CYTOGENETICAL STUDIES ON THE CHROMOSOMES OF GLAND CELLS OF THE SILKWORM WITH SPECIAL REFERENCE TO THE STRUCTURE AND BEHAVIOR THE SEX CHROMOSOMES

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1 JAPAN. J. GENETICS Vol. 52, No. 5: (1977) CYTOGENETICAL STUDIES ON THE CHROMOSOMES OF GLAND CELLS OF THE SILKWORM WITH SPECIAL SILK REFERENCE TO THE STRUCTURE AND BEHAVIOR OF THE SEX CHROMOSOMES SACHIKO IT01~ Department of Experimental Radiobiology, Faculty Hokkaido University, Sapporo of Veterinary 060 Medicine, Received April 28, 1977 It has been known that sex determination of the silkworm, Bombyx mori, is simply controlled by the Y chromosome (Hasimoto 1933a; Tazima 1944). The chromosome number of the silkworm is 56 and the formula for both sexes is XY for the female and XX for the male (Tanaka 1916). A pair of sex chromosomes (X and Y) attached to the nucleolus can be observed in the limited stage (pachytene-diplotene) in the differentiation to nurse cells in ovarian cells (Kawaguchi 1933). But it is difficult to identify cytologically the Y chromosome in either gametogenic or somatic cells. However, a condensed body can be observed in the nuclei of silk gland cells of the females. This is called `heteropicnotic mass' by Springhetti and Oddone (1961) or `female sex heterochromatin' by Gillot (1968). The author prefers to use a term of `sex-heterochromatin body' or `SB'. In the silkworm polyploids are easily induced by various experimental methods; for example, centrifugal force treatment of eggs (Tanaka and Kawaguchi 1932) or high temperature treatment of eggs (Hasimoto 1933b; Astaurov 1957). The behavior of chromosomes in meiosis of the polyploid female was investigated by many workers (Hasimoto 1933b, 1934, 1935; Kawaguchi 1934; Tazima 1944). It has been revealed that in meiosis the pairing of autosomes takes place by chance between any two of four homologues, but as to the sex chromosomes, a problem remains whether there is a preferential pairing between two kinds of sex chromosomes. The present study was undertaken with the hope of elucidating the correlation of the sex-heterochromatin body and the Y chromosome, using diploids as well as polyploid individuals produced by cold treatment of eggs. MATERIALS AND METHODS 1) Strains A wild type strain Chinese 108 of the silkworm was used for general observations 1) Sapporo Present 060. address: Department of Radiology, Faculty of Medicine, Hokkaido University,

2 328 S. ITo of the silk gland. The strains used as the material for observations of the sex-heterochromatin body (SB) in polyploids were TW and RE, the former carrying the recessive genotype chick (chocolate, ), and the latter being normal. In order to obtain further information on the correlation of the SB and Y chromosomes, two other strains were used; YP carrying a translocated ;Y chromosome marked with p+ (Normal marking, 2-0.0) and psa (Sable, 2-0.0) originally bred by Tazima (1941), and OS carrying the sex-linked translucent gene (os, 1-0.0). These strains were kindly supplied by the Sericulture Laboratory of Hokkaido University. 2) Induction of polyploids Polyploids were obtained by cold treatment of newly laid eggs (Tamazawa 1967). The methods were as follows. Females were placed on a paper (egg card) to oviposit for 30 min, which permitted to collect roughly synchronized eggs. About three-hourold eggs were reserved Tat -10 C for 24 h, and then kept at 25 C for 20 h. Finally they were immersed in about 5 N hydrochloric acid at 46 C for 5 min, rinsed, dried and then kept at 25 C. After about three days, serosa cells could be observed under a dissective microscope. Eggs having large-sized serosa cells, about 1.2 times larger in diameter than the normal ones, were sorted out and cultured in the usual way. Almost all specimens thus obtained were found to be tetraploids based on the evidences, i.e. chromosome observations of spermatogenic cells and that the female laid very large eggs. 3) Microscopic techniques For the observation of the structure of the silk gland dissected glands of larvae were fixed in Carnoy's fluid (ethanol 3: acetic acid 1) and embedded in paraffin. Sections were cut about 4 pm thick and stained with staining agents; Schiff's reagent (basic f uchsine), methyl green-pyronin and azur-b. The procedures of the staining were as follows. 1) Feulgen's reaction: Preparations were treated with Schiff's reagent for 2 h after hydrolysis in 1 N hydrochloric acid at 60 C for 10 min. 2) Methyl greenpyronin : Preparations were stained for 2 h in a mixture of 0.05% methyl green and 0.2% pyronin in acetate buffer (ph 4.2). 3) Azur-B: After digestion with DNase (0.002% in phosphate buffer, ph 7.5) for 3 h, preparations were stained for 3 h in a solution of 0.025% azur-b in Mcllvaine buffer (ph 4.0). Furthermore, dissected glands were squashed in 45% acetic acid after soaking in a cell dissociating solution which was a 9:1:4 mixture of 0.5% acetic acid, glycerin and distilled water, and then the cover slips were removed by rapid cooling with dryice. The cytochemical methods were also applied to squashed preparations. For electron microscopic observations, silk glands were doubly fixed in a solution of 6.25% glutaraldehyde and a solution of 1% osmium tetroxide in cacodylate buffer (ph 7.4). After dehydration with aceton series, materials were embedded in Epon mixture and sectioned. Specimens were stained with a solution of 1 % uranium acetate in water. The number of SB in the silk gland nuclei was determined in preparations squashed with acetic orcein (1% orcein in 45% acetic acid). Chromosomes of spermatogenic cells were observed in acetic orcein squashed preparations after treatment with water

