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1969 Spermatogenesis and Submicroscopic Structure of Spermatozoa in Heliothis Virescens (F) (: Noctuidae). Gong-tseh Chen Louisiana State University and Agricultural & Mechanical College

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Recommended Citation Chen, Gong-tseh, "Spermatogenesis and Submicroscopic Structure of Spermatozoa in Heliothis Virescens (F) (Lepidoptera: Noctuidae)." (1969). LSU Historical Dissertations and Theses. 1581. https://digitalcommons.lsu.edu/gradschool_disstheses/1581

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CHEN, Gong - tseh, 1935- SPERMATOGENESIS AND SUBMICROSCOPIC STRUCTURE OF SPERMATOZOA IN HELIOTHLS VIRESCENS (F.) (LEPIDOPTERA:NOCTUIDAE).

The Louisiana State University and Agricultural and Mechanical College, PfuD., 1969 Entomology

University Microfilms, Inc., Ann Arbor, Michigan SPERMATOGENESIS AND SUBMICROSCOPIC STRUCTURE OF SPERMATOZOA IN HELIOTHIS VIRESCENS (F.) (LEPIDOPTERA:NOCTUIDAE)

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

in

The Department of Entomology

by Gong-tseh Chen B. Sc., Taiwan Provincial Chung Hsing University, 1959 M. Sc.f National Taiwan University, 1963 M. Sc., McGill University, 1966 May, 1969 ACKNOWLEDGMENTS

The author wishes to express his sincere gratitude to Dr. J. B.

Graves under whose direction and supervision this research was con* ducted. The author is also grateful to Dr. L. D. Newsom, Dr. H. B.

Boudreaux, Dr. D. F. Clower and Dr. VJ. Blrchfield for their valuable advice and criticism.

Thiii study was supported in part by Cooperative State Research

ServiceUnited States Department of Agriculture Grant 616*15-17.

11 TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... 11

TABLE OF CONTENTS ...... ill

LIST OF PLATES ...... iv

ABSTRACT ...... vl

INTRODUCTION ...... 1

REVIEW OF LITERATURE ...... 3

Spermatogenesis ...... 3

Submlcroscoplc Structure of Sperm .... 10

MATERIALS AND METHODS ...... 15

OBSERVATIONS ...... 19

General Morphology of the Testes . . . . 19

Spermatogenesis ...... 20

Spermlogenesls ...... 24

Submlcroscoplc Structure of Spermatosoa . . . 27

DISCUSSION AND CONCLUSIONS ...... 34

General Morphology of the Testes .... 34

Spermatogenesis ...... 35

Submlcroscoplc Structure of Sperm .... 36

REFERENCES CITED ...... 38

APPENDIX - PLATES 1 through XIV ..... 47

VITA • 61

111 LIST OP PLATES

Plate Page

I Figures 1 through 8. 47

II Figures 9 through 16 .... 48

III Figures 17 through 24 .... 49

IV Figures 25 through 32 .... 50

V A schematic diagram of a spermatozoon of H. vlrescens...... 51

VI Cross-sections of sperm heads from region 1 (Plate V), sectioned with glass knife and stained with Reynold's lead citrate and uranyl acetate (X 11,300) ...... 52

VII Cross-sections of sperm heads from region 2 (Plate V), sectioned with glass knife and stained with Reynold's lead citrate and uranyl acetate (X 11,300) ...... 53

VIII Cross-sections of sperm heads from region 3 (Plate V), sectioned with glass knife and stained with Reynold's lead citrate and uranyl acetate (X 11,300) ...... 54

IX Cross-sections of spermatozoa from region 4 (Plate V), sectioned with glass knife and stained with Reynold's lead citrate and uranyl acetate (X 11,300) ...... 55

X Cross-sections of spermatozoa from region 5 (Plate V), sectioned with diamond knife and stained with lead hydroxide (X 11,300) . 56

XI Cross-sections of spermatozoa from region 6 (Plate V), sectioned with glass knife and stained with Reynold's lead citrate and uranyl acetate (X 11,300) ...... 57

XII Cross-sections of spermatozoa from regions 7 and 8 (Plate V), sectioned with glass knife and stained with Reynold's lead citrate and uranyl acetate (X 15,000) ...... 58

lv Plate Page

XIII Cross-sections of spermatozoa from regions 9 and 10 (Plate V), sectioned with glass knife and stained with Reynold's lead citrate and uranyl acetate (X 11,300). .... 39

XIV Slant-sectlons of spermatozoa through the mitochondrial strands, sectioned with diamond knife and stained with lead hydroxide (X 11,300) . . 60

v ABSTRACT

Spermatogenesis and the submlcroscoplc strut. vxe of spermatozoa

In Hellothis vlrescens (F.) were studied by using light and electron

microscopes, respectively.

In the larval stage of H . vlrescens the testes are paired organs

located dorsally near the alimentary canal In the fifth abdominal seg­

ment. They are yellowish, more or less kidney-shaped, and are situated

separately on each side of the dorsal line. Each testis consists of

four follicular chambers. During the larval stage, the two testes in­

crease in size, approach each other, and finally become fused into a

single spherical body in the prepupal stage.

The testes of the early fourth lnstar larvae contain primary and

secondary spermatogonia and primary spermatocytes. Maturation division

was observed first in the testes of late fourth lnstar larvae and con­

tinued through a part of pupal stage. Sperm bundles, both aupyrene and

apyrene, were observed in the testes of 3-day-old pupae. No evidence of

cell divisions, either mitotic or meiotic, was present in the testes 2 -

3 days before adults emerged. However, late-formed spermatids with axial vacuoles along their elongating tails were observed in the testes of 1 - day-old .

Thirty-one tetrold or dyadal chromosomes were observed during the

first or the second maturation division. Since the heterogametlc sex in

Lepldoptera is female, there must be 62 chromosomes In the diploid con­

dition. vi The structure of the spermatozoon as revealed by light microscopy

is typical of the , with neither a neck region nor a discrete

middle piece. The sperm head resembles a spear or a beak but its exact

length is difficult to determine without staining. However, after being

stained with aceto-carmine, the nucleus of the spermatozoon can be seen

easily as a two-segmented structure which measured about 11 microns in

length. The overall length of the spermatozoon, measured in the sperm

bundles, was about 520 microns.

A schematic diagram of a spermatozoon of (I. vlrescens was constructed

based on the information obtained from both light and electron microscopic

studies. Cross-sections of the sperm head revealed the electron-opaque nucleus which is internally homogenous. Transverse sections posterior

to the nucleus disclosed two parts: a flagellar and a mitochondrial complex. The flagellar complex consists of 9-outer-coarse fibrils,

9-perlpheral fibrillar doublets, 9-inner-monofibrils with spokes and

2-central-palred fibrils. They are arranged concentrically in a pattern similar to that reported by Fawcett (1962) in manmallan and by Andre

(1961) and Bawa (1964) in spermatozoa.

The mitochondrial complex is composed of two strands. They are unequal in diameter as well as in length. Structures similar to that of mitochondrial cristae were observed in both cross- and slant-sectlons.

Radial laminae and a cone-shaped structure were observed in the anterior part of the sperm. Both the radial laminae, which surround the anterior part of the sperm, and the cone-shaped structure are located outside the cell membrane of the sperm, and are probably derived from it.

vii INTRODUCTION

The Hellothia complex on cotton Includes the tobacco budworm,

H. vlrescens (F.), «n<* the bollworm, H. zea (Boddle). H. vlrescens

has been recognized as a cotton pest in Louisiana since 1949, al­

though Folsom (1936) collected it from cotton at Tallulah during

1934. According to Metcalf and Flint (1962), it attacks tobacco,

cotton, ground cherry, and other solanaceous plants as well as gera­

nium and ageratum. This species has become one of the major cotton

insect problems in Texas in recent years (Adklsson, 1964; Adkisson

and Nemec, 1965; 1966).

The principal means of controlling Heliothis has been through

the use of >the synthetic organic insecticides. Subsequently, several problems related to the use of insecticides have arisen. Insecticide- resistant strains of H. vlrescens have been reported from Texas, Mis­ sissippi, and Louisiana (Brazzel, 1963; Graves et: ,al., 1967; Pate and

Brazzel, 1964). In some areas of Texas, resistance has developed to the point that control of this rpecies is no longer feasible with

insecticides that are currently available. In addition, the effect of Insecticide residues upon wildlife, domestic , and humans is of increasing concern. Thus it is imperative that alternative means of controlling Heliothis be developed.

The eradication of the acrew-worm fly, Cochliomyla hominivorax

(Coquerel), from Curacao and the Southeastern United States was ac- t compllshed by releasing into the natural population laboratory-reared

1 2

males sterilized by gamma radiation. The success of this program has focused attention on the possibility of controlling other insect pests through the use of induced sterility (Knlpling, 1960; Lindquist,

1961).

Recently, El-Sayed (1968) completed iminary studies on the effects of gamma radiation on eggs, le. njpae, and adults of H. vlrescens. Soto and Graves (1967) ana ’ vith (1968) have evaluated most of the better known chemosterilan .gainst this species. All of these studies pointed out the urgent need for information concern* ing spermatogenesis in H. vlrescens. Consequently, the studies re­ ported herein were undertaken to develop information concerning the critical stage(s) in the development of this insect. Radiation or chemosterilants must be applied during spermatogenesis to be most effective.

The objectives of this Investigation were (1) to determine the time-sequence of spermatogenesis in H. vlrescens. and (2) to study the submlcroscoplc structure of its spermatozoa. REVIEW OF LITERATURE

Spermatogenesis

Cholodkovsky (1884) recognized that the testes of lepldopteran

Insects could be classified into four groups based on their morpho­ logy. In the most primitive condition as found in Hepialus. the two testes are completely separate and each testis is four-lobed present­ ing a digitate appearance. In Saturnia. the testes are also separate but they are rounded and three-lobed. In Lvcaena. the double testes are together in a single scrotum. However, in Pleris. the two testes are so closely united that they appear to be one round median gonad.

The latter type probably is prevalent in the higher Lepidoptera and is found in the western tiger swallow-tail, Papilio rutulus Bd. (Mun­ son, 1906); P. cresphontes Cramer, Danaus archjppus and Acronycta spp. (Cook, 1910); Heliothis zea (Boddle) (Callahan, 1958); Porthetria dispar (L.) (Goldschmidt, 1934 and Rule et al., 1965); Ostrinia nubi- lalis (Hllbner) (Chaudhury and Raun, 1966); and Diatraea saccharalis

(F.) (Virkki et al., 1969).

Early research on spermatogenesis in Lepidoptera was concerned mainly with the origin of the germ cells, the formation of the sperm

(with particular interest in the achromatic structures), and the cyto­ plasmic structures of the germ cells.

The apical cell was first observed by Spichardt (1886) In the testes of the sphlnx-, Smerinthus pppull. Verson (1889) found in the testes of silkworm, Bombvx mor1 (L,)t a large primitive cell

located at the distal end of the follicular chamber. He suggested

that this cell gave rise amitotically to all the formed cellular ele­

ments in the testis. Munson (1906) described the 'Grandmother Stem

Cell' in the testes of Pattilio rutulus and suggested that it origi­

nated as a germ cell and gave rise to the 'Mother Branch Cells' which

in turn gave rise to the primary spermatogonia. Apparently, Munson's

Grandmother Stem Cell and the apical cell are the same.

