Zenta Kajiura

Transgenic silkworms (Bombyx mori) produce recombinant spider dragline silk in cocoons

Spider dragline silk is a unique fibrous protein with a combination of tensile strength and elasticity, but the isolation of large amounts of silk from spiders is not feasible. In this study, we generated germline-transgenic silkworms (Bombyx mori) that spun cocoons containing recombinant spider silk. A piggyBac-based transformation vector was constructed that carried spider dragline silk (MaSp1) cDNA driven by the sericin 1 promoter. Silkworm eggs were injected with the vector, producing transgenic silkworms displaying DsRed fluorescence in their eyes. Genotyping analysis confirmed the integration of the MaSp1 gene into the genome of the transgenic silkworms, and silk protein analysis revealed its expression and secretion in the cocoon. Compared with wild-type silk, the recombinant silk displayed a higher tensile strength and elasticity. The results indicate the potential for producing recombinant spider silk in transgenic B. mori.

Myocyte enhancer factor 2 (MEF2) is a key modulator of the expression of the prothoracicotropic hormone gene in the silkworm, Bombyx mori.

Prothoracicotropic hormone (PTTH) plays a central role in controlling molting, metamorphosis, and diapause termination in insects by stimulating the prothoracic glands to synthesize and release the molting hormone, ecdysone. Using Autographa californica nucleopolyhedrovirus (AcNPV)‐mediated transient gene transfer into the central nervous sytem (CNS) of the silkworm, Bombyx mori, we identified two cis‐regulatory elements that participate in the decision and the enhancement of PTTH gene expression in PTTH‐producing neurosecretory cells (PTPCs). The cis‐element mediating the enhancement of PTTH gene expression binds the transcription factor Bombyx myocyte enhancer factor 2 (BmMEF2). The BmMEF2 gene was expressed in various tissues including the CNS. In brain, the BmMEF2 gene was expressed at elevated levels in two types of lateral neurosecretory cells, namely PTPCs and corazonin‐like immunoreactive lateral neurosecretory cells. Overexpression of BmMEF2 cDNA caused an increase in the transcription of PTTH. Therefore, BmMEF2 appears to be particularly important in the brain where it is responsible for the differentiation of lateral neurosecretory cells, including the enhancement of PTTH gene expression. This is the first report to identify a target gene of MEF2 in the invertebrate nervous system.

Identification of virus-specific polypeptides and translatable mRNAs in the isolated pupal abdomens of the silkworm, Bombyx mori, infected with nuclear polyhedrosis virus

Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that infection of the isolated pupal abdomens of the silkworm, Bombyx mori, with B. mori nuclear polyhedrosis virus (BmNPV) caused generation of a number of polypeptides with a concomitant decrease of cellular polypeptides. These generated polypeptides were not identified as viral structural polypeptides, but were characterized as the degradation products of cellular polypeptides, as evidenced by the reaction with specific antiserum against storage proteins 1 and 2. Immunoblot analysis using anti-BmNPV serum identified at least 14 virus-specific polypeptides in the BmNPV-structural polypeptides of the virus. In vitro translation and subsequent immunoprecipitation with anti-BmNPV serum showed that translation yielded at least 15 polypeptides at the expense of cellular polypeptides. Time-course experiments showed that the viral structural polypeptides and virus-specific translatable mRNAs were not detectable until 3 days postinoculation. On the basis of the fact that the isolated pupal abdomens are in an arrested state of development, the delayed onset of virus-specific macromolecule production in the infected isolated pupal abdomens, as compared with that in the developing intact pupae, implies that some cellular function associated with the pupal-adult development is an important prerequisite for the efficient commencement of BmNPV replication.

Induction of perfect superlarvae by the application of juvenile hormone analogue to starved larvae of the silkworm, Bombyx mori

Abstract Topical application of juvenile hormone analogue, methoprene, induced a supernumerary larval moult in the silkworm, Bombyx mori. The incidence changed greatly depending on developmental stages and physiological states of the methoprene-treated larvae. When methoprene was applied to feeding larvae, only those treatments from the middle of the 2nd instar until the middle of the 4th instar were effective. An 18-h starvation period from the beginning of the 4th instar and a dose of 1 μg of methoprene per larva were required for 100% incidence of the perfect superlarvae. Allatectomy had no effects on the induction of superlarvae by methoprene. The treated 4th-instar larvae ecdysed to the 5th instar without any delay compared to the controls, and underwent an additional larval ecdysis 4.5 days later. The induced 6th-instar larvae took 8.5 days until the onset of cocoon spinning. The induced superlarvae showed reduced growth rates but an increase of final mass due to prolonged feeding period. A sharp but reduced peak in ecdysteroid titre in the haemolymph appeared one and a half days prior to each larval ecdysis in the treated larvae, suggesting that methoprene provokes the extra larval moult through an additional release of ecdysteroids.

