The eupyrene-apyrene dichotomous spermatogenesis of Lepidoptera

DEVELOPMENTAL

68,515-524

BIOLOGY

(197%

The Eupyrene-Apyrene I. The

Relationship

with

Dichotomous Postembryonic

Juvenile RONIT Department

LEVIATAN’

of Biology,

Received

Hormone

April

Ben Gurion

Spermatogenesis

of Lepidoptera

Development

and the Role of the Decline

Titer

Pupation

AND

toward

MICHAEL University

13, 1978; accepted

in

FRIEDL~NDER of the Negeu,

in revised

form

Beer

September

Sheva,

Israel

8, 1978

The relationships between the stages of postembryonic development and the occurrence of eupyrene and apyrene spermatogenesis, and the effects of the decline of the juvenile hormone (J.H.) titer toward pupation in these processes, were studied in the carob moth, Ectomyelois ceratoniae. The accurate tuning of the spermatogenetic events was determined daily from the 2nd instar larva to the imago in squashes and electron microscope preparations of testes. Eupyrene spermatids elongate in two phases. In the fust, beginning in late 4th instar larva, only flagella elongate, while in the second, beginning in the mid 5th instar larva, both flagella and nuclei elongate. Apyrene spermatogenesis starts just after the beginning of the nuclear elongation of eupyrene spermatids, in the mid 5th instar larva and not in the pupa, as is commonly believed. Using ligatures, topical applications of a J.H. mimic, and testes transplantation, it was found that the nuclear elongation begins in the S-day-old eupyrene spermatid and cannot be induced earlier; the elongation is inhibited by high titer of the J.H. mimic. Elongation of the flagella, however, is unaffected by fluctuations of the J.H. titer. The onset of the apyrene spermatogenesis, which occurs in the verv earlv 5th instar larva or before. was found to be unrelated to the decline in the J.H. titer toward pupation. INTRODUCTION

Lepidopteran males produce two kinds of spermatozoa: typical-nucleated (eupyrene) and atypical-anucleated (apyrene) (Meves, 1903). Both kinds reach the spermatheca of the inseminated females, but only the eupyrene one fertilizes the eggs (Friedlander and Gitay, 1972). The function of the apyrene spermatozoa is still unclear. Apyrene spermatogenesis has a consistent pattern: The chromosomes form masses at meiotic metaphases and are irregularly distributed at anaphase, and the nuclei of the spermatids remain spherical until they are extruded from the cells; in the spermatozoa a truncate cone replaces the nucleus at the proximal end of centriole, and the mitochondria derivatives appear different from those of the eupyrene spermatozoa at the ultrastructural level (Friedlander and ’ Deceased.

Wahrman, 1970, 1971; Friedlander and Gitay, 1972; Friedlander and Miesel, 1977). Since this pattern appears to be a general feature of Lepidoptera, apyrene spermatogenesis should be considered an integral part of the normal spermatogenesis of this systematic group. However, the very existence of the apyrene spermatogenesis has been apparently overlooked in a great number of experimental works on different aspects of Lepidoptera spermatogenesis (Shen and Berryman, 1967; Kambysellis and Williams, 1971; Yagi and Fukushima, 1975; Mitsui et al., 1976). This omission may have resulted partially from problems of identification, since the apyrene spermatozoa form, after their nuclei are extruded, long bundles of flagella which appear very similar to those of the eupyrene spermatozoa at the resolving power of the light microscope. It is generally considered that the end of

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Copyright All rights

0 1979 by Academic of reproduction

Press, Inc.

in any form reserved.