The origin and function of the apical cell have been discussed

by various authors. Toyama (1894 a and b), Goldschmidt (1917 and

1931) and Takakusu (1924) suggested that the apical cell probably

had a somatic origin and functioned as a support for the young germ cells. Both La Valette St. George (1897) and Tichomirow (1898) dis­ agreed with Verson's idea and considered that the apical cell was probably a nutritive element. This suggestion was further extended by Grllnberg (1903) and supported by Buder (1913) and Kernewitz(1915).

All three of these authors contended that the apical cell probably originated from germ cell. Zick (1911) agreed with GrUnberg In that the apical cell provided nourishment for the spermatogonia but he also speculated that it probably exerted some influence over the development of the germ cells, since imnediately after its removal, the primary spermatogonia were ready to be transformed into more advanced stages. In a more recent study, Carson (1943) compared the apical cells from different sources of testes. He contended that the apical cell was characteristic of connective tissue cells and considered that it probably served as the support of the germ cells, but that its chief function was to provide the mitochondrial material to the latter. He also observed that In the testes of the sulphur

butterfly, Collas philodice Godart, the apical cell maintained its

function in the adult stage of the virgin males and degenerated soon

after copulation occurred. However, in the testes of Fleris brass 1-

cae (L.), Bombvx mori and Vanessa io (L.) , Carson (1945) observed

that the apical cell functioned only in the larval stage and became

degenerate as the Insect reached adulthood.

Apart from the cytoplasmic structures of the germ cells and

their changes during the process of cell divisions, the occurrence

of spermatogenesis within Lepidoptera varies from one species to an­

other. Munson (1906) found that in Papllio rutulus the cytocysts

which contained spermatocytes were formed about four months before

the emergence of the adult. The male was sexually mature about the

middle of its pupal period. In Danaus archippus, Cook (1910) ob­

served that the testes contained mature spermatozoa about 6 days

after pupation. In his monograph on Lymantrla, Goldschmidt (1934)

reported that spermatogenesis was completed before pupation in gypsy moth, Porthetria dispar. Santa and Otuka (1955) studied the occur­

rence of spermatogenesis in cabbage armyworm, Barathra brassicae L.

They found that before the first day of the fifth lnstar, germ cells

remained in the spermatogonial condition without any differentiation.

Thereafter, spermatogenesis proceeded rapidly and continued through

the sixth lnstar. Maturation division did not take place until the prepupal stage. At this time the testes had conjugated as a single capsule. These authors also reported that spermatids appeared in the

testes of 1-day-old pupae, and that after 14 days of pupation almost all the sex cells within the testes were converted into spermatids. 6

In the European corn borer, Ostrinia nubllalls. Cloutier and

Bock (1963) reported that in the testes of fourth instar larvae, the

germ colls did not advance beyond the stage of the primary spermato­

cytes. According to these authors, maturation division probably took

place as the larvae approached the molt to the fifth (last) instar or pharate fifth lnstar. Transforming spermatids were observed in the

testes of 12-day-old larvae and mature spermatozoa appeared in the

sperm tube as pupation occurred. Chaudhury and Raun (1966) also

studied the spermatogenesis and testicular development of the Euro­ pean corn borer. They found that in the testes of fourth Instar

larvae, the main cellular components were primary and secondary spermatogonia, and the primary spermatocytes. Meiotic divisions occurred during the late fourth or early fifth instar larvae, and the spermatids appeared at the latter stage. Mature spermatozoa were observed in the testes of fully grown larvae. These authors ascer­ tained that in the European corn borer, spermatogenesis ceased as the insect reached adulthood. Their findings coincided largely with those of Cloutier and Beck (1963).

Vivkki (1963) reported that in the testes of the sugarcane borer, Diatraea saccharalis. the primary spermatocytes appeared in 9- day-old larvae, and maturation division started in 11-day-old larvae, lnsnediately after pupation, he observed that the normal meiotic process terminated instead with an asynaptic one resulting in the formation of apyrene sperm. No maturation division was observed in the testes of 1-day-old moths and the spermatocytes were in the process of degeneration.

Rule et £l. (1965) contended that their observations of sperma- 7

togencsls in the gypsy moth were different in some details from those

findings reported by Goldschmidt (1934) in the same species. They

traced the development of the testes in the gypsy moth from first

instar larvae to adult moth males and found that the maturation di­ vision did not take place before the fourth instar. Spermatogonia were no longer in evidence in the testes at the prepupal stage, but the mature spermatozoa in bundles were observed in the testes of 4- to 6-day-old pupae. These facts implied that spermatogenesis ceased before ecloslon of adults.

Recently, Flint and Kressin (1967) while studying gamma irradi­ ation of the tobacco budworm briefly described spermatogenesis of the non-irradiated specimens. They found that the testes of the third instar larvae contained only spermatogonia and those of the fourth lnstar contained both gonial cells and primary spermatocytes. Bundles of spermatids appeared first in the testes of late fifth instar larvae.

Since the testes of 1-day-old moths contained primarily mature sperm, the authors concluded that the supply of sperm was not replenished in the adult stage.

Maklno (1951) recorded the chromosome numbers of about 325 lepl- dopteran species. However, only 11 species belonged to the family

Noctuidae. Eight of the eleven species possessed 31 chromosomes in the haploid condition. Of the remainder, one had 29 chromosomes in the haploid condition, another 30, and the third, 32.

The formation of flagellate sperm has been studied by a number of authors in a wide variety of animals. In regard to the origin of the cytoplasmic structures of the spermatozoa, there is general agreement. The acroaome, which with exception of the sperm of Thermobia domestica (Packard), (Bawa, 1964) is always anterior to

the sperm nucleus, la derived from Golgl complex (Bowen, 1920, 1922

a, c and d; Gresson, 1935; Dillon, 1950; Clayton jit _al., 1958;

Gatenby and Tahmlsian, 1959; Kaye, 1962; and Davey, 1965).

In the early works, the nebenkern was considered as partly

derived from the spindle fibers resulting from the second meiotic

division (Munson, 1906; and Cook, 1910). Mevea (Cook, 1910) claimed

that the formation of the nebenkern was Independent from the spindle

fibera but derived from the granules identical with the mitochondria

of Benda. This idea was confirmed and expanded by later works

(Bowen, 1922 b and d; Nath, 1925; Gresson, 1935; Dillon, 1950; Beams jet jil., 1954; Clayton jt al., 1958; Kaye, 1958 a; Gatenby and Tahmi-

sian, 1959; and Davey, 1965), and it is now understood that the

nebenkern Is formed from the condensation of mitochondrial materials within the spermatocytes during the second maturation division. As

the spermatids are in the process of transformation to sperm, the

nebenkern reforms into one or two mitochondrial strands of the mature

sperm.

The axial filament complex was considered to arise from the centrosomal substance by early Investigators (Paulmier, 1899; and

Cook, 1910). This Idea has been extended by further investigations of more recent authors. From their study of the kinetic apparatus in sperm cells by electron microscope, Sotelo and Trujillo-

Cendz (1958) suggested that the axial filament complex arises from one of the two centrloles which they termed the filament-forming centriole. Their opinion is shsred by many authors (Bowen, 1920;

Gresson, 1935; Dillon, 1950; Clayton et al., 1958; Gatenby and Tahmisian, 1959; and Davey, 1965).

In the sperm of Papilio rutulus (Munson, 1906), certain species of Saturnlldae and Papilionoldea (Cook, 1910), Stenophvlax stellus

Curt. (Trichoptera) (Gresson, 1935). Apis melllfera L. (Rothschild,

1955), Thermobia domestica (Bawa, 1964, Figs. 1 to 3) and fire ant,

Solenopsis saevisslma (Fr. Smith) (Thompson and Blum, 1967), neither a neck region nor a discrete midpiece has been identified. Davey

(1965) in his review of Insect reproduction states "Almost all of the Insects studied so far possess long, filamentous spermatozoa in which, by light microscopy at least, the head is but little differ* entiated from the tall". It is, perhaps, a general truth that there are no differentiated neck region and middle piece in the flagellated spermatozoa of insect species.

Apyrene spermatozoa were observed in the testes of Pierls bras* sicae (Gatenby, 1917), certain Saturniid species (Bowen, 1922 d),

Bombyx mor 1 (Machida, 1929), Tischeria an T.usticole 1 la Dup. (Knaben,

1931), and Diatraea saccharalls (Virkki, 1963). Bowen (1922 d) comnented that apyrene sperm probably resulted from the improper orientation of the nucleus and nebenkern causing their relative positions to be reversed. Virkki (1963) in his discussion stated that normal maturation division in the testes of D. saccharalls terminated JuBt after pupation, and that asynaptic meiotic cleavage occurred during the pupal stage resulting in the formation of apyrene spermatozoa. -Although pot all cytological studies of Lepidoptera have recorded such fact, it is believed that the formation of apyrene sperm during the pupal period is, perhaps, a general phenomenon among, lepidopteran species. The cytological changes and the process of cell divisions during

spermatogenesis are much the same within the animal kingdom. There*

fore, a review of this subject has not been undertaken.

Submicroscopic Structure of Sperm

Since Manton (1952) and Fawcett (1954) described the basic 9 + 2

fiber pattern of cilia and flagella among plants and animals, re­

spectively, by using the electron microscope, the structure of animal

sperm flagella has been studied extensively. However, Che structure

of spermatozoa in insect species, which have suffered from general

neglect, is less understood than that in the vertebrates (Grigg and

Hodge, 1949; Hodge, 1949; Randall and FriedlHnder, 1950; Fawcett,

1958 and 1962; Bradfield, 1955; Rothschild, 1958; Cleland and Roth­ schild, 1959; Serra, 1960; Telkktt et al.. 1961; Nicander and Bane,

1962; Bawa, 1963; and Bedford, 1963 and 1964), and some invertebrates

(Afzelius, 1955, 1959 and 1963; Bradfield, 1955; Colwin and Colwin,

1961; Shapiro et, a_l*, 1961; Satir, 1962; Silvelra and Porter, 1964;

Bonsdorff and TelkkH, 1965; and Shelanski and Taylor, 1968).

Most of the early electron microscopic studies of male Insect gametes were concerned with structure of the immature stages of the spermatozoa (Beams et al.. 1954; Tahmisian et al.. 1956; Gibbons and

Bradfield, 1957; Yasuzumi and Ishida, 1957; Dass and Ris, 1958; Gall and BJork, 1958; Kaye, 1958 a and b, 1962 and 1964; Gatenby and Tah­ misian, 1959; Andr£, 1959 and 1962; Meyer, 1964; Yasuzumi and Oura,

1964 ahd 1965; Kessel, 1966 and 1967; Phillips, 1966 a; and Tandler and Moribur, 1966). Little is known about the morphology of the mature spermatozoa of insects, even though there arc quite a few

papers dealing with the subject. These studies are contributed by

Dlugosz and Harrold (1952); Rothschild (1955); Yasuzumi et _al. (1958)

Andrd (1961); Daems e£ _al. (1963); Bairati and Bacetti (1964); Bawa

(1964); Makielski (1966); Phillips (1966 b, and 1969); Robison (1966)

Thompson and Blum (1967); and Ross and Robison (1969).

