The silkworm Bombyx mori L., a unique biological system with several means of artificial reproduction, was used in experimental analysis of some problems and possible developments in parthenogenesis and cloning. Results obtained on reinvestigating the cytological mechanism of ameiotic thermoparthenogenesis proved to be in good correspondence with Astaurov's original scheme based on his genetic data. The maternal genotype of the diploid female pronucleus as the basis for cloning is the result of the prevention of the first meiotic division and absence of crossing over in females. Heat shock treatment at thermoactivation is supposed to trigger off egg activation processes, simultaneously destroying modified synaptonemal complexes between homologues and the spindle fibers destined to perform the reductional division. These changes in egg microarchitecture, induced by the heat shock, were shown to be reversible unless fixed with abrupt cooling following the shock.
By definition, cloning is a closed genetic system of a single genotype. Corrections of the genotype through gene or chromosome recombination appear impossible unless outcrossing is used and new genetic material inevitably replaces some regions in the cloned genotype. This “contamination” with unknown genetic material can be excluded: ameiotic and meiotic types of parthenogenesis allow such corrections through “self-fertilization” and/or reversion of tetraploid eggs persisting in any clone to the diploid level. This procedure may sometimes result in decreasing of the ability to undergo thermoparthenogenesis, which can be compensated with ovary transplantation from the homozygous donor into best heterozygotic parthenoclones.
Parthenoclones and “self-fertilization” were used in the study of the ability to undergo spontaneous parthenogenesis. The latter can be increased through this type of inbreeding up to 23% hatching and we can soon expect elucidation of the factors causing egg activation during the process of egg laying, which, in turn, might eliminate the “principal” differences between spontaneous (natural) and artificial types of parthenogenesis. The molecular analysis of these factors and their genetic basis could be considered not only as solving the problem of parthenogenesis but also would give us new opportunities to control the earliest stages of individual development.
Gradual cooling of the egg (from temperatures above 40°C) soon after its artificial or natural activation produces a deactivating effect, which, in a definite sense, brings the egg back to the point before activation from where it can be reactivated in different ways. If cooling is quick enough (between 40°C and 0°C) these “non-activating” temperatures can effectively activate the egg. Deactivation and reactivation can be repeated and even performed in vivo and in situ, i. e., inside live moths at temperatures below 43°C. In the last case deactivated eggs were shown capable of normal fertilization.
A generalized concept of parthenogenetic engineering is proposed in the field of experimental cytogenetics dealing with the oocyte and producing new biological forms.

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cDNA of farnesyl pyrophosphate synthase (FPS; EC 2.5.1.10), a key enzyme in the formation of the sesquiterpene skeleton of the juvenile hormone (JH) was cloned from the silkworm Bombyx mori (BmFPS). BmFPS cDNA consists of 1743 nucleotides that encode 427 amino acids. Sequence analysis showed that BmFPS has two well-conserved FPS motifs of the five conserved in other FPSs; BmFPS shares 83% identity with the black cutworm moth Agrotis ipsilon, 49% with the fruit fly, Drosophila melanogaster, and 42% with the chicken, Gallus gallus. Analysis of BmFPS gene expression using RT-PCR revealed BmFPS to be distributed throughout tissues with some differences in magnitude. This contradicts our expectation that BmFPS is localized in the corpora allata. BmFPS could therefore catalyze isoprenylation of protein occurring in many tissues.

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Genomic Southern blotting analyses of Bombyx mori and B. mandarina were performed with the 5′-exons or 5′-flanking sequences of two members of genes A/B.L11 and A/B.L12. They represent multigene families encoding chorion proteins that are expressed in the middle period of choriogenesis. Many bands cross-hybridized with the probes in B. mandarina and in different strains and races of B. mori, supporting the idea that the middle chorion multigene families have remained essentially the same in the two species. Both the exon and the 5′-flanking probes exhibited similar patterns within the European stocks of B. mori, indicating that the examined DNA regions have been conserved. Patterns revealed in a Japanese and a Chinese strain showed distinct differences in band number and fragment size, indicating that the chorion gene families are not fixed within the domesticated silkworm. A greater number of fragments hybridized with the exon probes in B. mandarina than in B. mori; this may have relevance to the previous finding that B. mandarina has a large number of proteins with high cysteine content. The banding patterns for both the exon and 5′-flanking probes were markedly different between B. mori and B. mandarina, indicating that major evolutionary differences exist between the two species.

