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Progenies from some wild-caught females of Drosophila willistoni and three other sibling species are entirely female. The proclivity for production of unisexual female progeny by these flies was named the sex ratio (SR) trait and was originally thought to be genetic. However, experiments in the laboratory of Donald F. Poulson in the early 1960s demonstrated that this 'trait' was vertically transmitted and infectious, in that it could be artificially transferred by injection from infected females to non-infected females. Motile, helical micro-organisms were observed in females showing the trait. In 1979, the SR organisms were designated as group II in the informal spiroplasma classification system. The organisms proved to be extremely fastidious, but were eventually cultivated in a very complex cell-free medium (H-2) after initial co-cultivation with insect cells. Cultivation in the H-2 medium and the subsequent availability of a triply cloned strain (DW-1T) permitted comparative studies. Cells of strain DW-1T were helical, motile filaments 200-250 nm in diameter and were bound by a single trilaminar membrane. Cells plated on 1.8% Noble agar formed small satellite-free colonies 60-70 microns in diameter with dense centres and uneven edges. The temperature range for growth was 26-30 degrees C; optimum growth occurred at 30 degrees C, with a doubling time in H-2 medium of 15.8 h. The strain passed through filters with 220 nm, but not 100 nm, pores. Reciprocal serological comparisons of strain DW-1T with representatives of other spiroplasma groups showed an extensive pattern of one-way crossing when strain DW-1T was used as antigen. However, variable, usually low-level reciprocal cross-reactions were observed between strain DW-1T and representatives of group I sub-groups. The genome size of strain DW-1T was 2040 kbp, as determined by PFGE. The G + C content was 26 +/- 1 mol%, as determined by buoyant density and melting point methods. The serological and molecular data indicate that strain DW-1T is separated from group I representative strains sufficiently to justify retention of its group status. Continued group designation is also indicated by the ability of SR spiroplasmas to induce male lethality in Drosophila, their vertical transmissibility and their extremely fastidious growth requirements. Group II spiroplasmas, represented by strain DW-1T (ATCC 43153T), are designated Spiroplasma poulsonii.

We demonstrate the intracellular transport of secretory granules of a silk protein, fibroin, from the Golgi region to the apical cytoplasm with special reference to microtubule organization, electrolyte concentrations and the acidic intragranular pH of normal and mutant posterior silk gland cells, using the techniques of electrophysiological microelectrode and microprobe analysis and of light and electron microscopic autoradiography. The silk gland cells of a recessive mutant making only flimsy cocoons were defective in the microtubule systems, did not stain with an anti-tubulin antibody in immunofluorescent microscopy, and accumulated intracellular granules in the apical and basal cytoplasm. The increase in intracellular calcium concentration and levels of chloride secretion were also reduced in the mutant cells. A carboxylic ionophore, monensin, which collapsed the granular H+ gradient, induced the transport of chloride and an increase in the intracellular calcium concentration, while it blocked the intracellular transport of granules from the Golgi region to the apical cytoplasm in normal cells. Thus, we conclude that the H+ gradient across the membrane of secretory granules is responsible for the intracellular transport of the secretory granules along the microtubule systems in silk gland cells, while Ca2+ is thought to be required for the exocytosis of the granules.

Cells from Xenopus kidneys were cultured. Incorporations of 3H-uridine, 3H-thymidine and 14C-protein hydrolyzate were examined, and synthesized RNA was electrophoretically analyzed. The synthesis of RNA, DNA and protein was found to take place at different rates at different phases and the synthesis of ribosomal RNA, as well as other macromolecules, was quite active during the early logarithmic phase. The number and size of nucleoli varied markedly during culture, and these changes were correlated with the activity of cellular RNA synthesis.

Two preparations of trypsin inhibitor were purified from yolk, one from unincubated hen's eggs and the other from those at day 16 of incubation. Both were indistinguishable from albumen ovomucoid with respect to molecular weight, isoelectric point, peptide map after limited proteolysis, immunological reactivity and so forth. Although a gap in specific activity was observed, it was concluded that the yolk inhibitors were species of ovomucoid. This suggested a flow of ovomucoid from egg white to yolk during embryonic development.

The eggs of Bombyx mori, both in diapause and nondiapause, were subjected to cytological examination of nucleoli and measurement of RNA precursor incorporation (2 hours) into ribosomal RNA. In diapause eggs, the nucleoli were very small and the rate of ribosomal RNA synthesis was the lowest of the samples tested. Most cells in diapause possessed nuclei with one nucleolus. In contrast, the eggs activated from diapause by long chilling attained the largest size of nucleoli and the highest rate of ribosomal RNA synthesis. A significant proportion of the cell nuclei still had only one nucleolus at this stage. Three days after activation, the eggs exhibited intermediate levels in both the size of nucleoli and the rate of ribosomal RNA sythesis. At this stage, about half of the egg cell nuclei had two nucleoli.