516

DEVELOPMENTAL BIOLOGY

VOLUME 68.1979

the eupyrene spermatogenesis and the be- well. For electron microscopy, the testes ginning of the apyrene spermatogenesis co- were fixed for 3 hr in 3% glutaraldehyde in incide in the pupa (Sado, 1963; Tazima, 0.2 M phosphate buffer, pH 7.2, postfixed 1964; Holt and North, 1970; Riemann and in 1% OsOl in the same buffer for 1 hr, Thorson, 1971; Salama, 1976; Garbini and dehydrated, and embedded in Epon. The Imberski, 1977), which might indicate a thin sections were contrasted with alcoholic causal relationship between the shift from uranyl acetate and lead citrate. The presone kind of spermatogenesis to the other ence of all the stages of spermatogenesis and the decline of the juvenile hormone was confirmed at the ultrastructural level (J.H.) titer, which takes place before pu- with the help of the characteristic strucpation. It is, therefore, reasonable to expect tures distinguishing between the eupyrene that if the J.H. titer is prematurely lowered, and apyrene cells (Friedlander and Wahrapyrene spermatids might occur prema- man, 1970, 1971; Friedlander and Gitay, turely. On the other hand, the maintenance 1972). Ligatures were made between the of high J.H. titer at the time of its normal thorax and abdomen of 5th instar larvae at decline should interfere with the appear- different ages with thin hairs. Testes from ance of the apyrene spermatids. either the second half of 4th instar larvae In the present work, we checked this or ecdysing 5th instar larvae were dissected hypothesis in the carob moth Ectomyelois and transplanted either into pupating larceratoniae by (1) artificially decreasing the vae or into their isolated abdomens, thus J.H. level through neck ligatures of larvae avoiding a possible hormone release. The and transplantation of larval testes into hosts were anesthesized with COz before pupating larvae, and (2) topical applica- the implantations, and the wound was tions of a J.H. mimic to the 4th and early sealed with molted paraffin. Topical appli5th instar larvae. Before that, we studied cations were made on the abdomen with the spermatogenesis of this species with a the J.H. mimic Entecon ZR-512 4E (Ethyl special emphasis on the relationship be- 3,7,11-trimethyl dodeca-2,4-dienoate) from tween the different stages of the meiosis, Zoecon Corp., Palo Alto, Calif. The J.H. the differentiation of the two kinds of sper- mimic was dissolved in acetone at the conmatids, and the developmental stages of centrations indicated in each experiment. Control insects were treated with pure acethe postembryonic life. tone. To each larva, a l-p1 drop of either a MATERIALS AND METHODS solution of the J.H. mimic or pure acetone A culture of the carob moth Ectomyelois was applied. ceratoniae was reared in our laboratory on RESULTS an artificial medium at 25°C (Gothilf, 1968). Eupyrene and apyrene cells are found in The pigmentation of the head cuticle was used as an indicator of the age of the differdifferent cysts which appear distributed at ent instars. The head is white just after random within the testes. The course of the ecdysis and remains so for about 2 hr; sub- postembryonic development and its relasequently it gradually becomes brown and tion to different stages of the eupyrene and finally black. The stage of spermatogenesis apyrene spermatogenesis are summarized was studied daily in 10 or more individuals, in Figure 1. beginning at the ecdysis of the second instar Meiosis. The earliest spermatocytes at until the emergence of the adult. For light pachytene appear in mid 2nd instar larva, microscopy, the testes were squashed in 6-7 days after hatching. The testes of the ecdysing 4th instar larva contain numerous aceto-orcein and observed with Nomarski optics; thick sections of material, prepared spermatocytes at the diffuse stage bearing for electron microscopy, were studied as four developing flagella, as reported for

LEVIATAN

AND FRIEDL~NDER

Eupyrene-Apyrene

Spermatogenesis

517

I

m Eupyrene cells mu Apyrene cells

‘i;

Early spermotic with spheric0 nucleus

E Metophosc I I

I I

16 D 18 19 ?I

21 22 23 24 23 26 22 28 29 30 31 7

#

IV

L

I

I

I

I

I

I

I

I

I

I

I

I

AFTER

HATCHING

development and its relationship and apyrene spermatogenesis.