3 THE SEX CHROMOSOME OF SILK GLAND CELLS OF THE SILKWORM 329 for 30 min. RESULTS The silk gland consists of three parts, anterior, middle and posterior portions. In the silk gland cells, cytoplasmic division ceases during the embryonic stage, but endomitosis occurs throughout larval stage. The cells become very large in size, and highly polyploid nuclei are formed (Tanaka 1928). The nuclei are rectangular in form at the beginning of the third instar. Then ramification of the nucleus takes place within a few days. Many strands, about 0.3 pm thick, forming a network are observed in squashed preparations stained by Feulgen's reaction (Fig. 1). In squashed preparations of the silk gland, a spherical body, `sex-heterochromatin body (SB)', about 3 pm in diameter in the third instar, is found in all the nuclei of the female larvae (Fig. 1), but not in the male nuclei. The SB can be easily observed in acetic orcein squashed preparations during all larval stages. All the silk gland cells of the females have a single SB irrespective of the cell stage or the location of the cell on the gland. The SB is not found in the nucleus at any definite position in the squashed preparations. The SB is Feulgen-positive and stained with azur-b. In sectioned preparations the SB is not a homogeneous mass, but is surrounded by a layer of chromatin substances (Figs. 2 and 3) and this outside chromatin layer has a filamentous structure formed by coiled and folded fibrils, about 10.0 nm in diameter (Fig. 4). The chromosomes of spermatogonia and spermatocytes of male induced by cold treatment of eggs were observed. The representative figures of metaphase chromosomes of a spermatogonium of a diploid male and the induced tetraploid male are Fig. 1. A A nucleus network of silk gland cells structure of many squashed and stained by Feulgen's reaction. strands and densely stained SB (arrow).

4 330 S. ITo Fig. 2. SB in methyl green-pyronin stained preparation (about 7 'm thick section). Fig. 3. Fine structure of SB (about nm thick section). shown in Figs. 5 and 6. The chromosome numbers are 56 and 112, corresponding to 2n and 4n, respectively. The observations of 30 metaphase cells obtained from 20 larvae revealed that the modal number of the chromosomes of spermatogonia is 110, ranging from 103 to 118 (Fig. 7). These polyploids, therefore, were assumed to be tetraploid, although there were some variations in the number of chromosomes. Tetraploid females, which were mated to diploid males, laid large-sized eggs which were hatchable.

5 THE SEX CHROMOSOME OF SILK GLAND CELLS OF THE SILKWORM 331 Fig. 4. Fine structure of SB (about nm thick section). Fig. 5. Chromosomes of a spermatogonium of a diploid male. In the silk gland nuclei of 8) and there were no SB The two SBs are identical positions to each other, but two bodies fused together The number of the SB the induced tetraploid female, two SBs were observed (Fig. in the tetraploid male nuclei as observed in the diploid. in size. These two SBs in the female nuclei exist in near in some cells they make contact and on very rare occasions (Fig. 9). was counted in offspring larvae from the cross of tetraploid

6 332 S. ITO Fig. 6. Chromosomes of a spermatogonium of the in duced tetraploid male. Fig. 7. Distribution of the number 30 spermatogonia of induced of chromosomes in tetraploids. Fig. 8. Two SBs (arrows) of a nucleus of an induced tetraploid female.