Storage proteins, vitellogenin and vitellin of wild silkworms, Antheraea yamamai, Antheraea pernyi and their hybrids

Abstract 1. 1. Hemolymph proteins, fat body proteins and ovary proteins of Antheraea yamamai, Antheraea pernyi and their hybrids were analyzed by SDS-PAGE. A 83 kDa peptide increased remarkably in larval hemolymph, but a 75 kDa peptide increased only in the hemolymph of female larvae. 2. 2. Immunoblot analysis using antisera of Bombyx arylphorin and female-specific storage protein showed that the 83 kDa peptide was Antheraea arylphorin, the 75 kDa peptide was Antheraea female-specific storage protein. 3. 3. We found that a 36 kDa peptide was A. yamamai -specific serum protein, a 34 kDa peptide was A. pernyi -specific serum protein. These two peptides were accumulated in the hemolymph at constant level through larval instar and were detected in hybrids of A. yamamai and A. pernyi . 4. 4. A 185 kDa peptide appeared in the hemolymph of female pharate pupae of A. yamamai and was accumulated in the ovaries of pupae. A 210 kDa peptide appeared in hemolymph of female pharate pupae of A. pernyi . A 170 kDa peptide was accumulated in the ovaries of A. pernyi pupae. These three peptides were related to vitellogenin and vitellin as shown by immunoblot analysis using anti-BmVg serum. It was also found that these three peptides were accumulated in the ovaries of Antheraea F 1 hybrids. 5. 5. A new female-specific protein (24 kDa peptide) was discovered only in the female fat bodies.

Spermiogenesis of the testes of juvenile hormone-treated silkworm larvae, Bombyx mori

Abstract 1. 1. To examine changes in the percentages of testicular cells such as 1C cells (spermatids and spermatozoa), 2C cells (G1 somatic cells, spermatogonia and secondary spermatocytes), 2–4C cells (somatic and germinal cells in S phase) and 4C cells (G2 somatic cells and primary spermatocytes), we isolated nuclei from the testes of silkworm larvae and subjected them to flow cytometric analysis. 2. 2. In control testes, 1C cells appeared at day 0 of the fifth instar, increased gradually by spinning and increased steeply at 2 days later still spinning. The percentage of 2C cells decreased gradually after ecdysis to the fifth instar. The percentage of 4C cells increased from day 2 to day 5 of the fifth instar and decreased after day 11 of this instar. Cells in S phase remained constant through the fifth instar. 3. 3. An injection of juvenile hormone analog, methoprene, at day 0 of the fifth instar did not inhibit spermiogenesis, but resulted in increased 2C cells and decreased 4C cells dose-dependently. In contrast, the same treatment at day 2 of the fifth instar did not change the percentages of 1C, 2C and 4C cells of the testes at all, suggesting that the testes changed sensitivity to the hormone at larval development. 4. 4. Repeated injections of methoprene to induce the appearance of dauer larvae resulted in a complete block of the development of 1C cells.

A new female-specific fat body protein of Antheraea pernyi: purification, immunological properties, developmental profile and synthesis

Abstract A new female-specific protein has been discovered to be a major component of fat body proteins of the last larval instar of Antheraea pernyi and Antheraea yamamai . The new female-specific protein was purified to homogeneity by gel-permeation chromatographies, DE-52 cellulose column chromatography and hydroxyapatite column chromatography. The molecular weight of the protein was estimated to be 24,000 by SDS-PAGE and 43,000 by gel-permeation chromatography, suggesting that the protein is a homodimer protein. The protein was temporarily named “24 kDa peptide” (24K). Developmental changes in 24K titer were followed from the final larval ecdysis to adult emergence by immunoblotting with a specific antiserum against 24K. 24K appeared in the fat bodies at day 9 fifth instar and remained at an almost constant level up to adult emergence. Immunoblot analysis using antisera against Bm 30K proteins and BmLSP showed that the immunological properties of 24K were different from those of these proteins. In vitro culture to label fat body proteins with [ 35 S]-methionine showed that the synthesis of 24K occurred in only female fat bodies. The function of 24K remains unknown.

Long-term preservation of eri and ailanthus silkworms using frozen gonads

Cryopreservation of eri and ailanthus silkworms using frozen gonads was investigated. First, we evaluated the freeze tolerance of ovary and testis in the eri silkworm, which showed high tolerance. Mating between frozen ovary-transplanted females and frozen testis-transplanted males produced 163.0 eggs, yielding 105.7 larvae per moth. In a second experiment, we tested the use of the eri silkworm as a host insect for gonad transplantation from ailanthus silkworm donors. A high success ratio for laid and hatched eggs was demonstrated for ovary transplantation (97.8 and 51.3 eggs per moth, respectively). For testis transplantation, however, the average number of hatched larvae was low (12.0). Mating between host eri females and males in which both frozen ovary and testis of the ailanthus silkworm had been transplanted produced 6.4 fertilized eggs per host moth. Our success in using cross subspecies cryopreservation between these wild silkworms could lead to the alternative use of hosts between species in other insects.