contrast, the mRNA expression of Treh-2 remained at its initial level. Similarly, 20E upregulated Treh-1 expression but had no effect on Treh-2 expression. Taken together, these results suggest that an increase in the soluble trehalase activity at pupation is caused by upregulation of Treh-1 gene. Moreover, membrane-bound trehalase does not appear to be involved in the dynamic changes in the hemolymph trehalose concentration that occur during the larval-pupal transformation.

Juvenile hormone-mediated termination of larval diapause in the bamboo borer, Omphisa fuscidentalis.

Larvae of the bamboo borer, Omphisa fuscidentalis are in diapause for more than nine months (Singtripop, T., Wanichaneewa, S., Tsuzuki, S., Sakurai, S. 1999. Larval growth and diapause in a tropical moth, Omphisa fuscidentalis Hampson. Zool. Sci. 16, 725-733). To examine the endocrine mechanisms underlying this larval diapause, we assayed the responsiveness of the diapausing larvae to 20-hydroxyecdysone (20E) and a juvenile hormone analogue (JHA: S-methoprene). 20E injection caused the larvae to halt movement, followed by deposition of a pupal cuticle. Topical application of JHA induced pupation in a dose-dependent manner. JHA also induced pupation of the larvae whose brains were removed before JHA application. In those larvae, the prothoracic glands became active and competent to respond to brain extracts within seven days after JHA treatment, and the hemolymph ecdysteroid concentration began to increase 12 days after JHA application. These results indicate that JHA stimulates the prothoracic glands of diapausing Omphisa larvae, terminating larval diapause, in contrast with previous findings that JH inhibits the brain-prothoracic gland axis and thus maintains the larval diapause. Current results therefore suggest a novel regulatory mechanism for larval diapause in this species.

Nongenomic action of an insect steroid hormone in steroid-induced programmed cell death.

Programmed cell death (PCD) of the silkworm silk glands is triggered by the insect steroid hormone, 20-hydroxyecdysone (20E), and proceeds sequentially through cell shrinkage, nuclear condensation, DNA fragmentation, nuclear fragmentation and apoptotic body formation. A protein synthesis inhibitor, cycloheximide (CHX, 2 mM) induced a cell death that exhibited only nuclear and DNA fragmentation. A concentration of 0.2 mM CHX was ineffective at inducing the cell death when added alone, but in the presence of 20E, a cell death similar to that induced by 2 mM CHX was resulted with accompanying nuclear condensation. Since 2 and 0.2 mM CHX inhibited protein synthesis equally, the DNA and nuclear fragmentation appear to be mediated by a nongenomic action of 20E. In addition, we show a possible involvement of Ca2+-PKC-caspase-3 like protease pathway in the nongenomic action. The data suggest that 20E-induced PCD is accomplished through the integration of genomic and nongenomic actions.

Sensitivities to juvenile hormone and ecdysteroid in the diapause larvae of Omphisa fuscidentalis based on the hemolymph trehalose dynamics index

The final instar larva of the bamboo borer, Omphisa fuscidentalis, is in diapause for 9 months from September to the following June. Trehalose and ecdysteroid concentrations in hemolymph were measured through the larval diapause period and in the pupal stage. The ecdysteroid concentration remained low until November, followed by a gradual increase to about 30 ng/ml in May. The trehalose concentration remained at levels ranging between 40-50 mM until May, and decreased to an almost undetectable level after pupation. Since a juvenile hormone analogue (JHA), methoprene, is capable of terminating diapause by stimulating larval prothoracic glands, we examined its effects on ecdysteroid and trehalose concentrations in larvae in December and February. The hemolymph ecdysteroid increased more quickly in February than in December, indicating that the sensitivity of the prothoracic glands to JHA increased towards the end of diapause termination. Similarly, hemolymph trehalose in February decreased within a few days after JHA application, while in December the decrease occurred in the third week. Exogenous 20-hydroxyecdysone (20E) caused a decrease in trehalose concentration in a dose-dependent manner. The effective dose of 20E, however, did not change from January until April, implying that the sensitivity of tissue(s) to 20E may not change until the end of diapause. Taken together, our results suggest that the sensitivities of tissues to JH and 20E do not increase simultaneously with the progress of diapause development and that termination of larval diapause is not associated simply with the restoration of hormone deficiencies.

A novel member of the bombyxin gene family: structure and expression of bombyxin G1 gene, an insulin-related peptide gene of the silkmoth Bombyx mori

Abstract Bombyxin G1 gene, a novel insulin-related peptide gene of the silkmoth Bombyx mori, has been identified. The G1 gene encodes a precursor peptide which shows 41–56% and 28% sequence identities with preprobombyxins previously characterized and human preproinsulin, respectively. The G1 gene forms a pair with bombyxin C2 gene with opposite transcriptional orientation in a bombyxin gene cluster. The bombyxin G1 mRNA in Bombyx brain was shown to locate in four pairs of medial neurosecretory cells.