7 THE SEX CHROMOSOME OF SILK GLAND CELLS OF THE SILKWORM 333 Fig. 9. SB of the squashed silk gland nuclei preparation. of an induced tetraploid female in an acetic orcein Fig. 10. Method for obtaining polyploid from the cross, RE X TW. off springs female (F1 of RE and TW) to diploid male (TW), the mating scheme of which is shown in Fig. 10. As shown in Table 1, concerning the number of SB, there are two types of females, one has a single SB (named SB1) and the other has two SBs (5B2). The SB was never seen in nuclei of male off springs (SBO). Both of the ratio of male to female and that of normal to chocolate were 5:1. The ratio of SB1 to SB2 in female was 4:1, showing that SB2: SB1: SBO was 1:4:1 in total. In order to gain critical information on the correlation of the SB and Y chromosomes, crosses of YP, having a translocated Y chromosome marked with p+ and psa of 2-chromosome, and OS were carried out (Fig. 11). When tetraploid females were mated to os/os diploid males, triploid offsprings were all translucent in phenotype. The

8 334 S. ITO Table 1. Larval phenotypes and number RE X TW of SB in triploid off springs from cross, Fig. 11. Method for obtaining polyploid from the cross, YP x OS. off springs segregation of plain (p) and Sable with Normal marking (p+ and psa) was observed on the body color and the larval marking. Two characters, Sable and Normal marking, were linked in all the individuals without exception. Comparing the density of pigmentation of the offsprings with their mother, there were two types; some were denser in color than their mother, and the others were normal. The former was denoted

9 THE SEX CHROMOSOME OF SILK GLAND CELLS OF THE SILKWORM 335 Table 2. Larval phenotypes and number of SB in triploid offsprings from cross, YPxOS `Dense' and the latter `Pale'. Plain larvae were very rare, and this type was represented as `Plain'. As shown in Table 2, most of the offsprings were `Pale' (97.0%). Both SB2 and SBO are very low in percentage, almost 1 % each. If SBO were male, the percentage of the male must be also about 1%. The percentage of male in triploid off springs from the other batches of the same cross which were raised up to the pupal stage was almost similar to those mentioned above. DISCUSSION It is well-known that the ploidy of silk gland nuclei of the silkworm is high. It was reported that the DNA content of a nucleus in the silk gland cell of the 5th instar larvae was more than 200,000 times of that of a spermatogonium cell (Nakanishi et al. 1969). According to Nakanishi et al. (1969), until the third day of the third instar, the nuclei were rectangular in form, and at this stage, thick and distinct strands, about 0.5 pm in diameter, were seen running nearly parallel to the long axis of the nucleus, being about 60 in number which approximately corresponded to the diploid chromosome number of 56. In the present study, however, Feulgen-positive strands appeared to be network (Fig. 1). This may be due to different techniques. Furthermore, it was difficult to visualize distinct doubling in number of the strands in the silk gland nuclei of induced tetraploid. The sex-heterochromatin body (SB) is always present in the female of the silkworm, while it can never be found in the male. It is well-known in wide variety of animals that the number of sex-chromatin masses is one less than the number of X chromosomes (Barr 1966). The sex-chromatin masses have been observed in females whose sex chromosome is homozygotic, and the frequency of the occurrence of such sex-