other Lepidoptera species (Friedlander and Wahrman, 1970). We could not find any difference among spermatocytes before premetaphase, which might indicate the existence of two lines of spermatogenesis, eupyrene and apyrene. At the end of the 4th larval instar, 18 days after hatching and 2 days before molting, the spermatocytes enter the first metaphase of the eupyrene meiosis, which is characterized by regular arrangement of the bivalents at the equatorial plane of the spindle (Fig. 2A). Anaphase distribution (Fig. 2B) and telophases (Fig. 2C) are regular as well. Second metaphases and early eupyrene spermatids (Fig. 2G) appear 1 day later, the day before the end of the 4th instar larva. Therefore, the duration of the meiotic eupyrene prophase, from pachytene to premetaphase, is about 11-12 days. Eupyrene metaphases are present until the 2nd-3rd day of the pupal stage. The earliest apyrene metaphases (Fig. 2D), anaphases (Fig. 2E), and telophases (Fig. 2F), which are highly irregular like in other lepidopteran species (Friedlander and Wahrman, 1970), appear at the 7th day of the 5th instar larva. Apyrene metaphases are also found in the pupa and adults. The

I

I

I

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I

L

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33 34 36 36 37 38 39

I

V

DAYS FIG. 1. Course of the postembryonic spermatid differentiation of eupyrene

I

PUPA

AND INSTAR

to meiotic

metaphases

and stages of

resulting apyrene spermatids, which contain numerous micronuclei (Fig. 2H), appear immediately after the telophases (Friedlander and Miesel, 1977). Eupyrene spermatid differentiation. The differentiation of the eupyrene spermatid can be divided into two periods (Fig. 1). In the first, the flagella elongate, but the nuclei remain spherical (Fig. 3A), forming a crescent at the anterior end of the cyst. This period extends 5 days, from the last day of the 4th instar until the 4th day of the fifth instar. In the second period, the nuclei elongate gradually. At the beginning the nuclei are oval, a day later they become somewhat larger and vacuolated, and finally they appear lance-shaped (Fig. 3B). During the nuclear elongation, the flagella elongate too, and at the last third of the 5th instar the testes contain bundles of fully developed eupyrene spermatozoa. Apyrene spermatid differentiation. As mentioned before, the earliest apyrene metaphases occur at the 7th day of the 5th larval instar, and the earliest apyrene spermatids at the 8th day, both appearing 9 days after the corresponding stages of eupyrene differentiation. Apyrene spermatids

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FIG. 2. Eupyrene and apyrene meiotic divisions. The eupyrene metaphase (A), anaphase (B), and telephase (C) are regular, and the resulting spermatids are mononucleated (G). The chromosomes of the apyrene metaphase do not form an equatorial plate (D) and are irregularly distributed at anaphase (E). During apyrene telephase, only part of the chromosomes clusters at the poles (F), and the resulting spermatids (H) contain numerous micronuclei of different sizes.

contain several micronuclei that never elongate and that are finally extruded from the cells (Friedlander and Miesel, 1977). The flagella, however, elongate, and the micronuclei appear distributed along the amonemes before they are extruded (Figs. 3C

and D). Apyrene spermatogenesis continues in the pupa and the imago. Ligatures. Fifth instar larva males were neck-ligated at different times after ecdysis. The effects of the ligatures on the time of appearance of apyrene metaphases and the

LEVIATAN AND FRIEDL~NDER

Eupyrene-Apyrene

Spermatogenesis

Z

519

FIG. 3. Eupyrene and apyrene spermatid elongation. The nuclei of the eupyrene early spermatids are spherical and have the same diameter (A) while those of the comparable apyrene spermatids are irregular and display different diameters (C!). The nuclei of the eupyrene spermatids elongate during differentiation and remain at the anterior end of the cell (B), while the nuclei of the apyrene spermatid do not elongate and appear distributed along the flagella (D) x 1700.