In the house cricket, Acheta domesticus (L.), Kaye (1962) re­

ported that the acrosome is anterior to the sperm nucleus and con­

sisted of two hollow cones. The basal end of the anterior one is

invaginated into a cavity to accommondate the posterior one. In the

spermatids of Sciara coprophila Lint., the acrosome is also anterior

to the nucleus but its end extends posteriorly into the cavity of the

nucleus (Phillips, 1966 a). Tandler and Moribur (1966) observed that

the acrosome in the sperm of the water strider, Gerris remigis (Say),

is a tapering rod which measures about 2.5 mm in length. In contrast

to the usual situation of the acrosome being anterior to the sperm

nucleus, Bawa (1964) observed that in the spermatozoa of Thermobia

domestica, the acrosome is located at the basal end of the nucleus,

which runs nearly the whole length of the sperm. An unusual con­

dition was observed in the aflagellate sperm of an armored scale

insect, Farlatoria oleae (Colvde) (Robison, 1966) and a mealybug,

Pseudococcus obscurus (Ross and Robison, 1969), in that the acrosomal

organelles are lacking.

Apart from those non-flagellated insect spermatozoa (Robison,

1966; Ross and Robison, 1969) in which an organized nucleus cannot

be identified, the sp*. nucleus is, in general, a long, slender rod. infrastructures were ^ served within the nuclei of the spermatidB 12

from a wide variety of animals by a number of authors (Afzellus, 1955;

Grasse _et .al*, 1956; Rebhun, 1957; Dass and Ris, 1958; Kaye, 1958 b;

Yasuzumi and Oura, 1964 and 1965; and Kessel, 1966). With the ex­

ception of Thermobia domestica (Bawa, 1964), the nucleus of the mature

insect sperm Is free of Internal differentiation (Beams et al., 1954;

Yasuzumi and Ishida, 1957; Gall and Bjork, 1958; Yasuzumi et al..

1958; and Thompson and Blum, 1967).

Two-stranded mitochondria are described by a number of authors

in either spermatids or spermatozoa of a wide variety of insects

(Dlugosz and Harrold, 1952; Bradfield, 1955; Rothschild, 1955; Tahmi­

sian et .al., 1956; Clayton et .al., 1958; Yasuzumi .et a_l., 1958; Bawa,

1964; Meyer, 1964; Werner, 1964 and 1965; Tandler and Moribur, 1966;

Kessel, 1967; and Thoi i>son and Blum, 1967). However, in the sperm >

of the fungus gnat, Sclara coprophila. only one mitochondrial strand was observed (Makielski, 1966; and Phillips, 1966 a and b). The armored scale insect and mealybug studied by Robison (1966) and Ross and Robison (1969), respectively, possessed non-flagellate sperm which were devoid of mitochondrial organelles. With the exception of

Gerris remigis. in which the nebenkern derivatives of the sperm are modified into a helical sheath around the flagellum (Moribur, 1956), the mitochondrial strands extend alongside the flagellum in a linear fashion. Structures similar to that of mitochondrial crlstae were observed in the mature sperm of Murgantia (Hemiptera)(Kaye, 1958 a) and Drosophila (Meyer, 1964). In the fire ant, Thompson and Blum

(1967) observed that the spermatozoon beara a aeriea of oblique structures, repeating every 0.03 micron, that were believed to be characteristic of mitochondrial crlstae. 13

The 9 outermost coarse fibrils were reported by a number of authors in the spermatozoa of the Mammalia (Cleland and Rothschild,

1959; Fawcett, 1958, 1961 and 1962; Nlcander and Bane, 1962; Telkktt et al., 1961; and Bawa, 1963). In Insecta, these same features were observed in the sperm flagella of Drosophila and Gelastorrhinus (Ya­ suzumi ^t Al., 1958); Macroglossum stellatarum L. (Andr6, 1961);

Thermobia domestica (Bawa, 1964), and Solenopsis saevitsima (Thompson and Blum, 1967). Although Fawcett (1962) thought that these 9 coarse fibrils were characteristic of mammalian sperm flagella and were lack­

ing in the sperm of fish and many Invertebrates, It is believed that they are probably quite dommon In the flagellate insect sperm. Small arms in one member of each fibrillar doublet were observed by Afzellus

(1959); Gibbons and Grlmstone (1960); Andr£ (1961); Fawcett (1962); and Bawa (1964). Nine inner monofibrils around the central paired ones were described by Gibbons and Grlmstone (1960) in certain flagel­ lates, and by Andrd (1961) and Bawa (1964) in the sperm tails of

Insecta, although the latter claimed that they were the thickenings of the 9 spokes radiating from around the central pair.

The structure of radial laminae was described previously by Ya­ suzumi and Oura. (1965) as clear band derivatives in the spermatids of the silkworm and by Andrd (1961) as layered appendices in the sperm of Macroglossum (Sphingidae), The latter author gave practically no comnent on these structures other than that they belonged to the head region of the sperm. Yasuzumi and Oura (1965) speculated that these structures probably facilitate the activity of the spermatozoa.

Since these derivatives possess a highly ordered fine structure, they described them as being of a paracrystalline nature. The radial 14

laminar structures have not been observed in the spermatozoa of other animals, and they probably are found only around the anterior part of sperm of lepidopteran species.

A cone-shaped structure along with the radial laminae around the cell membrane in the anterior part of the sperm vas observed by AndrA

(1961) in the sperm of Macroglossum. However, AndrA did not speculate about this structure. A similar structure was found in the sperm of the fungus gnat, Sciara coprophila (Makielski, 1966; and Phillips,

1966 a and b), and it runs alongside the single mitochondrial strand and the axial filament which is composed of more than 70 pairs of microtubules. Both Makielski (1966) and Phillips (1966 a and b) sug­ gested that this structure is a part of the mitochondrial nebenkern.

Makielski designated it as the paracrystalline rod while Phillips termed it the crystalloid* Since this structure is located Inside the cell membrane of the sperm, it differs apparently from the cone- shaped structure found in MacroRlossum in ItB origin. The signifi­ cance of the occurrence of the cone-shaped structure is unknown.

Probably this structure serves as a backbone in the anterior part of the sperm in that it provides some rigidity to maintain a definite direction as the sperm undulates in the medium. MATERIALS AND METHODS

H. virescena collected from cotton in the vicinity of Transyl­

vania, Louisiana were cultured in the laboratory utilizing Berger's

(1963) techniques and diet with modifications as follows:

(1) Formaldehyde was eliminated from the diet;

(2) agar was reduced from 2,5 to 1.3 g. per 100 g. of diet; and (3) 2 ml of sodium ascorbate and 20% acetic acid were added into each per 100. g. of diet.

For the study of spermatogenesis, samples were taken from differ­ ent age groups of fourth and fifth instar larvae, pupae and adults.

Testes were removed from sampled individuals with the aid of a dis­ secting microscope to a 0.9% saline solution and fixed either in

Bouin's or in Flemming's strong fluid without acetic acid (Davenport,

1964). The treated tissues were dehydrated through a series of ethyl alcohol dilutions and then placed in xylene for clearing. An equal- parts mixture of xylene and paraffin was used as a preliminary step of embedding. The tissues were held in this mixture for two hours in a vacuum oven under 15 pounds of pressure at 60°C. They were then transferred into freshly melted paraffin for two hours under the same conditions for Infiltration. The tissues were finally embedded into o paraffin (melting-point 56 C) as blocks.

Serial sections about four to six microns in thickness were made by using an AO-815 model microtome with an attached microtome knife.

Ribbons obtained from sectioning were adhered onto thoroughly cleaned

15 16

slides (one end frosted) by utilizing Mayer*s egg albumen (Davenport, o 1964). The slides were thoroughly dried on a hot plate (40 C), then de-waxed, hydrated and stained with either Delafield's haematoxylln and eosin Y, or a modified Flemming's triple stain (Davenport, 1964).

The tissues on the slides were again dehydrated In ethanol dilutions and cleared in xylene before mounting In permount. The slides were dried on the hot-plate overnight. Information concerning the source of tissues, stage of insect development and serial numbers was placed on the frosted end of the slide by-using a pencil.

In order to compare the cytologlcal changes during spermato- and spermiogenesls, some of the tissues were examined inxnedlately after dissection without staining or fixing. Males of different stages

(exclusive of the first three Instars) were sampled, testes were removed by dissection and smeared on the slides with either a drop of 0.9% saline, 15% acetic acid (Cochram and Ross, 1967), or aceto- carmlne (Davenport, 1964). A cover-glass then was placed over the tissue and sealed with finger nail polish.

Living spermatozoa were studied also. Sperm samples were taken from testes, seminal vesicles, and the ductus ejaculatorius duplex and simplex of 1- and 3-day-old adults. Slides were prepared in the same manner as that described above.

All preparations of slides were examined by using an AO-Spencer phase-contrast microscope with an attached camera (EXA, IHAGEE DRES­

DEN). Micrographs were taken under different magnifications with

35 mm Panatomlc-X film. Positive prints were made by using Ilford photographic paper (Number 4) and Ilfoprlnt processor and enlarged non-uniformly to suitable alses. 17

In order to study the fine structure of the mature spermatozoon with the electron microscope, thin sections were made from testes of

H, virescens. Testes were removed from 3-day-old moths to 0.9%

saline, and then fixed for two hours in 3% glutaraldehyde made In

Sorenson's phosphate buffer without sucrose (pH 7.3) at 4°C (Pease,

1964). Cool Sorenson's phosphate buffer with sucrose (10 ml of 10% sucrose added to 90 ml of prepared buffer) was used to rinse the fixru tissue several times at 30-mlnute intervals. The tissue was o then post-fixed at 4 C for one hour in 1% osmium tetroxide in Soren­ son's buffer (Sorvall, 1967). A graded series of ethyl alcohol di­ lutions was used for dehydration. Propylene oxide served as an inter­ mediate solvent between the dehydrating agent and the embedding mixture. The treated material was finally embedded in Maraglas

(a mixture of 68% maraglas, 20% cardollte, 10% dibutyl phthalate and

2% benzyldlmethylamine). Thin sections which showed an interference o of silver color (about 600 to 900 A according to Sorvall, 1967) were sectioned with Porter-Blum MT-1 and MT-2 automatic Ultra-microtomes equipped with diamond and glass knives, respectively, and picked up with 300-mesh copper grids. In order to increase the contrast, the thin sections were either stained with 1% lead hydroxide for 20 minutes or double-stained with Reynolds lead citrate and 2% uranyl acetate for five and seven minutes, respectively (Sorvall, 1967).

Preparations of thin sections were examined with an EMU-3G

Electron Microscope with attached photographic apparatus. Pictures were taken under magnification of 11,300 and 15,000 by using 2 X 10 and 3% X 4 inch glass plates (manufactured by Kodak Company). Posi­ tive prints were made by using Ilford photographic paper (Number 4, 18

6^| X 11 inches) end Ilfoprint processor and enlarged uniformly to the desired size. OBSERVATIONS

In this study, the main interests were centered on (1) when the maturation division occurs; (2) whether or not spermatogenesis con­ tinues in the adult; and (3) the submicroscopic structure of the spermatozoon. The origin of the germ cells in the embryonic stage and the development of the testes before the fourth instar were not studied.