The effects of SRO (Sex-ratio organism; previously called SR spirochetes) infection on the viability of gynandromorphs of D. melanogaster were examined. A fraction of gynadromorphs, most of them with small areas of XO tissue, survived, but the majority were not viable when XO nuclei or cells are affected by the infecting SRO. Apparently the replacement of lethal XO nuclei or cells by adjacent XX ones does not take place. Some possibilities are discussed as to the time and the primary site of the lethal action of SRO.

Undegraded polysomes could be extracted from eggs of Bombyx mori by cutting egg shells with a blade in a buffer containing high salt. The polysome content as measured by this method increased steeply during post-diapause development, which was commenced by long term chilling followed by hot HCl treatment of diapausing eggs. At the 24th hr of the post-diapause development the polysome content became 2.6-times the level at 0 h. The alteration of the polysome content paralleled that of the incorporation of [14C]leucine into acid-insoluble fractions investigated by modified Takami's embryonic culture system.

The formation of segments of Bombyx larvae involves differentiation of legged and legless segments during embryogenesis. Observations by light microscopy of serial sections of developing embryos of Bombyx showed that the cell number of the ectodermal layers increased more rapidly in segments where legs were being extruded than in those where no appendages were formed. In the embryos of a homoeotic mutant for the E-pseudoallelic locus (about 0.0–VI), ETc/ETc, in which all the abdominal segments were legless, the cell number of the ectodermal layers did not increase as in normal embryos. These findings suggest that the ETc gene controls the cell number of the ectodermal layers in relation to the differentiation of abdominal segments.

The activity of ribosomal RNA (rRNA) genes as judged by nucleolar size and rRNA synthesis has been shown to depend upon the phase of diapause in the eggs of Bombyx mori. In the present study, we found that nucleolar size in diapausing eggs was enlarged at a very early stage during cold treatment, a procedure necessary for the termination of diapause. In contrast, the intrinsic capacity of ribosomal RNA synthesis in the chilled eggs, as examined at 25°C by radioactive precursor incorporation into rRNA, increased much later, in parallel with the break of diapause. The early phase of cold treatment is the period when the eggs undergo some important changes (the so-called diapause development), preparing for diapause termination. Thus we infer that the above mentioned increase in nucleolar size may be one of the features of diapause development.

The possible role of cytoplasmic sex ratio (SR) distortion in insect population control has been assessed experimentally. In Drosophila melanogaster the agent responsible for SR distortion is a male-killing spiroplasma and SR strains are characterized by females producing daughters but few, if any, sons. Populations were constructed of wildtype and SR females (of various male-kill effectivenesses) plus males, in different proportions. The frequency of females in the populations was monitored regularly. Under some circumstances the frequency of females increased but not to fixation or population collapse. In most instances the frequency of females declined (to as low as 25% in some cases), then went through cycles of increase and decrease, with gradually reduced amplitude over time. This pattern of fluctuations was repeatable over cages.Independently, it was shown that SR females persisted in the cages for the length of the experiments. The unexpected extremely low frequencies of females found in cages established with an excess of females, raised the possibility of distorting a population by SR releases, and then when the female frequency is at its lowest point to add sterile male or similar load inducers to suppress the population. The possible role of density dependence is discussed.

1. 1. On polyacrylamide gel electrophoresis, chymotrypsin inhibitors in the larval hemolymph of the silkworm were found as 15 electrophoretically distinct bands. 2. 2. One of them, CI-13 migrating fastest toward the anode, was purified from the hemolymph. 3. 3. The inhibitor was a monomeric protein with a molecular mass of 14,000 and showed stability for both heat and a wide range of pH. The isoelectric point was pH 4.1. 4. 4. CI-13 was able to inhibit chymotrypsin completely and trypsin slightly, but was ineffective against other proteinases such as papain, ficin, carboxypeptidase A, V8 proteinase, serratia peptidase, cocoonase and proteinases derived from gut juice of the silkworm.

Total RNA extracted from diapause eggs of Bombyx mori was found to be translatable in a cell-free heterologous protein synthesis system derived from wheat germ or rabbit reticulocyte lysate. This supports the idea that active mRNAs are contained in the polysomes of diapause eggs.

Cultured kidney cells of Xenopus laevis were pulse-labeled with [ 3 H]uridine for 10, 20 and 30 min during their logarithmic growth phase and then processed for autoradiography. The labeled cells were assigned into two categories, one- and two-nucleolated cells, and the rate of ribosomal RNA (rRNA) synthesis was measured by counting the number of grains in nucleoli. The results obtained revealed that a two-nucleolated cell incorporated significantly much more radioactivity into its nucleoli than did a one-nucleolated partner for all the periods examined. Cells of these different nucleolar types, however, contained essentially the same amount of rDNA (DNA complementary to rRNA) as estimated by in situ hybridization with [ 125 I]rRNA. Although it remains to be proved that the observed increase in incorporation represents the increased rate of rRNA synthesis in two-nucleolated cells, the present findings seem to be very interesting, since they might indicate that the activity of rRNA genes is in some way regulated or affected by their spatial relationship in a cell nucleus.