520

DEVELOPMENTAL BIOLOGY

beginning of the nuclear elongation of the eupyrene spermatids are presented in Table 1. We found no differences between the ligated larvae and the controls with respect to the appearance of both kinds of sperTABLE

1

ELONGATION OF THE NUCLEUS OF EUPYRENE SPERMATIDS AND THE APPEARANCE OF APYRENE SPERMATOZOA IN NECK-LIGATED ~TH INSTAR LARVAE~ Days between ecdysis and ligature

Davs 1

between

ligature

and dissection

2

3

4

6

8

4

SN EN

SN EN

SN EN

SN EN AP

SN EN AP

SN EN AP

2

SN

SN EN

SN EN

SN EN

SN EN AP

SN EN AP

0

SN

SN

SN

SN EN

SN EN

SN EN AP

SN

SN

SN

SN EN

SN EN

SN EN AP

Control

n Six testes were studied in each treatment at each tune. Control testes were dissected from unligated males of the same age. SN, eupyrene spermatids with spherical nucleus; EN, eupyrene spermatids with elongated nucleus; AP, early apyrene spermatids. TABLE

VOLUME 68,1979

matogenesis. In all treatments, the nuclear elongation of the eupyrene spermatids began 4 days after the ecdysis, and the earliest apyrene metaphases were found in larvae dissected 8 days after ecdysis, regardless of whether ligature was carried out or not and regardless of the age at which the ligatures were made. Testes transplantation from the 4th instar larvae into isolated abdomens of 5th instar spinizing larvae. The results of these experiments are presented in Table 2. We found that (1) the spermatocytes of the transplanted testes continued their regular development into both kinds of spermatids, irrespective of whether the most advanced meiotic stage at the time of the transplantation was pachytene or primary metaphase, and (2) spermatids appeared earlier in testes containing primary metaphases at the time of the transplantation than in those in which the spermatocytes were still at pachytene. (3) The stage of development achieved in the two kinds of spermatogenesis depended upon the time the testes remained in the host before dissection. The longer the time, the more advanced the stage of differentiation. Testes transplantation from 4th instar larvae into whole 5th instar spinning lar2

OCCURRENCE OF EUPYRENE AND APYRENE SPERMATOGENESIS IN ~TH INSTAR LARVA TESTES TRANSPLANTED INTO ISOLATED ABDOMENS OF ~TH INSTAR SPINNING LARVAE~ The most advanced spermatogenic stage at the transplantation

Days between dissection and transplantation

Number Pachytene

Pachytene

First

Metaphase

3 6 a 12 3 6 8 12

2 2 -

of testes which reach at dissection Spherical nucleus in eupyrene spermatids

the following

Elongating nucleus in eupyrene spermatids

stages

Apyrene spermatids

1

-

-

2 2 4 2

-

2 -

3 1 -

-

-

1

-

3

4

n One testis from each donor was dissected and used to determine the most advanced stage of spermatogenesis reached at the transplantation. The second testis of each donor was transplanted into an abdomen which was immediately ligated. These testes were dissected at different days after transplantation, as indicated.

LEVIATAN

AND FRIEDLANDER

Eupyrene-Apyrene

Spermatogenesis I

521

use. The larvae were allowed to pupate after the transplantation. The mortality was very high, but in the few surviving insects which pupated 2-3 days after transplantation the results were similar to those obtained in testes transplanted into isolated abdomens.

In the larvae which failed to pupate, we found that the nuclear elongation of the eupyrene spermatids was also concentration-dependent. The higher the concentration of the mimic, the lower the number of testes containing eupyrene spermatids with elongated nuclei. The elongation of the nuTopical application of the J.H. mimic to cleus was highly inhibited by 0.5% and com4th instar and early 5th instar larvae. The pletely inhibited by 1% J.H. mimic (Fig. effect on pupation of the topical application 4B). Nuclear elongation was also inhibited of the mimic to the 4th instar larvae is dose- by treating early 5th instar larvae with the dependent; the higher the concentration, same concentrations of the J.H. mimic. The the lower the number of pupating larvae. applications were effective, however, only Pupation was completely inhibited at the during the first 72 hr of the 5th instar larva. highest concentration used (1%) (Fig. 4A). Applications to older larvae had no effect A great number of larvae molted into an on nuclear elongation (Fig. 5). additional 6th larval instar. In the testes of Topical application of the J.H. mimic to all the larvae which failed to pupate even isolated abdomens of late 5th instar larvae testes transplanted from 5th 30 days after the application, we found containing apyrene spermatogenesis, characterized by instar ecdysing larvae. To compare the the appearance of irregular metaphase, effects of the J.H. mimic and the control multimicronucleated spermatids, and anu- solutions on testes of identical genetical cleated spermatozoa (Friedlander and Mie- background and age, each one of the two sel, 1977). Moreover, we also found that the testes of ecdysing 5th instar larvae was older the larva which failed to pupate, the transplanted to a different isolated abdolarger the number of cysts containing apy- men of spinning 5th instar larvae. One of rene cells. the abdomens was treated with 1% J.H.

Percent

of

J.H.

analog

Percent

of J. H. onalog

FIG. 4. Concentration effect of the juvenile hormone mimic applied to 4th instar larvae on pupation of the 5th instar larvae (A) and nuclear elongation of the eupyrene spermatids (B). (A) Groups of 50 larvae, 18-20 days old, were treated with the juvenile hormone mimic at the concentrations indicated. The larvae were allowed to pupate. (B) The larvae which did not pupate in experiment A were dissected, and the differentiation of the eupyrene and apyrene spermatids was studied. All the treated larvae displayed apyrene spermatogenesis. A l-4 drop was applied to each larva.

522

DEVELOPMENTAL BIOLOGY

-----

15

16 17 IS

I9120

21 22

23 24 25---

4thmcun

Days after hatching 00nly spmwtids having spherical nudeus pSpmatids having elangated nucleus

FIG. 5. Timing effect of the juvenile hormone mimic applied to the 4th and early 5th instar larvae on the nuclear elongation of eupyrene spermatids. The mimic (1%) was applied to groups of six larvae, at the times indicated. The larvae were dissected at the 5th day of the 5th instar, and the stage of nuclear elongation was studied. A 1-d drop was applied to each larva.

mimic, and the second, serving as a control, was treated with pure acetone. The applications of the analog were made, as in the previous experiments, at the time of transplantation (five pairs), or 2 days later (five pairs), the day before the nuclei normally begin to elongate, as indicated in Fig. 1. The presence of both, elongating nuclei in eupyrene cells and apyrene spermatids, was checked 10 days after transplantation, which corresponds to the beginning of nuclear elongation in nontreated (or transplanted) testes. In accordance with the previous experiments, we found that the nuclei of the eupyrene spermatids elongated at the usual time in the control testes, and that application of the J.H. mimic to the abdomens containing the second testis of the same donor, at transplantation or 48 hr later, inhibited nuclear elongation. Apyrene cells were found in both control and treated testes. DISCUSSION

Timetable of moth spermatogenesis. It is generally accepted that eupyrene spermatozoa derive from spermatocytes which end their meiotic divisions in the larva and early