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Recombinant human serum albumin (rHSA) was produced using the recombinant Bombyx mori nucleopolyhedrovirus (rNPV) vector in silkworm larvae. rHSA was purified by ion exchange chromatography, affinity chromatography, and gel filtration chromatography from the whole-body homogenate of infected 4th instar larvae. No impurities were detected in the purified rHSA by reverse-phase chromatography and SDS-polyacrylamide gel electrophoresis. We obtained 3.48 mg of mature rHSA (removed preprosequence) from 24 4th instar larvae on day 4 postinfection (1×10² PFU/larva). These results suggest that efficient rHSA mass production is possible using the rNPV vector and B. mori 4th instar larvae.

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In Bombyx mori a dominant, autosomal mutation was confirmed to produce loosely constructed, elliptic cocoons, which have a cotton-like texture. The mutant gene was named “flossy” with the symbol Fl after a previous achievement by Takasaki. Genetic analysis applying the genes q and obt as markers mapped the Fl locus to the 32.1 position on the seventh linkage group. The normal individuals segregating from Fl in a cross, using the q gene as a marker, gave tight cocoons with a peanut-like shape. No characteristics except the cocoon size and shape were different between Fl and normal individuals. It was concluded that the Fl gene function was related to the spinning behavior during the cocoon formation.

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Genetic analyses in Bombyx mori established a recessive, spontaneous mosaic mutation, in which various types of mosaicism appear with a mode of maternal inheritance. The relevant gene, named the “mosaic of Tanaka” (symbol mo-t), was found to be linked with the p^s gene, which belongs to the second linkage group, and was located at the map position of 38.3. The mo-t gene is independent of the previously known mosaic genes including mo and l-mo, whose loci have not been revealed to date.

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The natural heterogeneity of nucleopolyhedrovirus of Spodoptera litura (SpltMNPV) was investigated. Plaquepurified geographic variants of SpltMNPV from China, C2, C9, and C10, were compared with the Philippine clone, P7. Restriction endonuclease analyses revealed a similar overall restriction patterns among the clones except for the deletion and/or insertion of some restriction enzyme fragments with respect to P7. Light microscopy studies showed that all clones induced productive infection in CLS-79, Sf9, and SpLi cells, non-productive infection accompanied by typical NPV cytopathic effects in cell lines BmN-4, Se301, and Ld652Y, and no infection in Splm cells. Clones P7 and C10 induced higher degree of apoptosis in Ld652Y cells than clones C2 and C9. All clones produced the highest amount of budded virions in SpLi cells. The amount of BVs produced by C9 in CLS-79 and Sf9 cells at 72h postinfection was significantly higher than those produced by the other clones. DNA accumulation patterns of the clones differed among the permissive cell lines. All clones produced polyhedrin of the same molecular weight in CLS-79, Sf9, and SpLi cells. SpLi cell line supported the highest production of polyhedrin for all clones. Results showed that the clones obtained from China were closely related to the clone from the Philippines. Differences in the observed biological characters among the SpltMNPV clones can be attributed to the minor differences in genome organization among the genotypic variants. The implications of the existence of natural heterogeneity of NPVs were discussed.

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A software designated as 3DASBS has been used for the analysis of the silkworm spinning behaviour. However, its use has been limited to the expensive workstations that can run specialized 3D graphics libraries. In this paper, we report on the development of a new visualization system of the silkworm spinning behaviour based on 3DASBS that can be run on the widely used Windows 98/NT platform. It can display a silkworm body more realistically by overlapping spheres. Since all the procedures are written as object classes using an object-oriented programming language, the system can easily be extended by inheritance of the classes.

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