General Morphology of the Testes

In the larval stage of H. virescens. the testes are paired organs located dorsally near the alimentary canal in the fifth ab­ dominal segment. They are yellowish and more or less kidney-shaped, and are situated separately on each side of the dorsal line. Each testis consists of four follicular chambers, each of which is lined inside with an extremely thin membrane (TM) enclosing all the cellu­ lar elements within it, and outside with a layer of epithelium (EP) constituting the wall of the chamber (Plate I, Figures 1 and 2). The testis is then enclosed in a pigmented cellular layer, the peritoneal coat (PC), which is covered with few tracheae and tracheoles (Plate I,

Figures 1 and 2). Externally, each testis is constricted at its outer edge into four seml-annulae corresponding to each follicular chamber.

During the larval stage, the two testes increase in else, ap­ proach each other, and finally become fused into a single spherical

19 20

body at the prepupal stage or pharate pupal stage (Plate I, Figure 3).

Due to the vigorous differentiation of the cellular elements in both mitotic and meiotlc divisions during the fifth instar (resulting in the stretching of the follicular epithelium), the testicular wall becomes thinner and thinner (Plate 1, Figure 3, compare it with Figure

1). The testes during this stage appear to be soft, delicate and slack, and the peritoneal coat which is now covered with more tracheae becomes pale and translucent*

As the pupal stage progresses, the testes gradually become hard, tough, and compact. Upon the emergence of the adult, the male organ, which is covered with a densely woven tracheal network, attains its maximum size of about 3.0 mm in diameter. Copulation takes place usually on the second or third night after emergence. During this period of time, the sperm bundles are transferred to the seminal vesicles and the ductus ejaculatorius duplex and simplex, resulting in the distention of these portions. Meanwhile, the dimensions of the testes are reduced to some extent. It was observed that six days after emergence the testes shrank greatly and the tracheal network which covers the surface of the testes was easily separated from the peritoneal coat. The shrinkage of the testes may be due to (1) termi­ nation of cell divisions within the testes; (2) transference of sperm bundles from the testes to the seminal vesicles and ductus ejacula­ torius duplex and simplex; and (3) degeneration of cellular components within the testes.

Spermatogenesis 21

From a preliminary study, It was ascertained that in H. vlrescens.

the maturation division never took place before the fourth instar.

The first sign of melotic division observed was during the late fourth

or pharate fifth Instar (from a physiological point of view).

At the distal end of each follicular chamber lies a large nude*

ated mass of protoplasm known as the apical or Verson's cell (AP).

With its surrounding primary spermatogonia1 mass, the apical cell

forms a dense complex which occupies the apex of the follicular

chamber. Morphologically, the apical cell is merely a large nucleus

embedded in an unbounded protoplasmic mass which is associated and

conxnunicated with the nearest primary spermatogonia (PSP) through

the protoplasmic process from each of the latter (Plate 1, Figures 2

and 4). Its origin and function will be discussed later.

Because of the association between the apical cell and the prima-

ry spermatogonia by the protoplasmic strands from each of them, the

latter appear to be shaped as elongated spermatids. The nuclei of

spermatogonia are distally located with reference to the position of

the apical cell. The cytoplasm of each spermatogonium is elongated

toward and connected with the apical cell (Plate I, Figure 4). Im­

mediately after these primary spermatogonia dissociate from Verson*s

cell, they become rounded into spheres. Their nuclei are large and

spherical with little cytoplasm surrounding them. A prominent nu­

cleolus that stains deeply with Delafleld's haematoxylln is located

in the center of the chromatin network of each nucleus (Plate I,

Figure 5). Individual primary spermatogonia are at this stage crowded together against the wall of the follicular chamber.

After the first mitotic division of each primary spermatogonium 22

(Plate I, Figure 6), the resulting two daughter cells, termed second*

ary spermatogonia, remain attached to each other. Through a series

of mitotic divisions that alternate with the growing process, the two

secondary spermatogonia give rise to a cluster of cells forming a

gonadal cyst. Within the cyst, these cells, being somewhat larger,

appear in a rosette pattern, and a protoplasmic process extends

centrally from each of them. The whole cyst, being pressed and

pushed by the development of primary spermatogonia, moves further

inward In the follicular chamber. Some of the mitotic phases of the

secondary spermatogonia can be seen in Plate I, Figures 7 and 8, and

Plate II, Figure 9.

As the spermatogonia continue to multiply, they finally, in each cyst, attain a number of 64 cells which are now termed the primary spermatocytes (PSC). They no longer remain attached to one another centrally in the cyst by the protoplasmic processes. Instead, the cells are arranged peripherally and leave a cavity in the center of the cyst resembling a blastula of an embryo. During this stage of growth, the spermatocyte increases greatly in its size; in compari­ son with the spermatogonia, the cytoplasm of primary spermatocyte increases much more than the nucleus (Plate II, Figure 10).

With the exception of the stages later than the primary sperma­ tocytes, all stages of cellular elements were observed in the testes of early fourth instar larvae of H. vlrescens (Plate I, Figures 1 and 2). When the larvae reach the late fourth instar, the apparent change in the testicular chambers is that the first melotic division is taking place in some cytocysts (term adopted from Munson, 1906, meaning the cysts containing spermatocytes). The cytological changes 23

and the chromosomal behavior of the primary spermatocytes In this species during meiotic divisions follow the same patterns as those described in other male animals by early workers. Some of the first maturation division phases are shown in Plate II, Figures 11 through

16.

The chromosomes, which are now formed in tetroid bodies, can be easily counted in an equatorial plate in polar view of metaphase as pictured in Figures 12 and 13 (Plate II). It was found that in this insect there are 31 tetroid chromosomes in the dividing spermatocytes of first order. The intermediate stage between two successive di­ visions,was not observed. Immediately following the first meiotic division, the two cells resulting from each primary spermatocyte begin to divide again. The 31 dyads line up at the equatorial plate again with their centromeres at the equator (Plate III, Figures 17 and 18). With the division of centromeres, each dyadal chromatid is dissociated into two complete and separate chromosomes (Plate III,

Figure 19). As the two chromosomal groups move oppositely toward the polar ends where each group forms a chromosomal ring, the mito­ chondrial organelles form a bundle with a constriction at the middle part of it coinciding with the constriction of the dividing cell

(Plate III, Figures 20, 21 and 22). The resulting two cells from each secondary spermatocyte are now the spermatids. At this time, each cyst which was designated as spermatocyst by Munson (1906) contains 256 spermatids and the larva has already reached the fifth instar. Spermiogenesls

Upon the completion of the second meiotic division, the mitochon­ drial organelles of the daughter cells (or the spermatids) are con­ densed to form the nebenkern (NK). The nebenkern is positioned closely beside the nucleus whose size has become greatly reduced (Plate III, Fig­ ure 23). The reduction In size of the nucleus probably is due to the condensation of the chromatin material within the nucleus. An acroaomal element was not observed in the preparations of the early spermatids.

The nebenkern itself is composed of a membranous vesicle containing a thread-like material which forms a coiled spireme resembling the finger­ print (Plate III, Figures 23 and 24). While the spermatids are develop­ ing, the thread-like material within the nebenkern vesicle gradually be­ comes diminished. In the meantime, there appear some granules within the nebenkern vesicle of the spermatid, which probably resulted from the break-down of the thread-like material (Plate III, Figure 24, and Plate

IV, Figure 25). Also the relation between the nucleus and nebenkern is changed. Probably it is due to the elongation of the nebenkern vesicle and the spermatid itself. The result is that the anterior end of the nebenkern extends anteriorly and becomes attached to the nucleus at the place where it will become the posterior end of the nucleus (Plate III,

Figure 24, indicated by arrow only). At this time, the acrosome (AC) can be seen clearly as a dark-stained dot which is adjacent to the nucle­ us (Plate III, Figure 24). Examination of the cross-section of the spermatids revealed that the thread-like material is arranged in concen­ tric circles within the nebenkern vesicle (Plate IV, Figure 25). A septum, which probably becomes a part of the membranous components of the two mitochondrial strands of the spermatozoon, was observed in the middle part of the nebenkern In either cross- or longitudinal-sections

(Plate IV, Figures 25 and 26, at arrows). An eye-shaped structure con­

taining a dark-stained spot near the nebenkern, which can be seen In a

cross-section of the spermatid, Is probably the developing axial filament

complex (AFC) (Plate IV, Figure 25).

Gradually, -the acrosome shifts its position anteriorly to the nucle­

us and the granular material within the nebenkern also Increases. The

nucleus becomes enclosed in the anterior part of the nebenkern. This is

probably due to the extension of the nebenkern, resulting in half of the

nucleus being enclosed in the anterior part of the former (Plate IV, Fig­

ure 26). This confirmed the observation made on the electron microscopic

preparations of spermatozoa, in which it was observed that the basal

end of the nucleus extends posteriorly into the cavity created by the in­ completely fused anterior ends of the two mitochondrial strands (Plate V, region 4, and Plate IX, indicated by arrow). The nucleus (NU) becomes condensed into a chromatin mass as shown in Plate IV, Figure 27 and then extends both anteriorly and posteriorly to form a two-segmented struc­ ture (Plate IV, Figure 28). Meanwhile the spermatid with its inner con­ tents elongates posteriorly resulting in a thread-like immature sperma­ tozoon with numerous axial vacuoles along its longitudinal axis (Plate IV,

Figure 29, indicated by arrow). In 3-day-old pupae, sperm bundles have already been formed in the testes.

It was found that in the testes of H. vlrescens. cell divisions were terminated about two to three days before the emergence of the adults.

Both fresh material and paraffin section preparations of testes from newly emerged males were examined. Although the apical cell, which is surrounded by degenerated spermatogonia and cytocysts, was observed, 26

no dividing cells were found. Nevertheless, late-formed spermatids

with axial vacuoles along their elongating tails were observed in the

testes of 1-day-old moths. In the testes, the mature spermatozoa re­

mained in intact cysts as sperm bundles. Two types of sperm bundles

were found in the testes of this species: aupyrene (AU) and apyrene

(AR). The former, which have a huge hyaline cyst cell forming a cap

covering the head region of sperm, contains nucleated spermatozoa

and measures about 550 microns in length. The latter, which is rod­

shaped and compact, measures about one-fifth to one-sixth the length of

the aupyrene forms and contains non-nucleated spermatozoa (Plate IV,

Figure 30). Sperm bundles were not found in the seminal vesicles or

the parts posterior to them until two to three days after emergence.

At this time, the male moths are ready for mating. Whether or not

there is some morphological change of spermatozoa within the sperm

bundles as they are transferred from testes to simlnal vesicles or the

parts posterior to them is difficult to determine. However, there is

some morphological change in the sperm bundle. It was observed that

the huge hyaline cyst cell in the head region of the sperm bundle was greatly reduced as the sperm bundles reached the ductus ejaculatorius duplex and simplex.

Counts of 8perm were made from the cross sections of the sperm bundles. It was determined that each sperm bundle contains exactly

256 spermatozoa resulting from one single primary spermatogonium.

Apparently, to obtain this number, eight cell division! beginning with 27

one single primary spermatogonium, are necessary. This includes six

mitotic and two meiotic divisions.