10 336 S. ITO chromatin masses has been almost 50-80%, although variable in species. The W chromatin of females whose sex chromosome was heterozygotic was observed in snakes by Kay-Chaudhuri et al. (1970) and its frequency was 30-80% in the female. Furthermore, the sex-chromatin in Lepidoptera was reviewed by Traut and Mosbacher (1968). The SB in spherical shape can be clearly observed in the silk gland nuclei of the silkworm, being almost 100% in the female and 0% in the male. In this respect the existence of the SB in the silkworm is a remarkable phenomenon, and this willl make it easy to discriminate sex in somatic cells. It has been known that sex determination in the silkworm is simply controlled by the Y chromosome. According to many investigations with the use of polyploids (Hasimoto 1933b, Kawaguchi 1934), experimentally produced tetraploid females laid larger eggs than those laid by diploid ones when crossed to diploid males. Characteristics of the females obtained from cooled eggs were the same as previously reported on tetraploid females. The chromosomes of spermatogenic cells had a modal value of tetraploid. Unfortunately, it was impossible to follow the successive generations, since they were sterile when crossed to normal females. From these results, it was concluded that these induced individuals were tetraploids. Induced tetraploid females had two SBs in the silk gland nuclei (Figs. 8 and 9). Observation of gonads and the number of the SB in triploid off springs of the crosses shown in Fig. 10, revealed that females had one or two SBs and males had no SB. It may be regarded that the individual is female when it had at least a single SB in the silk gland nuclei. Based on the data obtained in the experiments (Table 1), the ratio of normal to chocolate was estimated to be 5:1. Moreover, the segregation ratio of the number of the SB in triploid offsprings was SB2 1: SB1 4: SBO 1. If the number of the SB was equal to that of the Y chromosome, the constitution of the sex chromosome of the tetraploid mother must be considered to be XXYY. Since in tetraploid silkworm females `pairing' of autosomes appears to occur by chance between two of four homologues in meiosis (Hasimoto 1933b; Kawaguchi 1934), the genetic constitution of autosomes must be A+A+A~hAch. Whereas, sex chromosomes appear to behave differently from those autosomes. Two possibilities have been postulated on the behavior of sex chromosomes in meiosis of the tetraploid female. Hasimoto (1935) and Tazima (1944) assumed that there was preferential conjugation of two X chromosomes, while Kawaguchi (1934) postulated that pairing occurred at random between four homologues. On the basis of the number of the SB, the following four cases of the behavior of sex chromosomes in meiosis can be considered. 1) When the constitution of the sex chromosome is XXYY and there is sortative pairing between X and X or Y and Y (Fig. 12) and the homologues separate and migrate toward opposite poles at anaphase-i, a half of eggs is XX and others YY. 2) If separation of the paired homologues takes place at anaphase-ii, the eggs must be only XY. 3) Pairing occurs between X and Y (Fig. 13), and separation of paired chromosomes takes place at anaphase-i, eggs would be formed with the ratio of XX 1: YY 1. 4) If separation would occur at anaphase-ii, the ratio of the eggs must be XX 1: XY 2: YY 1. However, those possibilities do not actually support the observed ratio of the SB, 1:4:1.

11 THE SEX CHROMOSOME OF SILK GLAND CELLS OF THE SILKWORM 337 Fig. 12. Mode of pairing of sex chromosomes Paired chromosomes separate toward and in anaphase-ii (right). (X-X; Y-Y) opposite poles in anaphase-i (left) Fig. 13. Mode of pairing of sex chromosomes (X-Y; X-Y). Table 3 Ratios of eggs having various consittutions of sex chromosomes supposed behavior of sex chromosomes in meiosis of tetraploid females from the If random pairing occurs, 1/3 of pairing is X-X; Y-Y and others (2/3) being X-Y; X Y, then the seperation of homologues may take place at anaphase-ii and the eggs will be produced in the ratio of XX 1:XY 4:YY 1 (Table 3). This ratio of eggs having different sex chromosome constituents is consistent with the observed segregation ratio shown in Table 1. It is, therefore, assumed that there is random pairing between four homologues, and separation of the paired homologues takes place at anaphase-ii in meiosis of the tetraploid female. The interpretation, however, does not satisfactorily explain the findings made by previous workers. During continuation of a series of experiments on tetraploid females, Hasimoto (1933b) obtained an evidence that two X chromosomes undergo separation invariably. By thermal treatment of Fl eggs of females X+Y mated to d-translucent males (X dx d), he obtained tetraploid females, whose genotype was assumed to be