Eggs of Bombyx mori are aroused from diapause by long-term chilling and develop when transferred to 25°C. During the first 20 hr of post-diapause development, the polysome content and the presumed rate of protein synthesis increase about 3-fold, while the ribosome content and the total RNA content increase only 1.1-fold. In this study, total RNAs were extracted from chilled eggs (termed 0 hr of development), and post-diapause eggs at 10 and 20 hr of development. The RNAs were purified further by high pressure liquid chromatography to remove RNA-like oligonucleotides. On translation in a protein-synthesizing system derived from wheat germ with a subsaturating amount of RNA, no difference was found in the relative amounts of translatable mRNA activity at 10 and 20 hr of development from that at 0 hr. Moreover, the translation products of the different RNA preparations in a rabbit reticulocyte lysate system appeared very similar when separated by gel electrophoresis and located by fluorography. These facts suggest that protein synthesis in early post-diapause development is controlled at a translational level.

The mucous glands of Bombyx pupae secrete glue proteins which attach deposited eggs to the mounting sheet. A mutant of a dominant gene, named no glue (Ng), produces nonadhesive eggs which have a low capacity for glue-protein synthesis. In the present study it was shown that the mucous glands of Ng silkworms showed rapid degradation of mRNA as well as rRNA during development; this may cause the low capacity for glue-protein synthesis in the mutant organ. In contrast, the mucous glands of normal silkworms showed a significant increase in content of RNA's until the maximum rate of glue-protein synthesis was achieved. The degradation of RNA in the Ng mucous gland was inhibited by actinomycin D injected into the body fluid. Thus it is supposed that the Ng gene codes for a presumptive controller RNA, which would be the mediator of RNA instability in the mucous glands of Ng pupae.

The surface patterns and the structures of transverse sections of the egg-shells of mutant Grey egg alleles in the silkworm, Bombyx mori, have been examined with the scanning electron microscope. The principal surface pattern of the egg-shell is a network of hexagons or pentagons. The shapes and the sizes of the polygons are different among the strains of Grey egg alleles. The micropyle on the anterior end is surrounded with a pattern like a single-petaled flower. These patterns do not so much differ among strains of Grey egg alleles. Small pores, aeropyles, open at each corner of the polygons, on the knobs which rest in every polygon, or at the furrow between the knobs. The location of the aeropyles differs among different strains. The structures of transverse sections of the egg-shell are composed of the outer, middle, and inner layers. Grey egg alleles affect mainly the formation of the middle layer in the egg-shells and cause an abnormal lamellar structure in the layer. The gene interactions between Grey egg alleles are also noted in the formation of the middle layer.

Fifth-instar larve with genotypes +/leml and lem/leml, which had segregated from a cross between +/leml and lem/leml, were used as material. The ovaries of the larvae of both types were exchanged. Of 630 larvae thus operated upon, only 3 survived and had offspring.The female moths produced from these larvae were mated with male moths of the genotype lem/leml. The eggs laid were incubated and larvae homozygous for leml gene were examined.When the +/leml larvae were implanted with ovaries of lem/leml, the homozygous leml embryos which developed from the transplanted eggs hatched out, as the apparently normal ones do, as black-coloured larvae, but they changed into distinctly yellowish larvae which died immediately after the first moulting.On the other hand, the leml homozygous larvae emerging from eggs laid by the lem/leml moth which had received a +/leml ovary by transplantation developed into yellowish-brown young larvae, then died within the egg.From these results it may be concluded that the stage of death of the lethal larvae is not determined by the genotype of the embryo (+/leml or lem/leml) but by that of the mother's body in which the eggs develop.

The dominant mutant “Retarded growth” (symbol Rg) was found as a gamma-ray induced phenomenon. It is characterized by a departure from the normal type representing distinctly delayed larval growth. The homozygote is completely lethal in embryonic stage.Linkage test showed the Rg locus to be in the third chromosome at 24.9. Further crosses with the recessive gene sm(N) gave without exception pseudo-dominantly manifested sm(N) character in Rg females. While the crosses with allelic sm(S) gave sm(S) character in only a part of Rg females, which is explained as due to a position effect. It is concluded from these results that Rg is not a gene mutation but a deletion of intercalary chromosomal segment. The left break is at 24.9 and the right break is just left by the sm(S) locus at 41.8. The deficit is about 17 units of the length of the third chromosome.

1. 1. RNAs from unfertilized and fertilized eggs (entering diapause or activated by acid treatment) of Bombyx mori showed variable translation activities in a rabbit reticulocyte lysate system. 2. 2. However, the translation products of the RNAs were similar when separated by electrophoresis and located by fluorography. 3. 3. The major set of maternal mRNAs, and/or comparable ones, may predominantly exist in the eggs tested.

Interference between two "sex-ratio" agents—that is, treponemalike spirochetes—of different origin, one from Drosophila willistoni and the other from D. nebulosa, was demonstrated by experiments in vivo and in vitro. When the two sex-ratio agents were combined in females of the Oregon-R strain of D. melanogaster, expression of the sex-ratio condition was temporarily interrupted. Several lines of evidence indicate that a substance produced by the sex-ratio agent of D. nebulosa may kill or incapacitate the sex-ratio agent of D. willistoni.