Living spermatozoa were measured in sperm bundles obtained from

the testes of 1-day-old moths and the ejaculatorius simplex of 3-day-

old moths (Plate IV, Figure 30). The overall length of the spermatozoon

was about 520 microns. This was very close to the 500 microns measure­

ment given by Callahan and Casclo (1963) for the sperm bundles obtained

from seminal vesicles and lower vas deferens of H. zea (Boddie). The

sperm head with its projecting acrosome resembles a spear or a beak

(Plate IV, Figure 31), but its exact length was difficult to determine

externally. However, after the fresh material is stained with aceto-

carmlne, the nucleus of the spermatozoon can be easily seen. It was

composed of two segments with the anterior part measuring about 4.5 microns and the posterior part about 5.5 microns in length. A ring or a nodular structure (at arrows), which is about one micron in length,

joins the two segments together and the total length of the nucleus measured about 11 microns (Plate IV, Figure 32). Neither a neck region nor a discrete middle piece was observed in the sperm of this species.

Submlcroscopic Structure of Spermatozoa

Since a complete serial section of a spermatozoon was not obtained in either cross- or longitudinal-section, the descriptions and interpretations that are made are based on fragmental observations.

A schematic diagram of a spermatozoon of J|. virescens which is based upon the Information obtained from both light and electron microscopic studies, is presented in Plate V. In order to facilitate explanations of the observations, numbers are used to represent different 28 regions of the spermatozoon and correspond to the electron micro* graphs.

The acrosome which projects from the anterior end of the sperm head was not observed in the thin section preparations in the present study. However, it is assumed that the acrosome originates deep within the head with its base extending probably into the anterior end of the nucleus (Plate V, AC).

It was found that the sperm nucleus consists of two segments jointed together by a ring or nodular structure. A cross-section of the sperm in region 1 anterior to the nodular structure revealed that the nucleus was electron-opaque with homogeneous material in its center.

The nucleus, surrounded by a laminar structure (Plate VI), measured about 0.17 X 0.53 microns in diameter.

A cross-section of region 2, through the area of the nodular structure, disclosed that an electron transparent cleft is located near the center of the nucleus (Plate VII). The cross-section of the sperm head and the nucleus measured about 0.9 and 0.26 X 0.40 microns in diameter, respectively. It was observed from transverse sections through the region just below or behind the modular struc­ ture that the anterior end of the axial filament complex lies immediately next to the nucleus. However, in this area, the mitochondrial strands

(MS) and the radial laminae (RL) were not observed. This strongly suggests that the axial filament complex, which is parallel and con­ tiguous with the nucleus, originates from just below or behind the nodular structure. The more posterior parts of the sperm head in region

3 exhibited homogeneous nuclear contents which were intensely electron- opaque (Plate VIII). The radial laminar structures, which surround 29

but a e outside the common membrane of the axirl filament com­

plex and the nucleus, were observed. This region, with Its surround­

ing radial laminar structures measured about 0.08 microns in diameter

(Plate VIII). The axial filament complex and the nucleus measured

about 0.26 and 0.20 X 0.42 microns in diameter, respectively. A . ound

structure (a cone-shaped structure would be more proper if you visualise

it three-dimenslonally), less electron-opaque than the nucleus, . s

located outside of but attached to the cell membrane on the side be­

tween the axial filament complex and the nucleus. This structure is

referred to as the cone-shaped structure (CSS).(Plates VIII, IX, and X).

Its diameter decreases gradually from its anterior to the posterior end along the longitudinal axis of the spermatozoon. Its diameter measured

from about 0.25 microns anteriorly (Plate VIII) to about 0.0885 microns posteriorly (Plate X). Its function and chemical nature s l c unknown.

Since its appearance or disappearance is concomitant with the radial

laminae and both structures are located outside the cell membrane, it may be that both structures have a common origin.

Cross-sections of the sperm from the basal area of the head in region 4 reveal that it consists of two parts: a mitochondrial moiety and a flagellar moiety, both of which are enclosed in a common cell mem­ brane which is surrounded by radial structures (Plate IX).

The mitochondrial moiety is composed of two strands, unequal in diameter. They are derivatives of the nebenkern of the spermatids. The two strands are fused together at their anterior parts and separate grad­ ually posteriorly along the longitudinal axis of the sperm. Anteriorly, the two fused mitochondrial strands terminate into rounded blunt ends 30

forming a space between them. This space accommodates the basal end of the nucleus. The cross-sections of the sperm In region 4 dis­ closed an intensely electron-opaque area between the juncture of the two mitochondrial strands (Plate IX, indicated by arrow). This electron* opaque area is probably the base of the nucleus. The fact that the nu­ cleus extends posteriorly between the mitochondrial strands was confirmed by the light microscopic studies of spermatids in an earlier section

(Plate IV, Figure 26). The cristae of mitochondria (CR) can be seen clearly in the transverse sections from the anterior part of the sperm

(Plate IX). Slant sections of the sperm in the anterior part reveal that the mitochondrial strands partially bear a series of oblique and parallel lines (Plate XIV). Apparently, they are the mitochondrial cristae. This strongly suggests that in each mitochondrial strand, the cristae run helically around the longitudinal axis. The space between the oblique lines and that between the cristae in cross-sections of mitochondrial strands (Plates IX and XIV) measured about the same, 0.033 microns.

The flagellar moiety consists of the axial filament complex which constitutes the main part of the locomotor organ of the sperm. Cross- sections of the sperm from region 3 to region 8 reveal the structures of the axial filament complex. It is composed of a plasmic sheath, which encloses three 9-dotted circles arranged in a concentric pattern. A pair of additional structures was observed in the center of these concen­ tric circles. Apparently, these structures are characteristic of either tubules or fibrils, depending upon whether or not their interior are solid. They are parallel to each other as well as to the longitudinal axis of the sperm. The entire filament complex probably takes its origin 31

just below or behind the nodular structure of the sperm nucleus and

extends alongside the mitochondrial strands posteriorly to the sperm

tall. The central paired fibrils (CPF) are probably connected to each

other by a semi-circular sheath (Plates X and XX). Cross-sections of

the sperm anterior to region 7 reveal that the Interior of the central

paired fibrils is more electron-opaque than that from the cross-sections

posterior to region 7 (Plate XII). The two central fibrils are about

0.0083 microns apart and each measures about 0.0166 microns in diameter.

Immediately surrounding the two central fibrils are nine electron-opaque

inner monofibrls (IMF). From each monofibril, a spoke structure

extends centrally attaching to the central paired fibrillar complex

and outwardly probably connecting with the arm-bearing subunit of each

fibrillar doublet. The monofibrils are enclosed by nine fibrillar

doublets (FD) (Plate X). Small arms (AR) project from one member of

each doublet towards the adjacent doublet. A difference In the density was observed between the two subunits of each doublet. The interior of

the armed subunit was slightly denser than the armless subunit (Plate V,

regions 3 through 8 and Plates X and XII). Each member of the fibrillar doublets measured about 0.0165 microns In diameter. The space between

two adjacent doublets was about 0.0230 microns. Immediately inside the plasmic sheath of the axial filament complex, nine coarse fibrils (OF) each measuring about 0.025 microns In diameter were observed around the peripheral doublets (Plate V f regions 3 through 3, and Plates VIII to

XII). Like the central paired fibrils, the Interior of these coarse fibrils is intensely electron-opaque at the anterior region of the spermatosoon. They are practically solid rods (Plates VIII to X).

However, in the cross-sections of the sperm posterior to region 5, these fibrils are less electron-opaque in their interiors and it is probably

more proper to consider them as tubules than fibrils in this region

(Plate XII). The interval between two adjacent coarse fibrils measured

about 0.05 microns, and the distance from the outermost coarse fibril to

the central pair was about 0.051 to 0.083 microns.

The unique structure of radial laminae, which in cross-sections re­

sembles stralted muscle fibers, was observed around the cell membrane in

the anterior part of the sperm in region 3 through 5. These structures

are probably derivatives of the cell membrane as is the cone-shaped

structure. Their chemical nature and function are unknown. In cross-

section of the sperm, the radial laminae measure about 0.066 to 0.11

microns in length and 0.022 to 0.033 microns in width.

Proceeding posteriorly from the anterior part of the sperm, some of

the sperm structures diminish or become greatly reduced. Cross-sections

of sperm in the part posterior to region 5 disclosed that the radial

laminae and the cone-shaped structure were no longer observed (Plates XI

and XII). Concomitant with the disappearance of the cristae, the dimen­

sions of the two separated mitochondrial strands are reduced greatly.

However, the axial filament complex remains more or less constant in

its diameter throughout the length of the sperm.

A single or sometimes a pair of structures can be seen in the area between the axial filament complex and the two mitochondrial strands

(Plate XI, at arrows). These are probably discrete microtubules (MT) observed only in the tail part of the sperm.

Near the posterior end of the sperm tail, only one mitochondrial strand was observed in the cross-sections (Plate XII, MS), which suggests that the two mitochondrial strands are not equal in length. The sperm 33

tail (Plate V, region 8), consisted of the axial filament complex only

(Plate XII, at arrow). The fibrils end one by one (Plate XIII) and

the tip of the tall is finally ended in a solid piece (Plate XIII, indicated by arrow). DISCUSSION AND CONCLUSIONS

General Morphology of the Testea

Based on Cholodkovsky’s (1884) classification system, the testes

of H. virescens apparently belong to the Plerla group. It appears that

In most higher Lepldoptera, the two testes are 4-chambered and separately

located on each side of the dorsal line In the fifth abdominal segment

during the larval stage and become fused into a single median organ in

the prepupal stage. In £. virescens. the testes are kidney-shaped and

composed cf four follicular chambers. They are separate during larval

life but become fused as a single median organ at the prepupal stage.

The embryonic and early larval stages of H. virescens were not

studied. Hence, the origin of the apical cell cannot be discussed. As

to its function, the apical cell probably serves as a supporting mechanism of, and provides the mitochondrial material to, its associated

germ cells (Carson, 1945). It was observed that in H. virescens.

spermatogenesis terminated two to turee days before the emergence of

the adult, and the withering of the apical cell was concomitant with

the termination of the cell divisions in the testes. Although, the apical cell was observed occasionally In the imaginal testes of this species, It was functionless since It was surrounded only by some de­ generated primary spermatogonia and cysts*

34 35

Spermatogenesis

Observations on the occurrence of spermatogenesis in H. virescens

generally agreed with the finds reported by Flint and Kressin (1967).

However, it was observed in the present study that the maturation divi­

sion occurred in the testes of the late fourth (or pharate fifth) in­

star larvae. Sperm bundles, both aupyrene and apyrene, were observed in

the testes of 3-day-old pupae. Also it was found that both mitotic

and meiotic divisions terminated wtihin the testes two to three days before the adults emerged but some late-formed spermatids with axial vacuoles along their elongating tails were observed in the testes of

1-day-old moths. Apparently, the process of spermatogenesis in relation to the developmental stages varies from one species to another among

Lepldoptera. This is due to the fact that the duration of the develop­ ment stages of different species varies. It is not unexpected that the occurrence of spermantogenesis is not completely synchronized within a species since the developmental rate varies from one Individual to another. The termination of spermatogenesis iomediately before or after emergence of the adults is probably a general case among lepidopteran species.