12 338 S. ITo X+X dyy. These tetraploid females gave a 5:1 sex ratio when back-crossed to diploid. X dx d males. The segregation of d-translucent to normal occurred, however, according to 1:1 ratio in both sexes. The results indicated that the tetraploid female possessed two X's in addition to two Y's. From these findings Hasimoto (1933b, 1934) considered that pairing occurs between two X's but not necessarily between two Y's. By using a translocation strain (Y p+ psa), Tazima (1944) induced a tetraploid females with two Y's, having the constitution X $X dy p+ psay p+ psa Half of these females were back-crossed to diploid sex-linked translucent males and the other half to d- translucent males. The results showed that the tetraploid mothers carrying two Y's had both X S and X d, and that dense pigmented YY triploid daughters possessed one of those two X's from the tetraploid mother and one X from the diploid father. In the present study, the frequency of SBO in triploid offspring from tetraploid females having similar genetic constitution to those used by Tazima (1944) was only 0.89% as shown in Table 2, showing clear difference from 17.0% obtained in case of tetraploids having free Y's, (RE x TW hybrids in Table 1). The difference of the result may presumably be due to the characteristics of the strains. In the experiment of Tazima (1944), the frequency of the male was 6.5%. Although this value is higher than the data in the present study, it is much lower than the data reported by the other authors, 17.1% (Hasimoto 1933b), or 21.6% (Kawaguchi 1934). This raises the question of why the frequency of the male was so low. A possible speculation was that preferential conjugation between two Y chromosomes was apt to occur by an increased affinity of the translocated 2-chromosome fragment (Tazima 1944). The incidence of exceptional larvae in the present experiment, `Dense' with one SB and `Pale' without SB, suggests a possibility of elimination of the translocated fragments (Tazima 1944). But, such an event has never occurred so far in the diploid strain of YP. Tetraploid females obtained in the present crosses, YP x OS, must have been X $X $Y p+, psay, p+, psa, because triploid offsprings were all translucent. These induced females could have been produced by the doubling of a fertilized nucleus in the cleavage stage (Takizawa and Tamazawa 1977, in press). Since both X's are marked with the same gene, no information on the behavior of X chromosomes could be obtained in the present experiment. It seems that some following questions have still remained on the hypothesis of the preferential conjugation of X chromosomes. In Hasimoto's (1933b) experiment, there were three batches in which triploid offsprings could not be considered to give a ratio of + d 5 : + ' 1: od 5 : od 1, because the d-translucent males were extremely rare. In Tazima's (1944) experiment `Dense' females were less than males of complementary type. He postulated that `Dense' individuals might be less viable. But such a decrease of viability was never found in SB2 larvae (Table 1) in the present experiment (RE x TW). Although, in the crosses, YP x OS, the percentage of `Dense' females was less than 1 % (Table 2). Observing a sex ratio with a variation of wide range in triploid offsprings, Hasimoto (1941) inferred that the cause of variation lay in the irregular distribution of the Y chromosome (non-disjunction of Y's) in the meiosis of the tetraploid females. It seems that the ratio of SB2 1: SB1 4: SBO 1 may be explained.

13 THE SEX CHROMOSOME OF SILK GLAND CELLS OF THE SILKWORM 339 that non-disjunction of Y chromosomes occurred in one of three cases (Table 1). The low percentage of SB2 and SBO in the crosses, YP X OS, represents that the frequency was very low (Table 2). W-chromatin of the snakes had a characteristic of a late replication of DNA and was formed due to the condensation of the W (Y) chromosome (Ray-Chaudhuri et al. 1970). Gillot (1968) showed that the DNA synthesis of the female sex-heterochromatin, which corresponded to the SB in the present study, and that of the autosomes of the silkworm was asynchronous. Although the correlation between phenotypic expression and the appearance of SB is not necessarily complete, it may be assumed that the SB corresponds to the Y chromosome. The SB was also observed in the female nucleus of a Malpighian tubule of the silkworm. The present findings will contribute to investigations of sex determination as well as the mechanisms of sex mosaicism. SUMMARY 1) The structure of the nuclei in the silk gland cells of the silkworm, Bombyx mori, was investigated with special reference to the fine structure of the sex-heterochromatin body (SB), using both diploids and experimentally induced polyploids. 2) Microscopic observations revealed that the SB was not a homogeneous mass, but it was surrounded by a layer of chromatin substances formed by the coiling and folding of fibrils. 3) The SB was clearly observed in the silk gland nuclei in acetic orcein squashed preparations, being 100% in the female (SB1) and 0% in the male (SBO) in diploids. In the tetraploids, the female nucleus had two SBs (SB2), and the male nucleus contained no SB (SBO). 4) On the observation of the segregation of the number of the SB in triploid offsprings from the backcross of the tetraploid female, it was revealed that the ratio of SB2: SB1: SBO was 1:4:1. The behavior of sex chromosomes in meiosis of the tetraploid female was considered according to the above results. 5) Using triploid off springs from the strain having a translocated Y chromosome marked with visible genes, it is apparent that the SB is corresponding to a Y chromosome. ACKNOWLEDGMENTS The author wishes to express her sincere thanks to Professor Giichi Yoshii for making this study possible and to Dr. Yuh H. Nakanishi for his continued support and for his kind advice in the preparation of the manuscript. Also hearty thanks are due to Professor Shigeru Ohba, Tokyo Metropolitan University, and Dr. Sayaka Nakai, National Institute of Radiological Sciences, for their valuable criticisms. She desires to express her obligation to Dr. Susumu Tamazawa, Faculty of Agriculture, Hokkaido University, who kindly supplied materials for the author's use.