VOLUME 68.1979

pupa, while apyrene spermatozoa derive from spermatocytes which divide in late pupa and imago (Sado, 1963;Tazima; 1964). However, in disagreement with this idea, we found both apyrene metaphases and spermatids already in mid 5th instar larva of the carob moth. In view of this finding, it appears worthwhile to determine whether interspecific differences exist in the timetable of the apyrene spermatogenesis (Sado, 1963; Tazima, 1964; Holt and North, 1970; Riemann and Thorson, 1971; Salama, 1976). In fact, most of the reports on the absence of apyrene spermatogenesis in the 5th instar larva are based on light microscope observations. Apyrene spermatozoa, however, are difficult to identify under the resolution of the light microscope, especially after the nuclei have been extruded from the cells. We would like to stress that we confirmed the presence of apyrene spermatids in the 5th instar larva of the carob moth at the ultrastructural level (Fig. 6). Spermatid elongation. The elongation in the carob moth comprises two phases. During the first, which extends throughout the first 5 days of spermatid differentiation, the flagellum elongates, while the nucleus remain spherical. During the second phase both the flagellum and the nucleus elongate (Fig. 1). The preparation for the first phase begins in the primary spermatocyte, which already bears four elongating flagella, to be distributed among the four resulting spermatids (Friedlander and Wahrman, 1970). The word “elongation,” however, has apparently been used indiscriminately to indicate elongation of the whole spermatid, without considering whether the nucleus and flagellum elongate at different times (Shen and Berryman, 1967; Kambysellis and Williams, 1971; Yagi and Fukushima, 1975; Mitsui et al., 1976; Salama, 1976; Garbini and Imberski, 1977). This lack of distinction between the two processes can cause confusion in the final analysis of the control of eupyrene spermatid differentiation, since the factors acting in flagella elon-

LEVIATAN

FIG.

6. Electron

micrograph

AND

FRIEDLANDER

of a cyst of 5th instar

Eupyrene-Apyrene

larva

gation are already active in the first spermatocytes (found in the 3rd instar larva), while those acting in nuclear elongation are active from the mid-spermatid (found in the 5th instar larva) on. The fact that in apyrene spermatids the flagella elongate while the nuclei never do clearly indicates that two independent mechanisms control the elongation of flagella and nuclei. Accordingly, it appeark that the elongation of the eupyrene nucleus is triggered by the decline of the J.H. titer, which takes place during preparation for pupation and could be inhibited by maintaining J.H. titer at a high level, while the elongation of the flagella is unaffected by the high titer of the exogeneous J.H. mimic. The level of the J.H. titer allowing nuclear elongation is ap-

testes containing

Spermatogenesis I

apyrene

late spermatids.

523

x 20,000.

parently attained 72 hr after the ecdysis of the 5th instar larva, since the J.H. mimic was ineffective in inhibiting nuclear elongation when applied after this time. Decline of the J.H. titer alone, however, is insufficient for inducing the nucleus to elongate. The elongation could not be induced prematurely either in 5th instar larvae which were ligated at ecdysis or in the testes of the 4th instar larvae which were transplanted to pupating larvae or their isolated abdomens. Therefore, it can be concluded that preparatory processes for nuclear elongation should be completed during these first 5 days of eupyrene spermatid differentiation, and that the decline of the J.H. titer is necessary for their subsequent expression.

524

DEVELOPMENTAL

BIOLOGY

The eupyrene and apyrene spermatozoa appear to be derived from the same kind of bipotential cells, probably primary spermatocytes. We did not find any difference between the two types of cells before meiotic premetaphases which might indicate the existence of two differentiating lines, nor did we find any preferential location of the apyrene cysts, which might indicate either functionaltopographical diversification between apyrene and eupyrene cell, or the existence of morphogenetic gradients inducing apyrene spermatogenesis. The cessation of the eupyrene meiosis and the beginning of the apyrene one may be related, since eupyrene metaphases appear earlier than the apyrene metaphases and disappear after the onset of the apyrene metaphases. This shift from one to the other type of spermatogenesis probably occurs in the 4th larval instar or earlier, before the J.H. titer declines in the 5th larval instar toward pupation. This should be so since the duration of the meiotic prophase from pachytene to metaphase is about 11-12 days, and the earliest apyrene metaphase appears 7 days after the ecdysis of the 5th larval instar. Accordingly, the factor causing the onset of the apyrene spermatogenesis is unrelated to the decline of J.H. titer in the 5th larval instar. Apyrene spermatogenesis occurs in the additional 6th larval instar induced by the exogenous J.H. mimic. Moreover, the normal timetable of the apyrene spermatogenesis is undisturbed by either applications of the J.H. mimic to the 4th and 5th larval instars or testes transplantation from the 4th larval instar to pupating larva. Apyrene

We thank tions and for for providing G. Raziel for

differentiation.

Dr. S. Applebaum for his helpful suggesproviding the J.H. mimic, Dr. S. Gothilf the carob moths for our culture, and Mr. the skillful printing of the micrographs. REFERENCES

FRIEDL~NDER, M., and GITAY, H. (1972). The fate of the normal-anucleated spermatozoa in inseminated females of the silkworm Bombyx mori. J. Morphol. 138, 121-12s.

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68,1979

FRIEDLXNDER, M., and MIESEL, S. (1977). Spermatid anucleation during the normal atypical spermatogenesis of the warehouse moth Ephestia cautella. J. Submicrosc. Cytol. 9, 173-185. FRIEDL~NDER, M., and WAHRMAN, J. (1970). The spindle as a basal body distributor. A study in the meiosis of the male silkworm Bombyx mori. J. Cell Sci. 7, 65-89. FRIEDLANDER, M., and WAHRMAN, J. (1971). The number of centrioles in insect sperm. A study in two kinds of differentiating silkworm spermatids. J. Morphol. 134, 383-387. GARBINI, C. P., and IMBERSKI, R. B. (1977). Spermatogenesis in Ephestia kuhniella (Lepidoptera, Pyralididae). Trans. Amer. Microsc. Sot. 96, 189-203. GOTHILF, S. (1968). The biology of the carob moth (Ectomylois ceratoniae Zell) in Israel. I. Mass culture on artificial diet. Is. J. Entomol. 3, 109-118. HOLT, G. G., and NORTH, D. T. (1970). Spermatogenesis in the cabbage looper, Z’richoplusia ni (Lepidoptera: Noctuidae). Ann. Entomol. Sot. Amer. 63, 501-507. KAMBYSELLIS, M. P., and WILLIAMS, C. M. (1971). In vitro development of insect tissues. I. A macromolecular factor prerequisite for silkworm spermatogenesis. Biol. Bull. 141, 527-539. MEVES, F. (1903). Ueber oligopyrene und apyrene Spermien und uber inre Enstehlung, nach Beobachtungen an Paludina and Pygaera. Arch. Mikrosk. Anat. Entwicklungsmech. 61, l-82. MITSUI, T., NOBUSAWA, C., FUKAMI, J., and FUKUNAGA, K. (1976). Effect of ecdysterone and C&-juvenile hormone on spermiogenesis in the silkworm larva, Bombyx mori. L. Appl. Entomol. 2001. 11, 344-355. RIEMANN, J. G., and THORSON, B. J. (1971). Sperm maturation in the male and female genital tract of Anugusta kiihniellu (Lepidoptera: Pyralididae). Int. J. Insect Morphol. Embryol. 1, 11-19. SADO, T. (1963). Spermatogenesis of the silkworm and its bearing on radiation induced sterelity. J. Fat. Agr. Kyushu Univ. 12,359-386. SALAMA, H. S. (1976). Spermatogenesis and testicular development in the gypsy moth Porthetria &spar L. 2. Angew. Entomol. 81,102-110. SHEN, S. K., and BERRYMAN, A. A. (1967). The male reproductive system and spermatogenesis of the european pine shoot moth Rhyucioniu buoliunu (Lepidoptera: Olethreutidae), with observation on the effect of gamma irradiation. Ann. Entomol. Sot. Amer. 60,767-774. TAZIMA, Y. (1964). “The Genetics of the Silkworm.” Logos, London, and Academic Press, New York and London. YAGI, S., and FUKUSHIMA, T. (1975). Hormonal effect on cultivated insect tissues. II. Effect of juvenile hormone on spermiogenesis of the silkworm Bombyx mori. L. in vitro (Lepidoptera: Bombycidae). Appl. Entomol. Zool. 10,77-83.