In H. virescens, 31 tetroid or dyadal chromosomes were observed during the first or the second maturation division. Since the hetero- gametic sex in Lepldoptera is female, there must be 62 chromosomes in the diploid condition.

The observations of sperm formation in H. virescens were similar to those of early studies (Bowen, 1922 d, for example). No new findings were observed except that of the two-segmented sperm nucleus. However, 36

the significance of this condition is unknown. A neck region or discrete middle piece was not found In the spermatozoa of H. virescens.

However, neither has been reported in any Insect species.

Submicroscopic Structure of Sperm

The submicroscopic structure of spermatozoa of H. virescens was similar to the structure of flagellate Insect spermatozoa recorded in early works. The acrosome was not observed in the thin section prepara* tions of this investigation. However, it appears that the acrosome assumes a position anterior to the sperm nucleus. Its basal end probably extends posteriorly into the anterior end of the sperm nucleus. Such an arrangement of the acrosome is by no means an unusual one, since it was observed in the spermatids of the fungus gnat, Sciara coprophila Lint.

(Phillips, 1966 a). It was assumed, based on Figures 1, 4 and S of

Thompson and Blum (1967), that such an acrosomal arrangement probably also existed in the spermatozoa of the fire ant Solenopsis saevlssima

(Fr. Smith).

Beams ejt al, (1954) and Yasuzuml et_ al,. (1958) stated that the cristae of the spermatozoan mitochondria diminished as the spermatids transformed into their final forms. However, in the present work, structures which appeared as a series of oblique parallel lines were observed on the surface of mitochondrial strands (Plate XIV). These structures were also observed in the cross sections of the spermatozoan mitochondria (Plate IX). Apparently, they are similar to those reported by early authors (Kaye, 1958 a; Meyer, 1964; and Thompson and Blum, 1967), and are believed to be characteristic of mitochondrial cristae. 37

The fibrillar elements of the sperm flagelium in H. virescens are

arranged in a fashion similar to that described by Fawcett (1962) in

mammals and by Andre (1961) and Bawa (1964) in insects. In the anterior

part of H. virescens sperm, the interior of both the 9-coarse and the

central-paired fibrils was Intensely electron-opaque, This condition

was especially true for the 9-coarse fibrils in that the interior was

practically solid presenting a rod appearance. On the other hand, in the

posterior part of the sperm, they were clearly tubular in nature. Per­

haps, the significance of this condition is that in the anterior part of

the sperm, the 9-coarse fibrils provide some rigidity to malntaing a

definite direction of the sperm head as the spermatozoon undulates in

the medium. The tubular condition of these fibrils in the posterior

end of the sperm provides some flexibility to facilitate the movement

of the sperm.

The radial laminae observed in the present investigation are

apparently homologous with those observed by Yasuzumi and Oura (1965)

and Andre (1961) in other lepidopteran species. These structures which

are located in the anterior part of the sperm, probably increase the

surface area in the head region where the main parts of the mitochon­

drial nebenkern are located. This possibly facilitates the diffusion of

the nutritive materials from the surrounding medium of the spermatozoa to

the energy generator, the stranded mitochondria.

The significance of the occurrence of the cone-shaped structure is

unknown. Probably this structure serves as a backbone in the anterior region of the sperm providing a ileal support as the sperm moves.

The cone-shaped structure probably shares the origin of the radial

laminae and the latter is paracrystalline in nature (Yasuzumi and Oura,

1965). DEFERENCES CITED

Adkisson, P. L. 1964. Comparative effectiveness of several insecticides for controlling bollworms and tobacco budworms. Texas Agr. Expt. Sta. Misc. Publ. 709. 13 pp.

Adkisson, P. L., and S. J. Nemec. 1965. Efficiency of certain insecticides for killing bollworms and tobacco budworms. Texas Agr. Expt. Sta. PR-2357. 11 pp.

Adklsson, P. L., and S. J. Nemec. 1966. Comparative effectiveness of certain insecticides for killing bollworms and tobacco budworms. Texas Agr. Expt. Sta. B-1048(Feb.). 4 pp.

Afzelius, B. A. 1955. The fine structure of the sea urchin spermatozoa as revealed by the electron microscope. Z. Zellforsch. 42:134-148.

Afzelius, B. A. 1959. Electron microscopy of the sperm tail. Results obtained with a new fixative. J. Biophys. Biochem. Cytol. 5:269-278.

Afzelius, B. A. 1963. Cilia and flagella that do not conform to the 9 + 2 pattern. 1. Aberrant members within normal populations. J. Ultrastr. Res. 9:381-392.

Andr£, J. 1959. Etude au microscope electronique de 1'evolution du chondriome pendant la spermatogenese du paplllon du chou Pieris brassicae. Ann. Scl. Nat. Zool. Biol. Anim. 21:283-307.

Andrd, J. 1961. Sur quelques details nouvellement connus de 1'ultra­ structure des organites vibratiles. J. Ultrastr. Res. 5:86-108.

AndrA, J. 1962. Contribution a la connassance du chondriome. Etude de ses modifications ultrastructurales pendant la spermatogenese. J. Ultrastr. Res. Suppl. 3:1-185.

Bairati, A., Jr., and B. Baccettl. 1964. Observation on the ultra­ structure of the male germ cells of Drosophila melanogaster and Dacus oleae. Electron Microscopy. Biol. B:441-442.

Bawa, S. R. 1963. Outer coarse fibers of the mammalian sperm tall - An electron microscope study. J. Ultrastr. Res. 9:475-483.

Bawa, S. R. 1964. Electron microscope study of spermatogenesis in a fire-brat insect, Thermobia domestlea (Pack.). I. Mature spermatozoon. J. Cell Biol. 23:431-446.

38 39

Beams, H. W., T. N. Tahmisian, R. L. Devine, and L. E. Roth. 1954. Phase-contraat and electron microscope studies on the nebenkern, a mitochondrial body in the spermatids of the grasshopper. Biol. Bull. 107:47-56.

Bedford, J. M. 1963. Morphological reaction of spermatozoa in the female reproductive tract of the rabbit. J. Reprod. and Fert. 6:245-255.

Bedford, J. M. 1964. Fine structure of the sperm head in ejaculate and uterine spermatozoa of the rabbit. J. Reprod. and Fert. 7:221-228.

Benkwith, K. B,, Jr. 1968. Effect of selected chemosterilants on mating competiveness and longevity of the tobacco budworm.Heliothis virescens (F.). Thesis, Louisiana State University. 66 pp.

Berger, R. S. 1963. Laboratory techniques for rearing Heliothis species on artificial medium. USDA.-ARS-33-84. 4 pp.

Bonsdorff, C. H. V., and A. TelkkH. 1965. The spermatozoon flagella In Dlphyllobothriuim latum (Fish tapeworm). 2. Zellforsch. 66:643-648.

Bowen, R. H. 1920. Studies on insect spermatogenesis. 1. The history of the cytoplasmic components of the sperm in Hemiptera. Biol. Bull. 39:316-362.

Bowen, R. H. 1922 a. Studies on insect spermatogenesis. II. The components of the spermatid and their role in the formation of the sperm in Hemiptera. J. Morphol. 37:79-193.

Bowen, R. H. 1922 b. Studies on insect spermatogenesis. III. On the structure of the nebenkern in the Insect spermatid and the origin of nebenkern patterns. Biol. Bull. 42:53-80.

Bowen, R. H. 1922 c. On the idiosome, Golgi apparatus, and acrosome in the male germ cells. Anat. Rec. 24:159-180.

Bowen, R. H. 1922 d. Studies on insect spermatogenesis. V. Formation of the sperm in Lepldoptera. Quart. J. Microscop. Sci. 66:595-626.

Bradfield, J. R. G. 1955. The fiber patterns In animal flagella and cilia. Symp. Soc. Expt. Biol. 9:309-334.

Brazzel, J, R. 1963. Resistance to DDT in Heliothis virescens. J. Econ. Entomol. 56:571-574.

Buder, J. E. 1915. Die Spermatogenese von Dellephlla eunhorbiae (L.). Arch. f. Zellforsch. 14:26-78.

Callahan, F. S. 1958. Serial morphology as a technique for determi­ nation of reproductive patterns in corn earvorm, Heliothis zea (Boddia). Ann. Entomol. Soc. Auer. 51:413-428. 40

Callahan, p. S., and T. Cascio. 1963. Histology of the reproductive tracts and transmission of sperm In the corn earworm, Heliothis zea. Ann. Entomol. Soc. Amer. 56:535-536.

Carson, H. L. 1945. A comparative study of the apical cell of the insect testis. J. Morphol. 77:141-161.

Chaundhury, M. F. B., and E. S. Raun. 1966. Spermatogenesis and testicular development of the European corn borer, Ostrinia nubilalls (Lepldoptera: Pyraustidae). Ann. Entomol. Soc. Amer. 59:1157-1159.

Cholodkovsky, N. 1884. Ueber die Hoden der Lepidopteren. Zool. Anz. 7:564-568.

Clayton, B. P., K. Deutsch, and B. M. Joradn-Luke. 1958. The spermatid of the house-cricket, Acheta domesticus. Quart. J. Microscop. Sci. 99:15-23.

Cleland, K. W. and Lord Rothschild. 1959. The bandicoot spermatozoon: an electron microscope study of the tall. Roy. Soc. London, Proc. (B) 150:24-42.

Cloutier, E. J., and S. D. Beck. 1963. Spermatogenesis and diapause in the European corn borer.Ostrinia nubilalis. Ann. Entomol. Soc. Amer. 56:253-255.

Cochram, D« G., and M. H. Ross. 1967. Preliminary studies of the chromosomes of twelve cockroach species (Blattaria: Blattidae, Blattellidae, Blaberidae). Ann. Entomol. Soc. Amer. 60:1265-1272.

Colwin, A. L., and L. H. Colwin. 1961. Fine structure of the sperma­ tozoon of Hydroides hexagonus (Annelida), with special reference to the acrosome region. J. Biophys. Biochem. Cytol. 10:211-230.

Cook, M. H. 1910. Spermatogenesis in Lepldoptera. Proc. Acad. Nat. Sci. Philadelphia. 62:294-327.

Daems, W. T., J. P. Persijn, and A. D. Tates. 1963. Fine structural localization of ATPase activity in mature sperm of Drosophila melanogaster. Expt. Cell Res. 32-163-167.

Dass, C. M. S., and H. Ris. 1958. Submicroscopic organization of nucleus during spermatogenesis in the grasshopper. J. Biophys. Biochem. Cytol. 4:129-132.

Davenport, H. A. 1964. Histological and Histochemical Technics. W, B. Saunders Co. Philadelphia, London. 401 pp.

Devey, K. G. 1965. Reproduction in the Insects. Oliver and Boyd. London. 96 pp. 41

Dillon, L. S. 1950. A review of spermiogenesis in insects, with a study of the process in Hippiscua rugosus under the phase microscope. Texas J. Sci. 2:419-430.

Dlugosz, J., and J. W. Harrold. 1952. The spermatozoon of spider beetle Flinus tectus B., as seen by light and electron microscope. Proc. Roy. Soc, Edinburgh (B). 64:353-366.