14 340 S. ITo LITERATURE CITED Astaurov, B. L., 1957 High temperature as a tool for controlling development and sex determination: A review of studies in artificial parthenogenesis, androgenesis and elimination of embryonic diapause in the silkworm, Bombyx mori L. Proc. Zool. Soc. Calcutta, Mookerjee Memor. Vol Barr, M. L., 1966 Correlation between sex chromatin patterns and sex chromosome complexes in man. In "The sex chromatin" (K. L. Moore. ed.) pp W. B. Saunders Co. Philadelphia. Gillot, S., 1968 Heterogeneites fonctionelles dans 1'ADN de noyaux geants. Etude autoradiographique sur la glande sericigene de Bombyx mori L. Exptl. Cell Res. 50: Hasimoto, H., 1933a The role of the W chromosome in the sex determination of Bombyx mori. Japan. J. Genetics 8: (in Japanese) Hasimoto, H., 1933b Genetical studies on the tetraploid female in the silkworm. Bull. Seric. Exp. Sta. Jap. 8: (in Japanese) Hasimoto, H., 1934 Genetical studies on the tetraploid female in the silkworm. III. Behavior of the Z chromosome. Bull. Seric. Exp. Sta. Jap. 8: (in Japanese) Hasimoto, H., 1935 Segregation of sex chromosomes in tetraploid females of the silkworm. Bot. Zool. 3: (in Japanese) Hasimoto, H., 1941 Genetical studies on the tetraploid female of the silkworm. IV. The embryonal mortality and sex ratio in the F1 hybrid of a tetraploid female with a diploid male. Bull. Seric. Exp. Sta. Jap. 10: (in Japanese) Kawaguchi, E., 1933 Die Heteropyknose der Geschlechtschromosomen bei Lepidopteren. Cytologia 4: Kawaguchi, E., 1934 Chromosome behavior in tetraploid female of the silkworm. J. Seric. Sci. Jap. 5: (in Japanese) Nakanishi, Y. H., H. Kato, and S. Utsumi, 1969 Polytene chromosomes in silk gland cells of the silkworm, Bombyx mori. Experientia 25: Ray-Chaudhuri, S. P., L. Singh, and T. Sharma, 1970 Sexual dimorphism in somatic interphase nuclei of snakes. Cytogenetics 9: Springhetti, A., and P. Oddone, 1961 Osservazioni biometriche sulla massa eteropicnotica nei seritteri del Bombyx mori L. Atti VII Congresso Annuale Soc. Italiana Genetica Agraria. Genet. Agrar. 14: Takizawa, Y., and S. Tamazawa, 1977 On the polyploid induced by supercooling treatment of the eggs of the silkworm, Bombyx mori L. The relation between the enlargement of the serosa cells and the polyploidy. Mem. Fac. Agric. Hokkaido Univ. (in press). (in Japanese) Tamazawa, S., 1967 Polyploid silkworms induced by supercooling treatment on eggs. Proc. 21st Cong. Tohoku Branch of Seric. Soc. Jap. p. 14. (in Japanese) Tanaka, Y., 1916 Genetic studies in the silkworm. J. Coll. Agric. Sapporo. 7: Tanaka, Y., 1928 "The anatomy of the silkworm". Meibun-Do, Tokyo. (in Japanese) Tanaka, Y., and E. Kawaguchi, 1932 On the triploid silkworm produced by the centrifugal force treatment. Japan. J. Genetics 7: (in Japanese) Tazima, Y., 1941 A simple method of sex discrimination by means of larval markings in Bombyx mori. J. Seric. Sci. Jap. 12: (in Japanese) Tazima, Y., 1944 Studies on chromosome aberrations in the silkworm. II. Translocation involving second and W-chromosomes. Bull. Seric. Exp. Sta. Jap. 12: (in Japanese) Traut, W., and G. C. Mosbacher, 1968 Geschlechtschromatin bei Lepidopteren. Chromosoma 25: