Jpn. J. Genet. (1981) 56, pp. 249-256

Studies

on the increase induced

from

in egg size in tetraploid

a normal

and a giant-egg

silkworms strains

BY Naoko KAWAMURAand Tohru NAKADA Laboratory

of Sericology, Faculty of Agriculture, University, Sapporo, Hokkaido 060

Hokkaido

(Received November 21, 1980) ABSTRACT Deposition of large-size eggs by tetraploid females of the silkworm has been reported by many investigators, while no detailed analyses have been carried out on the cause of the increase in egg size due to chromosome doubling. In this insect, a sex-linked gene called `Giant egg (Ge)' is known, and the diploid females with Ge-gene lay remarkably large-size eggs. In the present study, tetraploid females of Fl hybrids which do or do not carry Ge-gene were induced by applying a low-temperature treatment, and the effect of the chromosome doubling was compared to that of the doubling of Ge-gene on egg size. Eggs of the tetraploid females for both hybrids showed a remarkable increase in size, although the increment was relatively small in the females with the double Ge-gene dose. Furthermore, especially large eggs laid by some of Ge-progeny were all unfertilized. The results suggested the possibility of a limitation of egg size increase in the silkworm.

1. INTRODUCTION It has been reported by many investigators on silkworms that large eggs are deposited by tetraploid females, which are induced artificially by applying various chemical or physical treatments to eggs at early developmental stages (Hasimoto 1933; Kawaguchi 1936; Astaurov 1967; Tazima and Onuma 1967; Tamazawa 1977). However, it is not evident why such changes are induced by doubling chromosome sets. In the silkworm, a dominant gene called `Giant egg (Ge)', located in the Zchromosome, is known and the females with Ge-gene lay eggs much larger than those of normal strains. By using Ge-gene, the present study was carried out to examine whether to the eggs of tetraploid females with double Ge-gene dose can increase the egg size in the same ratio as those of tetraploid females of normal strains which have no Ge-gene, or whether the doubling of the gene has any other effects to the egg size. 2. MATERIALS

AND METHODS

The strains of the silkworm (Bombyx mori) used in the present study were re9 (red egg, striped silkworm; re/re, ps/ps), Tw1(white egg 2, plain silkworm;

250

N. KAWAMURA

and

T. NAKADA

Figs. 1-5. Eggs of TR and TG groups. Eggs of average size in each group are shown. ear: 1 mm.

Increase

in egg size in

tetraploid

251

silkworms

w2/w2, pip) and Ge (red egg, giant egg, normal marked, knobbed; re/re, Ge/Ge, +pi+p, K/K). Tetraploid silkworms were induced by a low-temperature treatment following the method of Takizawa and Tamazawa (1968). The eggs from the age of 120 to 150 minutes after laying were subjected to a low temperature of -10°C for 24 hours and then returned to the regular condition of 26° C. Among cold-treated eggs, those with large nuclei in serosa cells were picked up as presumed tetraploids and those with small nuclei were regarded as diploids (Kawamura 1979). In the present study, F1 eggs from a cross between strains of a white egg and a red egg were cold-treated, because serosa nuclei of white or red eggs can not be observed, while those nuclei of F1 are black and easily discernible. The ploidy of female individuals was determined by the mode of segregation of egg color in their offspring by backcrossing them to re9 males. If they were tetraploids, they may cause segregation of black and red eggs to a ratio of 5:1, while in case of diploids they may have a 1:1 ratio. Measurement of egg size was carried out in six experimental groups described below. 1. TR-untreated 2n: Eggs deposited by untreated control 2n females from Twi X re9 cross 2. TR-treated 2n: Eggs deposited by 2n females from cold-treated eggs of Twi X re9 cross 3. TR-4n: Eggs deposited by 4n females from cold-treated eggs of Twl X re9 cross 4. TG-untreated 2n: Eggs deposited by untreated control 2n females from Twi X Ge cross 5. TG-treated 2n: Eggs deposited by 2n females from cold-treated eggs of Twi X Ge cross 6. TG-4n: Eggs deposited by 4n females from cold-treated eggs of Twi X Ge cross Egg size was measured as follows: Eggs and a micrometer (total length, 1 mm) were photographed and enlarged to the same magnification on Fuji negative paper. Squares corresponding to 1 mm2 were cut from the enlarged Fig. 1. Eggs deposited by a 2n female from untreated eggs of Twi (TR-untreated 2n). Fig. 2. Eggs deposited by a 4n female from cold-treated eggs of Twi (TR-4n). Fig. 3. Eggs deposited by a 2n female from untreated eggs of Twi (TG-untreated 2n). Fig. 4. Eggs deposited by a 4n female from cold-treated eggs of Twi (TG-4n). Fig. 5. Unfertilized eggs deposited by a female of TG-4n group. Fig. 6. Oviduct of a female of TG-4n group which failed oviposition.

x re9

cross

x re9

cross

x Ge

cross

x Ge

cross

252

N. KAWAMURA

and

T. NAKADA

photographs and weighed to make a standard unit (661 mg ± 0.18, n = 41) with Shimazu Digibalance D 1003 and Digiprinter DP 60 D, and then the enlarged photographs of eggs were also cut and weighed. The weight of the photographs of eggs was converted to area by using the standard unit. Calculation for statistical analysis was performed with use of a computor, HITAC-M180, in the Computing Center of the Hokkaido University. 3, RESULTS Egg size in the six experimental

groups

As shown in Figures 1 and 3, the diploid females with Ge-gene (TG-2n) laid eggs about 1.4 times larger than the diploid females which do not carry the gene (TR-2n). In both TR and TG groups, diploid females from cold-treated or untreated eggs did not show significant difference in their egg size, whereas eggs of the tetraploid females showed a remarkable increase in size (Figs. 1-4). Three types of females appeared in TG-4n group, that is, eight of these laid large-size eggs that were normally fertilized (Fig. 4) and three laid larger eggs than the former, though all were unfertilized (Fig. 5). The third type was the females which did not lay eggs. After dissection, it was found that the ovarian tubes were full of eggs and appeared quite normal, but a few eggs were obstracted in the oviducts (Fig. 6). Variations group

of egg size among female individuals

within the same experimental

For measurement of egg size, 16 female individuals were used in each experimental group and 20 eggs were selected from each batch by random sampling. The results are shown in Table 1 (TR groups) and Table 2 (TG groups), which are summarized in Table 3. From the values of standard error (SE) and coefficient of variation (CV) , variations of egg size among eggs in the same batch were found to be considerably small, i, e., the size of eggs laid by a female were rather uniform in both TR and TG. Difference in egg size among untreated-2n,

treated-2n

and ~n groups

Table 4 shows an analysis of variance of the values in Table 1 and 2 . From the statistical analysis, high significance was observed among the groups (Factor A), which was considered to be due to the difference of ploidy of mothers, i. e., 4n > 2n. The difference between the strains (Factor B) was also significant, for it showed a difference in egg size between TR and TG groups . Furthermore, a difference in the rate of increase in egg size caused by the doubling of ploidy was obtained in the interaction between the factors.

ln('rerLgp Table

Table

Values

in

in P,aa 'izp

in tetrriminirl,

Size of eggs laid by Fl females

1.

2.

ei,ik~a,nrrn, c

of the cross Twl ~ X re9

Size of eggs laid by F, females of the cross Twl

are those

of unfertilized

eggs.

xGe

253

(TR)

(TG)

254

N. KAWAMURA Table

3.

and

Summary

T. NAKADA

of Table 1 and 2

` Significant at 1% level .

TR groups: In the three groups, the mean values were almost equal between TR-untreated 2n and TR-treated 2n, but that of TR-4n was evidently larger than 2n. The rate of increase in egg size in TR-4n was approximately 22%. TG groups: The mean values of three groups showed the same tendency as TR groups, i. e., TG-untreated 2n =. TG-treated 2n< TG-4n. The rate of egg size increase observed in TG-4n was 17% in the case where only fertilized 4n eggs were considered, whereas it was 20% when unfertilized 4n eggs were included. Difference of the rate between TR and TG was found significant. 4. DISCUSSION Although the increase in egg size in tetraploid female silkworms was observed by many early investigators (Hasimoto 1933, Kawaguchi 1936, and others), the mechanism of the increase in egg size by doubling of chromosome sets was not clear at all. By measuring diameters of eggs, Kawaguchi (1937), Sato and Chino (1937) and Astaurov (1940) showed that the length of the diameter of eggs laid by tetraploid silkworms was 1.12 to 1.13 times of that of diploid eggs. In the present study, measurement of area of an egg was performed at the maximum optical section and the egg size was compared

Increase

in egg size in tetraploid

silkworms

255

between those laid by diploid and tetraploid females. As diploid females of a giant egg strain are known to deposit eggs of remarkably large size, the effect of doubling of Ge-gene was studied. Ge-gene locates in Z-chromosome and F1 females from the cross between Ge male and normal female possess ZG8Win diploid and ZGeZGeWWin tetraploid. As shown in the tables, in each six group of TR and TG, the size of eggs laid by the same female was comparatively uniform, while relatively large variations were seen among batches. Comparison of the egg size between diploid and tetraploid females showed a remarkable increase in both groups, the increase rate being 22% for TR group and 17% for TG group, and this difference in the rate was statistically significant. Contrary to the authors' expectation, the smaller increase rate was seen in the TG-4n individuals with double Ge-gene dose. However, some females of TG-4n (Table 2, TG-4n-9,10, 11) laid unfertilized eggs of larger size than the normally fertilized eggs of the same group, and other females failed even to oviposit, which was probably due to an enlargement of eggs. It is considered that there is a limit of egg size increase and failure in fertilization, as well as in oviposition, results in extremely enlarged eggs. The relatively small rate of egg size increase observed in TG-4n group might have been due to this limitation, although it is not known, at this stage, why such a failure in fertilization occurred. Tetraploidy is far more common in plants than in animals. In plants, it is generally accepted that chromosome doubling produces an increase in the size of some organs such as sepals, petals, anthers, few-seeded fruits and seeds. Increase in size of an individual cell is considered the most general effect. In polyploid animals, Fankhauser and Griffiths (1939) observed an increase in the size of cells and nuclei in the newt, along with a decrease in number, and showed that a karyo-plasmic ratio was constant. Quantitative studies dealing with the size and number of cells in tetraploid silkworms were carried out by Kawaguchi (1936, 1937) in spermatocytes and silkgland cells. He observed that the number of silkgland cells in tetraploid silkworms decreased, while their size was much larger than that of diploid cells. Kawamura (1979) confirmed this phenomenon in serosa cells of F1 eggs from a cross between tetraploids. Increase in egg size in tetraploid silkworms may have been caused by the same mechanism which resulted in an increase in the size of seeds in tetraploid plants. The mechanism of deposition of large-size eggs by diploid Ge females is not evident. Further cytological studies are necessary on the oogenesis of tetraploid females of normal strains and diploid females of the Ge strain. The authors wish to express their thanks to Prof. Hokkaido University for his constant encouragement

T. Nakajima of Laboratory in the present study.

of Sericology,

256

N. KAWAMURA

and

T. NAKADA

REFERENCES AsTAUROV,B. L. (1940) Artificial parthenogenesis in the silkworm (Bombyx mori) . An experimental study. USSR Acad. Press, Moscow (R, ER) ASTAUROV,B. L. (1967) Experimental alterations of the developmental cytogenetic mechanisms in mulberry silkworms: Artificial parthenogenesis, polyploidy, gynogenesis and androgenesis. Advance in Morphogenesis 6,199-257. FANKHAUSER,G. and GRIFFITHS, R. B. (1939) Induction of triploidy and haploidy in the newt, Triturus viridescens, by cold treatment of unsegmented eggs. Proc. Natl. Acad. Sci. USA 25, 233-238. HASIMOTO,H. (1933) Genetical studies on the tetraploid female in the silkworm. Bull. Imp. Sericult. Exp. Stat. 8, 359-381. KAWAGUCHI,E. (1936) Der Einfluss der Eierbehandlung mit Zentrifugierung auf die Vererbung bei dem Seidenspinner. I. Uber experimentelle Auslosung der polyploiden Mutation. J. Fac. Agr. Hokkaido Imp. Univ. 38,111-133. KAWAGUCHI,E. (1937) Economic characters of polyploidy in the silkworm. J. Sericult. Sci. Japan 8,121-126. KAWAMURA,N. (1979) Polyploidy and size of serosa nuclei and cells in eggs of the silkworm, Bombyx mori. J. Sericult. Sci. Japan 48, 28-36. SATO. H. and CHINO, K. (1937) Biometrical analysis of triploidy in the silkworm. J. Sericult. Sci. Japan 8,107-120. TAKIZAWA,Y. and TAMAZAWA,S. (1968) A relation between polyploidy and size of serosa cells of silkworm eggs induced by the supercooling method. J. Sericult. Sci. Japan 37, 248. TAMAZAWA,S. (1977) On the polyploid induced by supercooling treatment of the eggs of the silkworm, Bombyx mori. I. Relation between enlargement of serosa cells and the polyploidy . Mem. Fac. Agr. Hokkaido Univ. 10, 272-283. TAZIMA,Y, and ONUMA,A. (1967) Experimental induction of androgenesis, gynogenesis and polyploid in Bombyx mori by treatment with CO2 gas. J. Sericult. Sci. Japan 36, 286-292.