Pupal commitment and its hormonal control in wing imaginal discs

The timing of pupal commitment of the forewing imaginal discs of the silkworm, Bombyx mori, was determined by a transplantation assay using fourth instar larvae. The wing discs were not pupally committed at the time of ecdysis to the fifth instar. Pupal commitment began shortly after the ecdysis and was completed in 14 h. When the discs of newly molted larvae (0-h discs) were cultured in medium containing no hormone, they were pupally committed in 26 h. In vitro exposure of 0-h discs to 20-hydroxyecdysone accelerated the progression of pupal commitment. Methoprene, a juvenile hormone analog (JHA), did not suppress the change in commitment in vitro at physiological concentrations. Thus the wing discs at the time of the molt have lost their sensitivity to JH, and 20E is not a prerequisite for completion of pupal commitment. These results suggest that the change in commitment in the forewing discs may begin before the last larval molt.     

Ecdysteroid-induced programmed cell death and cell proliferation during pupal wing development of the silkworm, Bombyx mori

The wing margin of adult wings of Lepidoptera is defined by the position of a "bordering lacuna"(BL). During adult wing development, cell proliferation and scale formation proximal to this lacuna and programmed cell death distal to the lacuna are generally observed. To determine the effect of 20-hydroxyecdysone (20E) on these events, we cultured the silkworm pupal wings with or without 20E and analyzed regional specificity for cell death by the TUNEL method and cell proliferation by 5-bromodeoxyuridine labeling. Programmed cell death was induced by 20E after 5 days of culture and was detected only in the region distal to BL. Cell proliferation after 1 day of culture and scale formation after 5 days of culture were also inducible by 20E and detected in the region proximal to BL. These results suggest that two types of pupal wing cells, which are divided by the position of the BL, respond to ecdysteroid in different manners. Higher concentrations of 20E (more than 1,000 ng/ml) repressed the scale formation, while such repression could not be observed in the peripheral cell death even with 5,000 ng/ml 20E. The ecdysteroid may work both as a trigger to make the wing margin and scales and as a developmental timer to arrange these cellular responses.     

Shotgun proteomic analysis of wing discs from the domesticated silkworm (Bombyx mori) during metamorphosis

Proteomic profiles from the wing discs of silkworms at the larval, pupal, and adult moth stages were determined using shotgun proteomics and MS sequencing. We identified 241, 218, and 223 proteins from the larval, pupal, and adult moth stages, respectively, of which 139 were shared by all three stages. In addition, there were 55, 37, and 43 specific proteins identified at the larval, pupal, and adult moth stages, respectively. More metabolic enzymes were identified among the specific proteins expressed in the wing disc of larvae compared with pupae and moths. The identification of FKBP45 and the chitinase-like protein EN03 as two proteins solely expressed at the larval stage indicate these two proteins may be involved in the immunological functions of larvae. The myosin heavy chain was identified in the pupal wing disc, suggesting its involvement in the formation of wing muscle. Some proteins, such as proteasome alpha 3 subunits and ribosomal proteins, specifically identified from the moth stage may be involved in the degradation of old cuticle proteins and new cuticle protein synthesis. Gene ontology analysis of proteins specific to each of these three stages enabled their association with cellular component, molecular function, and biological process categories. The analysis of similarities and differences in these identified proteins will greatly further our understanding of wing disc development in silkworm and other insects.     

Reciprocal transplantation of wing discs between a wing deficient mutant (fl) and wild type of the silkworm, Bombyx mori

The wingless mutant flügellos ( fl ) of the silkworm lacks all four wings. Although wing discs of the fl seem to develop normally until the fourth larval instar, wing morphogenesis stops after the fourth larval ecdysis, probably caused by aberrant expression of an unidentified factor, referred to as fl . To characterize factor fl , the wing discs dissected from the wild-type (WT) and fl larvae were transplanted into other larvae and developmental changes of the discs were examined. When the wing disc from a WT larva was transplanted into another WT larva and allowed to grow until emergence, a small wing appeared that was covered with scales. Thus, the transplanted wing discs can develop autonomously, form scales and evert from adult skin. The WT wing discs transplanted into the fl larvae also developed at a high rate. However, the fl wing discs transplanted into the WT larvae did not develop during the larval to pupal developmental stages. These data suggest that the fl gene product (factor fl) works in the wing disc cells during wing morphogenesis. Its function cannot be complemented by hemolymph in the WT larva. It is also implied that the level of humoral factors and hormones required for wing morphogenesis are normally maintained in the fl larva.     

A scaleless wings mutant associated with tracheal system developmental deficiency in wing discs in the silkworm, Bombyx mori

A mutant of Bombyx mori has wings with few scales and is named scaleless. We investigated the morphology of this mutant and found that it had many fewer wing scales than the corresponding wild type (WT) silkworm and that the remaining scales were smaller in shape with fewer furcations. Reciprocal transplantation of wing discs between scaleless and WT revealed that the WT wing disc could develop into a small wing with scales after transplantation into a scaleless larva; however, the scaleless wing disc developed into a small wing without scales in a WT larva. Upon dissection of WT and scaleless wing discs at different stages from the fifth instar larva to adulthood, no obvious differences were found before pupation. However, after pupation, tracheae produced from WT wing veins extended to the lacunae between the veins and formed a network on the second day after pupation, whereas this did not happen in scaleless. At the same time, no marked difference in adult body tracheal development was found between the mutant and wild type. Furthermore, if the surface of a wing disc was cut and its veins injured, the resulting wing also had fewer scales than the corresponding WT. Also, we found that higher partial pressure of O2 could rescue the loss of scales in scaleless. These data suggest that the factors affecting the growth of scales were not produced in the hemolymph, but in the wing disc itself. It is also implied that wing scale development is dependent on the correct organization of the tracheal system in the wing disc.     

Comparative transcriptome analysis of wing discs from Bombyx mori and Bombyx mandarina

The insect wing is developed from the wing imaginal disc which is designed from the embryonic ectoderm. To get insight into gene expression profiles in wing discs of Bombyx mori during metamorphosis, we compared the gene expression in the wing between B. mori and B. mandarina moth through RNA-seq. Out of total valid reads identified from the 5th day of 5th instar larvae of silkworm (L5), 7th day of pupae (P7), 1st day of moth (M1) and 1st day of wild silkworm moth (WM1), 20,092,004, 29,251,647, 24,654,695 and 19,753,089 reads were mapped to the mRNA reference sequences of silkworm, respectively. 9229, 7048, 9268 and 6701 differentially expressed genes (DEGs) were respectively recorded in P7 vs L5, M1 vs P7, M1 vs L5 and WM1 vs M1. Further, the peroxisome, ribosome, endocytosis and oxidative phosphorylation pathways were significantly regulated in the metamorphosis of the silkworm. Our study identified 16 orthologous genes with a positive selection from M1, which might be subjected to artificial selection in the domestication of B. mori and would play vital roles in the flight of B. mandarina.     

Death commitment in the anterior silk gland of the silkworm, Bombyx mori

The insect steroid hormone, 20-hydroxyecdysone (20E) triggers the programmed cell death (PCD) of the anterior silk glands (ASGs) of the silkworm, Bombyx mori. We tried to determine the time of commitment to die (death commitment) by examining ASG responses to 20E and juvenile hormone analogue (JHA) in vivo as well as in vitro. The ASGs obtained late on day 6 of the fifth instar completed PCD when cultured with 20E, while the ASGs obtained on day 4 and cultured with 20E did not undergo PCD. The ASGs became competent to respond to 20E at mid-day 5. The ASGs with responsiveness to 20E were not sensitive to JHA, indicating that the ASGs were committed to die before becoming capable of responding to 20E. Topical application of JHA on day 4 suppressed 20E-induced PCD, but that on day 5 failed to do so, indicating that the death commitment might occur between day 4 and 5. We also determined the time of death commitment after allatectomy of the fourth instar larvae, a procedure that induced the precocious PCD. Timed application of JHA and culture of ASGs with 20E in the presence of JHA showed that the ASGs had lost their sensitivity to JHA between 72 and 96 h after allatectomy, i.e. 24-48 h before precocious gut purge in the allatectomized larvae. This result is similar to that obtained in the fifth instar. We conclude that the cellular commitment to die takes place one day before the ASGs become competent to respond to 20E.     

Adenylate cyclase in prothoracic glands during the last larval instar of the silkworm, Bombyx mori

We have previously reported that the absence of prothoracicotropic hormone (PTTH) signal transduction during the early last larval instar of Bombyx mori plays a role in leading to very low ecdysteroid levels in the hemolymph, inactivation of the corpora allata, as well as larval-pupal transformation. In the present study, adenylate cyclase was characterized in crude preparations of prothoracic gland cell membranes in an effort to localize the cause of refractoriness to PTTH. It was found that cyclase activity of the prothoracic glands from the day 6 last instar showed activation responses to fluoride, a guanine nucleotide analogue, as well as calmodulin (CaM) in dose-dependent fashions. The additive effects of day 5 prothoracic gland adenylate cyclase stimulation by fluoride and CaM imply that there may exist Gs protein-dependent and CaM-dependent forms of adenylate cyclase. For day 1 last instar prothoracic glands, which showed no response to stimulation by PTTH in either cAMP generation or ecdysteroidogenesis, adenylate cyclase activity exhibited far less responsiveness to Ca(2+)/CaM than did that from day 5 glands. These findings suggest that day 1 prothoracic glands may possess some lesions in the receptor-Ca(2+) influx-adenylate cyclase signal transduction pathway and these impairments in PTTH signal transduction may be, at least in part, responsible for decreased ecdysteroidogenesis.     

Hormonal control of ovarian development in the silkworm,Bombyx mori

Hormonal control of ovarian development was examined in Bombyx mori. The weight of the ovary increased suddenly by 3 days after pupal ecdysis, and vitellogenin could be immunologically detected in the ovary at that time. The ecdysteroid titers during pupal-adult development, quantified by radioimmunoassay, increased from day 0 to day 2. Ovarian development was arrested for a long period in brainless pupae and isolated pupal abdomens. Injection of 20-hydroxyecdysone into such preparations stimulated development of the ovaries, and vitellogenin could be detected in ovaries 2 days after injection. The results suggest that 20-hydroxyecdysone acts by stimulating the growth of ovary.     

Structure and expression of mRNA for a pupal cuticle protein of the silkworm,Bombyx mori

Electrophoretic and immunoblot analyses of proteins extracted from the salt-washed integuments of the silkworm Bombyx mori demonstrated that the pupal cuticle contains structural proteins distinct from those present in the larval cuticle. The cDNA clone encoding a pupal cuticle protein was isolated from the cDNA library constructed from epidermal mRNA of pharate pupae. Northern blot hybridization by use of a cDNA probe provided evidence that mRNA for the pupal cuticle protein accumulate in integument during larval-pupal transformation, though temporal rise of the mRNA level was also noticed at the stages of larval molting. Primary structure of the pupal cuticle protein was deduced from the nucleotide sequence of cDNA. The cloned mRNA sequence encodes a 27 kDa protein rich in alanine and proline, containing characteristic repeats of Ala-Pro-Ala-His-Gln-(Asp/Ser)-Trp-Asn sequence in the carboxyl-proximal domain. The sequence (Ile/Val)-(Leu/Ala)-(Asp/Glu)-Thr-Pro-Glu-Val-Ala-(Gln/Ala)-Ala-Arg-Ala-Ala-His-(Leu/Ile)-(Ala/Ser)-Ala-(Leu/His) occurs in three hydrophobic domains of the molecule.     

Proteomic profiling of the hemolymph at the fifth instar of the silkworm Bombyx mori

Abstract Two‐dimensional gel electrophoresis (2‐DE) followed by matrix‐assisted laser desorption ionization – time‐of‐flight/time‐of‐flight mass spectrometry (MS) analysis were used to charaterize the hemolymph proteomic profiles of the silkworm, Bombyx mori. At days 4 (V4) and 5 (V5) of the fifth (final) instar, when the larvae were at the fast‐growing stage, we found dramatic changes in spots representing proteins having an approximate molecular weight (MW) of 30 kDa. Of these spots, four 30K proteins were highly up‐regulated, implying a close association with the growth and development of B. mori larvae. To understand the molecular basis and underlying mechanisms involved in development and metamorphosis, the proteome of whole hemolymph at V5 was analyzed using shotgun liquid chromatography tandem mass spectrometry with an LTQ‐Orbitrap. A total of 108 proteins were identified without any false discovery hits. These proteins were involved in a variety of cellular functions, including metabolism, development, nutrient transport and reserve, and defense response. Gene ontology analysis showed that 3.4% of these proteins had nutrient reservoir activities and 5.7% were involved in the response to stimulus. Pathway analysis revealed that 22 proteins with common targets were involved in various cellular processes such as immunity, differentiation, proliferation and metamorphosis. These results suggested that some key factors such as the 30K proteins in hemolymph play important roles in B. mori growth and development. Moreover, the multiple functions of hemolymph may be operated by a complex biological network.     

Nutritional and hormonal effects on storage proteins in the silkworm,Bombyx mori L.

Starvation of 48 h old fifth instar larvae depressed storage protein titres initially for 48 h but retained the levels comparable to control thereafter, possibly due to nutrients obtained during the 48 h feeding after fourth ecdysis. After an initial decline ligated larvae accumulated maximum storage proteins in haemolymph. This is because of inhibitory juvenile hormone titre at the basal level besides the appropriate release of 20-hydroxyecdysone from the ectopic source(s). Injection of methoprene (10 Μg/larva) repressed accumulation of storage proteins while 20-hydroxyecdysone (10 Μg/larva) increased the same. P-soyatose injection to starved and ligated larvae accelerated storage protein accumulation in haemolymph, signalling nutrient indispensability for initiation of storage protein synthesis at the appropriate time of last instar development inBombyx mori.     

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.