El-Sayed, E. I. 1968. Some effects of gamma radiation on the tobacco budworm, Heliothis virescens (Fabricius). Dissertation, Louisiana State University. 62 pp.

Fawcett, D. W. 1954. A study of epithelial cilia and sperm flagella with the electron microscope. Laryngoscope. 64:557-567.

Fawcett, D. W. 1958. The structure of the mammalian spermatozoon. Intern. Rev. Cytol. 7:195-234.

Fawcett, D. W. 1961. Cilia and flagella. IN: J. Brachet and A. E. Mirsky (ed.) The Cell. 2:217-297.

Fawcett, D. W. 1962. Sperm tail structure in relation to the mechanism of movement, 147-170. IN_: D. W. Bishop (ed.) Spermatozoan Motility. Amer. Assoc. Adv. Sci. Publ. No. 72.

Flint, H. M . , and E. L. Kressin. 1967. Gamma irradiation of pupae of the tobacco budworm. J. Econ. Entomol. 60:1655-1659.

Folsom, J. W. 1936. Notes on little-known cotton insects. J. Econ. Entomol. 29:283-285.

Gall, J. G., and L. B. Bjork. 1958. The spermatid nucleus in two species of grasshopper. J. Biophys. Biochem. Cytol. 4:479-484.

Gatenby, J. B. 1917. The degenerate (apyrene) sperm formation of moths as an index to the inter-relationship of the various bodies of the spermatozoon. Quart. J. Microscop. Sci. 62:465-488.

Gatenby, J. B., and T. N. Tahmlsian. 1959. Centriole adjunct, centrioles, mitochondria, and ergastoplasm in orthopteran spermatogenesis. La Cellule. 60:105-134.

Gibbons, I. R., and J. R. G. Bradfield. 1957. The fine structure of nuclei during sperm maturation in the locust. J. Biophys. Biochem, Cytol. 3:133-140.

Gibbons, I. R., and A. V. Grlmstone. 1960. On flagellar structure in certain flagellates. J. Biophys. Biochem. Cytol. 7:697-716.

Goldschmidt, R. 1917. Versuche zur Spermatogenese in Vitro. Arch. f. Zellforsch. 14:421-450. 42

Goldschmidt, R. 1931. Neue Untersuchungen ueber die Umwandlung der Gonaden bei intersexuellen Lymantria dispar (L.). Zeit. wiss. Biol. Abt. D. Roux* Arch. f. Entwicklungsmech. Organ. 124:618- 653.

Goldschmidt, R. 1934. Lymantria. Bibliographia Genetics. 11:1*185.

Grasse, P., P. N. Carasso, and P. Favard. 1956. Les ultrastructures cellulaires au cours de la spermiogenese de l'escargot (Helix gomatla L.)': evolution des chromosomes, du chondriome, de l’appareil de Golgi, etc. Ann. Sci. Nat. Zool. Biol. Animal. 18:339-380.

Graves, J. B., D. F. Clover, and J. R. Bradley, Jr. 1967. Resistance of the tobacco budworm to several insecticides in Louisiana, J. Econ. Entomol. 60:887-888.

Gresson, R. A. R. 1935. The spermatogenesis of Stenophylas stellatus Curt. (Trichoptera). Quart. J. Microscop. Sci. 78:311-327.

Grigg, G. W. , and A. J. Hodge. 1949. Electron microscopic studies of spermatozoa. 1. The morphology of the spermatozoa of the common domestic fowl (Gallus domesticus) . Austral. J. Sci. Res. (B) 2:271-286.

Gruenberg, K. 1949. Untersuchungen ueber die Keim- and Naehrzellen in den Hoden und Ovarien der Lepidoptern. Zeit. f. wiss. Zool. 74:327-395.

Hodge, A. J. 1949. Electron microscopic studies of spermatozoa. II. The morphology of the human spermatozoon. Austral. J. Sci. Res. (B) 2:368-378.

Kaye, J. S. 1958 a. Changes in the fine structure of mitchondria during spermatogenesis. J. Morphol. 102:347-400.

Kaye, J. S. 1958 b. Changes in the fine structure of nuclei during spermiogenesis. J. Morphol. 103:311-321.

Kaye, J. S. 1962. Acrosome formation in the house cricket. J. Cell. Biol. 12:411-431.

Kaye, J. S. 1964. The fine structure of flagella in spermatids of the house cricket. J. Cell Biol. 22:710-714.

Kernewitz, Br. 1915. Spermiogenese bei Lepidopteren mit besonderer Berussichtigung der chromosomen. Arch. f. Naturgesch., Abt. A. 81:1-34.

Kessel, R. G. 1966. The association between microtubules and nuclei during spermiogenesis In the dragonfly. J. Ultrastruct. Res. 16:293-319. 43

Kessel, R. G. 1967. An electron microscope study of spermlogenesls in the grasshopper with particular reference to the development of microtubular systems during differentiation. J. Ultrastruct. Res. 18:677-694

Knaben, N. 1931. Spermatogenese bei Tischerla angusticolella Dup. Z. Zcllforsch. raikr. Anat. 13:290-323.

Knipling, E. F. 1960. The eradication of the screw-vorm fly. Sci. Amer. 203:54-61.

La Valette St. George. 1897. Zur Samen- und Eibildung beim Seidenspinner (Bombyx mori). Arch. f. mikr. Anat. 50:751-766.

Lindquist, A. W. 1961. New ways to control Insects. Pest Control. 29:9-19. 236-240.

Machida. J. 1929. Eine experimentelle Untersuchung ueber die apyrenen Spermatozoon des Seidenspinners Bombyx mori (L.) Z. Zellforsch. mikr. Anat. 9:466-510.

Maklelskl, S. K. 1966. The structure and maturation of the spermatozoa of Sclara coprophila. J. Morphol. 118:11-23

Makino, S. 1951. An Atlas of the Chromosome Numbers in Animals. Iowa University Press. Ames. Iowa. 290 pp.

Manton, I. 1952. The fine structure of plant cilia. Symposia Soc. Expt. Biol. 6:306-319.

Metcalf. C. L., and W. P. Flint. 1962. Destructive and Useful Insects. 4th Ed. McGraw-Hill Book Co. 1087 pp.

Meyer, G. 1964. Die parakristallinen Korper in den Spermienschwarmen von Drosophila Z. Zellforsch. 6:762-784.

Moribur, L. C. 1956. A cytochemical study of hemipteran spermatogensla. J. Morphol. .99:271-327.

Munson, J. P. 1906. Spermatogenesis of the butterfly, Papillo rutulus. Proc. Boston Soc. Nat. Hist. 33:43-124.

Nath, V. 1925. Mitochondria and sperm-tail formation, with particular reference to moths, scorpions, and centipedes. Quart. J. Microscop. Sci. 69:643-659.

Nlcander, L . , and A. Bane. 1962. Fine structure of boar spermatozoon. Z. Zellforsch. 57:390-405.

Pate, T. L., and J. R. Brazzel. 1964. Resistance of bollworms to poison tested. Mississippi Farm Res. 27(4):1-3.

Paulmier, F. C. 1899. Spermatogenesis of Anasa tritis. J. Morphol. Suppl. 15-223-270. 44

Pease, D. C. 1964. Histological Techniques for Electron Microscopy. Second Ed. Academic Press. New York, London. 381 pp.

Phillips, D. M. 1966 a. Observations on spermiogenesis in the fun­ gus gnat Sciara coprophila. J. Cell Biol. 30:477-497.

Phillips, D. M. 1966 b. Fine structure of Sciara coprophila sperm. J. Cell Biol. 30:499-517.

Phillips, D. M. 1969. Exceptions to prevailing pattern of tubules (9+9+2) in the sperm flagella of certain insect species. J. Cell Biol. 40:28-43.

Randall, J. T. and M. H. G. Friendlander. 1950. The microstructure of ram spermatozoa. Expt. Cell Res. 1:1-32.

Rebhun, L. I. 1957. Nuclear changes during spermiogenesis in a pol- monate snail. J. Biophys. Biochem. Cytol. 3:509-524.

Robinson, W. G. 1966. Microtubules in relation to the motility of a sperm syncytium in an armored scale Insect. J. Cell Biol. 29:251-265.

Ross, J., and W. G. Robinson, Jr. 1969. Unusual microtubular patterns and three-dimensional movement of mealbug sperm and sperm bundles. J. Cell Biol. 40:426-445.

Rothschild, Lord. 1955. The spermatozoa of the honeybee. Trans. Roy. Entomol. Soc. London. 107:289-294.

Rothschild, Lord. 1958. The human spermatozoon. Brit. Med. J. No. 5066:301.

Rule, H. D., P. A. Godwin, and W. E. Haters. 1965. Irradiation effects on spermatogenesis in the gypsy moth, Porthetrla dispar (L.). J. Insect Physiol. 11:369-378.

Santa, H., and M. Otuka. 1955. Studies on the diapause in the cabbage armyworm Barathra brassicae L. IV. Development of the male sex cells under the condition inducing diapause or non-diapause. Bull. Natl. Inst. Agric. Sci. (Tokyo) (C) No. 5:58-72

Satir, P. 1962. On the evolutionary stability of the 9 + 2 pattern. J. Cell Biol. 12:181-184.

Serra, J. A. 1960. Why flagella and cilia have 1 + 9 pairs of fibres. Expt. Cell Rea. 20:395-400.

Shapiro, J. E., B. R. Hershenov, and G. S. Tulloch. 1961. The fine structure of Haematoloechus spermatozoon tail. J. Biophys. Biochem.

Shelanskl, M. L., and E. W. Taylor. 1968. Properties of the protein subunit of central-pair and out-doublet microtubules of aea urchin flagella. J. Cell. Biol. 38:304-315. 45

Silveira, M., and K. R. Porter. 1964. The spermatozoid of flatworms and their microtubular systems. Protoplasma. 59:240*265.

Sorvall, I. 1967. Thin Sectioning and Associated Technics for Electron Microscopy. Second Ed. with Supple. Ivan Sorvall Inc. Norwalk, - Conn. 123 pp.

Sotelo, R. J., and O. Trujille-Cenoz. 1958. Electron microscope study of the kinetic apparatus in animal sperm cells. Z. Zellforsch. 48:565-601.

Soto, P. E., and J. B. Graves. 1967. Chemosterilization of bollworms and tobacco budvorms. J. Econ. Entomol. 60:550-553.

Spichardt, C. 1886. Beitrag zu der Entwicklung der mannlichen Genitalien and ihrer Ausfuhrungsvorgange bei Lepldoptern. Verb, naturh. Ver. preuss. Rheinl. 43:1-34.

Tahmlsian, T. N. , E. L. Powers, and R. L. Devine. 1956. L.ght and electron microscope studies of morphological changes of mitochon­ drial during spermatogenesis in the grasshopper. J. Biophys. Biochem. Cytol. 2:325-330.

Takakusu, S. 1924. Beebachtungen ueber die Spermatogenese in vltre. Zelt. f. Zellen- und Gewebelehre. 1:22-28.

Tar.dler, B., and L. B. Moribur. 1966. Microtubular structures associated with the acrosome during spermiogenesis in the water-strider, Gerris remigis (Say). J. Ultrastr. Res. 14:391-404.

Telkka, A., D. W. Fawcett and K. A. Christensen. 1961. Further observations on the structure of the mammalian sperm tall. Anat. Rec. 141:231-245.

Thompson, T. E., and M. S. Blum. 1967. Structure and Behavior of Spermatozoa of the Fire Ant Solenopsis saevissima (Hymenoptera: Formicidae). Ann. Entomol. Soc. Amer. 60:632-642.

Tlchomlrow, A. 1898. Zur Anatomie des Insektenhodens. Zool. Anz. 21:623-630.

Toyama, K. 1894 a. Preliminary note on the spermatogenesis of Bombyx mori. Zool. Anz. 17:20-24.

Toyama, K. 1894 b. On the spermatogenesis of the silkworm. Bull. Coll. Agric. Imp. Univ. Japan. 2:125-157.

Verson, E. 1889. Zur Spermatogenese. Zool. Anz. 12:100-103.

Virkkl, N. 1963. Gametogenesls in sugarcane borer moth, Dlatraea saccharalis (F.) (Crambldae). Univ. Puerto Rico. J. Agr. 47:102-137. 46

Virkki, N., D. W. Walker, and C. L. Rodriguez. 1969. Excision of gonads from Immature Dlatraea. Ann. Entomol, Soc. Amer. 62:457-458.

Werner, G. 1964. Ueber die Stllung von Akrosom and Zentriol beim Spermium des Silberfiscbchens, Lepisma saccharine L. Electron Microscopy. Biol, B:443-444,

Werner, G. 1965. Unterschungen ueber die Spermiogenese beim Sandlaufer, Cicindela campestris L. Z. Zellforsch. 66:255-275.

Yasuzumi, G., W. Fukimura, and H. Ishida. 1958. Spermatogenesis in animals revealed by electron microscopy. V. Spermatid differentia­ tion of Drosophila and grasshopper. Expt. Cell Res. 14:268-285.

Yasuzumi, G., and H. Ishida. 1957. Spermatogenesis in animals as revealed by electron microscopy. II. Submicroscopic structure of developing spermatid nuclei of grasshopper. J. Biophys. Biochem. Cytol. 3:663-668.

Yasuzumi,G., and C. Oura. 1964. Spermatogenesis in animals as revealed by electron microscopy. XIII. Formation of a tubular structure and two bands in the developing spermatid of the silkworm, Bombyx mori Linne. Z. Zellforsch. 64:210-226.

Yasuzumi, G., and C. Oura. 1965. Spermatogenesis in animals as re­ vealed by electron microscopy. XIV. The fine structure of the clear band and tubular structure in late stages of development of spermatids of the silkworm, Bombyx mori Linne. Z. Zellforsch. 66:182-196.

Zick, K. 1911. Die postembryonale Entwicklung des mannlichen. Genitalapparates der Schmetterlinge. Zeit. wiss. Zool, 98:430-477. APPENDIX PLATE I

Figure 1 Horizontal-section of testis, fourth instar larva, Bouin's fixative and Delafield's haematoxylin (X 250). EP: Epithelium; PC: Peritoneal Coat TM: Thin Membrane.

Figure 2 Sagittal-section of testis, fourth instar larva, Bouin's fixative and Delafield's haematoxylin (X 250). AP: Apical Cell; EP: Epithelium; PC: Peritoneal Coat; TM: Thin Membrane.

Figure 3 Cross-section of testis, prepupal stage, Flemming's fixative and modified Flemming's triple stain (X 250).

Figure 4 Apical cell with associated primary spermatogonia (PS), fourth instar testis, Flemming’s fixative and modified Flemming's triple stain (X 1250).

Figure 5 Individual spermatogonia, fourth instar testis, Bouin's fixative and Delafield's haematoxylin (X 1250).

Figure 6 Mitotic telophase of primary spermatogonia, fourth instar testis, fresh material stained with aceto-carmine (X 1250).

Figure 7. Mitotic prophase of second spermatogonia, fifth Instar testis, fresh material stained with aceto-carmine (X 1250).

Figure 8. Mitotic metaphase of secondary spermatogonium, fourth instar testis, fresh material stained with aceto-carmine (X 1250).

PLATE II

Figure 9. Miotic telophase of secondary spermatogenia, fifth instar testis, fresh material stained with aceto-carmine (X 1250).

Figure 10. Firest meiotic metaphase, fifth instar testis, fixed in Flemming's solution and stained with a modified Flemming's triple stain (X 537).

Figure 11. Early diplotene stage, fifth instar testis, fresh material stained with aceto-carmine (X 1250).

Figure 12. First meiotic metaphase, equatorial plate in polar view, immediately after pupation, fresh material stained with aceto-carmine (X 1250).

Figure 13. First meiotic metaphase fifth instar testis, fresh material stained with aceto-carmine (X 1250).

Figure 14. First meiotic metaphase and second meiotic metaphase (indicated by an arrow), fifth instar testis, fresh material stained with aceto-carmine (X 1250).

Figure 15. First meiotic late anaphase, fifth instar testis, fresh material stained with aceto-carmine (X 1250).

Figure 16. First and second (indicated by an arrow) meiotic telophases, fifth instar testis, fresh material stained with aceto-carmine (X 537). 48 PLATE III

Figure 17. Second meiotic mctaphase, equatorial plate in polar view, fifth instar testis, fresh material stained with aceto-carmine (X 1250).

Figure 18. Second meiotic metaphase, fifth instar testis, fresh material stained with aceto-carmine (X 1250).

Figure 19. Second meiotic anaphase, fifth instar testis, fresh material stained with aceto-carmine (X 1250).

Figure 20. Second meiotic telophase with dividing nebenkern, fifth instar testis, fresh material stained with aceto-carmine (X 1250).

Figure 21. Dividing nebenkern of secondary spermacytes, fifth instar testis, fresh material in 0.9% saline, (X 250).

Figure 22. Second meiotic telophase with dividing nebenkern, prepupal testis, fixed in Flemming's solution and stained with modified Flemming's triple stain (X 1250).

Figure 23. Early stage of spermatid with spireme nebenkern (NK), immediately after pupation, fresh material in 15% acetic acid, (X 1250).

Figure 24. Slant sections of early spermatids showing the acrosome (AC), and nebenkern (NK). An arrow indicates the position of nucleus with relation to the nebenkern. Prepupal stage fixed in Flemming's solution and stained with modifed Flemming's triple stain (X 1250). 49 PLATE IV

Figure 25. Cross- and slant-scctions of spermatids, pre­ pupal stage fixed in Flemming's solution and stained with a modified Flemming's triple stain (X 1250). AFC: Axial Filament Complex

Figure 26. Longitudinal sections of spermatids, pre­ pupal stage fixed in Flemming's solution and stained with modified Flemming's triple stain (X 1250) AC: Acrosome

Figure 27. Some early and late spermatids from the testes of newly pupated H. virescens, fresh material stained with aceto-carmine (X 537). NU: Nucleus

Figure 28. Late stage of spermatids with elongated nuclei immediately after pupation, fresh material stained with aceto-carmine (X 1250).

Figure 29. Late stage of spermatids with axial vacuoles (at arrow) along elongated tails, 1-day-old pupa, fresh material in 0.9% saline (X 537).

Figure 30. Aupyrene (AU) and apyrene (AR) sperm bundles from testes of 1-day-old moth, fresh material in 0.9% saline (X 125).

Figure 31. Cross- and slant-sections of sperm bundles from testes of 6-day-old moth, fixed in Flemming's solution and stained with modified Flemming's triple stain (X 1250).

Figure 32. Spermatozoan nuclei from 1-day-old moth testes fresh material stained with aceto-carmine (X 1250). 50 PLATE V

A schematic diagram of a spermatozoon of H. virescens. Numbers 1 to 10 represent different regions corresponding to the cross sections of spermatozoa shown in Plates VI through XIII.

AC: Aerosome AFC: Axial Filament Complex AR: Arm(s) CF : Coarse Fibril CPF: Central Paired Fibrils CR: Cristae of Mitochondrion CSS: Cone-shaped Structure FD: Fibrillar Doublet IMF: Innter Monofibril MS: Mitochondrial Strand(s) MT: Microtubule(s) NU: Nucleus RL: Radial Laminae 51

AC- 1 -NU * 05 a j .

CSS

0 4 4 a j . NU RL

. 0 4 a j ,

0 4 a j , MS

MS- RL

■ 0 4 4 a j . AFC- 6 MT-

0 2 7 a j . -AFC 7 -MS

CPF- . 022 a j FD- IMF- AR- CF-

021 a j

J 9 o 0-26 M Q 3 aj 10 PLATE VI

Cross-sections of sperm heads from region 1 (Plate V), sectioned with glass knife and stained with Reynold's lead citrate and uranyl acetate (X 11,300), 52 ?IATE VII

Cross-sections of sperm heads from region 2 (Plate V), sectioned with glass knife and stained with Reynold's lead citrate and uranyl acetate (X 11,300). 53 PLATE VIII

Cross-sections of sperm heads from region 3 (Plate sectioned with glass knife and stained with Reynold lead citrate and uranyl acetate (X 11,300).

PLATE IX

Cross-sections of spermatozoa from region 4 (Plate V), sectioned with glass knife and stained with Reynold's lead citrate and uranyl acetate (X 11,300). Arrow indicates the posterior end of the sperm nucleus. 55 PLATE X

Cross-sections of spermatozoa from region 5 (Plate sectioned with diamond knife and stained with lead hydroxide (X 11,300). 56 PLATE XI

Cross-sections of spermatozoa from region 6 (Plate V), sectioned with glass knife and stained with Reynold's lead citrate and uranyl acetate (X 11,300). Arrows indicate microtubules. 57 PLATE XII

Cross -sect ions of spermatozoa from regions 7 and 8 (Plate V), sectioned with glass knife and stained with Reynold's lead citrate and uranyl acetate (X 15,000). Arrow Indicates the filament complex.

PLATE XIII

Cross-sections of spermatozoa from regions 9 and 10 (Plate V) , sectioned with glass knife and stained with Reynold's lead citrate and uranyl acetate (X 11,300). Arrow indicates the solid end piece of the sperm tail. 59 PLATE XIV

Slant-sections of spermatozoa through the mitochondrial strands, sectioned with diamond knife and stained with lead hydroxide (X 11,300). 60 VITA.

Gong-tseh Chen wat born at Hupei, China, on August 4, 1935.

He was graduated from Fprovincial Pintung Agricultural Vocational

High School, Taiwan, Ch.na, in 1955. In the same year he enrolled in Taiwan Provincial ChungHsing University where he receive! his

B. Sc. degree from the department of Entomology and Phytopathology in 1959. He received his M. Sc. degree in Entomology and Plant

Pathology from National Taiwan University in 1963. He also received his second M. Sc. degree: in Entomology from McGill University, Canada, in 1966.

He entered Louisiana State University in 1966 and presently is a candidate for the degree of Doctor of Philosophy in Entomology.

61 EXAMINATION AND THESIS REPORT

Candidate: Gong-tseh Chen

Major Field: Entomology

Title of Thesis: Spermatogenesis and submicroscopic structure of spermatozoa in Heliothis virescens (Lepidoptera:Noctuidae)

Major ifessor and Chairman

Dean of the Graduate School

EXAMINING COMMITTEE:

Date of Examination:

May 15. 1969