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LSU Historical Dissertations and Theses
Graduate School
1969
Spermatogenesis and Submicroscopic Structure of
Spermatozoa in Heliothis Virescens (F)
(Lepidoptera: 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.
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70-228
C H E N , Gong - tse h , 1935SPERMATOGENESIS AND SUBMICROSCOPIC
STRUCTURE OF SPERMATOZOA IN HELIOTHLS
VIRESCENS ( F . ) (LEPIDOPTERA:NOCTUIDAE).
T he L ouisiana State U n iversity and A gricultural and
M echanical C ollege, P fuD ., 1969
Entom ology
U niversity Microfilms, Inc., A nn A rbor, M ichigan
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
S e r v i c e U n i t e d States Department of Agriculture Grant 616*15-17.
11
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS
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TABLE OF CONTENTS
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1
LIST OF PLATES
ABSTRACT
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INTRODUCTION
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REVIEW OF LITERATURE
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3
Spermatogenesis
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Submlcroscoplc Structure of Sperm
MATERIALS AND METHODS .
OBSERVATIONS
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10
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General Morphology of the Testes
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Spermatogenesis
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Spermlogenesls
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Submlcroscoplc Structure of Spermatosoa
DISCUSSION AND CONCLUSIONS
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General Morphology of the Testes
Spermatogenesis
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APPENDIX - PLATES 1 through XIV
VITA
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Submlcroscoplc Structure of Sperm
REFERENCES CITED
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36
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61
111
LIST OP PLATES
Plate
Page
I
Figures 1 through 8.
II
Figures 9 through 16
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48
III
Figures 17 through 24
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49
IV
Figures 25 through 32
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V
VI
VII
VIII
IX
X
XI
XII
47
A schematic diagram of a spermatozoon of
H. vlrescens.
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51
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) .
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52
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) .
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53
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) .
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54
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)
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55
Cross-sections of spermatozoa from region 5
(Plate V), sectioned with diamond knife and
stained with lead hydroxide (X 11,300)
.
56
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) .
.
.
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.
57
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) .
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58
lv
Plate
XIII
XIV
Page
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).
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39
Slant-sectlons of spermatozoa through the
mitochondrial strands, sectioned with diamond knife
and stained with lead hydroxide (X 11,300) .
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60
v
ABSTRACT
Spermatogenesis and the submlcroscoplc strut. v xe 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.
four follicular chambers.
Each testis consists of
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 moths.
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 Insects, 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.
to the nucleus disclosed two parts:
complex.
Transverse sections posterior
a flagellar and a mitochondrial
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 Insect spermatozoa.
The mitochondrial complex is composed of two strands.
unequal in diameter as well as in length.
They are
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 animals, 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 act
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
effects of gamma radiation on eggs, le.
vlrescens.
Soto and Graves (1967) ana ’
most of the better known chemosterilan
iminary studies on the
njpae, and adults of H.
vith (1968) have evaluated
.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.
are together in a single scrotum.
In Lvcaena. the double testes
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 nubilalis (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-moth, 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.
prepupal stage.
capsule.
Maturation division did not take place until the
At this time the testes had conjugated as a single
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 9day-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 4to 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 lepldopteran species.
Noctuidae.
However, only 11 species belonged to the family
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 a l., 1958; Kaye, 1958 a; Gatenby and Tahmisian, 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 animal sperm cells by electron microscope, Sotelo and TrujilloCendz (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 1la 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.
spermatids of Sciara
In the
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.
about this structure.
However, AndrA did not speculate
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 coneshaped 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,
1964).
o
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.
dried on the hot-plate overnight.
The slides were
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 acetocarmlne (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 n u d e *
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 primary 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).
changed.
Also the relation between the nucleus and nebenkern is
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.
nucleus becomes enclosed in the anterior part of the nebenkern.
The
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.
were found in the testes of this species:
(AR).
Two types of sperm bundles
aupyrene (AU) and apyrene
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.
some morphological change in the sperm bundle.
However, there is
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-dayold moths (Plate IV, Figure 30).
was about 520 microns.
The overall length of the spermatozoon
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 electronopaque (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 b e ­
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.
of the nucleus.
This space accommodates the basal end
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).
opaque area is probably the base of the nucleus.
This electron*
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.
two adjacent doublets was about 0.0230 microns.
The space between
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
the testes of 3-day-old pupae.
in
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.
gametic sex in Lepldoptera is female, there
Since the hetero-
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
sperm in Hemiptera. J. Morphol.
37:79-193.
the
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
the male germ
c. On the idiosome, Golgi apparatus, and acrosome in
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
insect testis. J. Morphol. 77:141-161.
apical cell of the
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.
7:564-568.
1884.
Ueber die Hoden der Lepidopteren.
Zool. Anz.
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.
Sci. Philadelphia. 62:294-327.
Proc. Acad. Nat.
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.
W, B. Saunders Co.
Devey, K. G.
London.
Histological and Histochemical Technics.
Philadelphia, London. 401 pp.
1965. Reproduction in the Insects.
96 pp.
Oliver and Boyd.
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.
Mirsky (ed.) The Cell. 2:217-297.
IN:
J. Brachet and A. E.
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.
Entomol.
1936. Notes on little-known cotton insects.
29:283-285.
J. Econ.
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.
Zellforsch.
14:421-450.
Arch. f.
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.
124:618653.
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.
spermatogenesis.
Changes in the fine structure of mitchondria during
J. Morphol.
102:347-400.
Kaye,
J. S.
1958 b.
spermiogenesis.
Changes in the fine structure of nuclei during
J. Morphol.
103:311-321.
Kaye,
J. S.
Biol.
Kaye,
J. S.
1964.
house cricket.
1962. Acrosome formation in the house cricket.
12:411-431.
J. Cell.
The fine structure of flagella in spermatids of the
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.
Amer. 203:54-61.
The eradication of the screw-vorm fly. Sci.
La Valette St. George.
1897. Zur Samen- und Eibildung beim
Seidenspinner (Bombyx mori). Arch. f. mikr. Anat. 50:751-766.
Lindquist, A. W.
1961.
29:9-19. 236-240.
New ways to control Insects.
Pest Control.
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.
Expt. Biol. 6:306-319.
Symposia Soc.
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.
Z. Zellforsch. 57:390-405.
Fine structure of boar spermatozoon.
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.
Suppl. 15-223-270.
J. Morphol.
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 polmonate 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.
Entomol. Soc. London.
107:289-294.
Rothschild, Lord.
5066:301.
1958.
The human spermatozoon.
Trans. Roy.
Brit. Med. J. No.
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
J. Cell Biol.
12:181-184.
Serra, J. A. 1960. Why flagella and cilia have 1 + 9
Expt. Cell Rea. 20:395-400.
Shapiro, J. E., B. R. Hershenov, and G. S. Tulloch.
structure of Haematoloechus spermatozoon tail.
pattern.
pairs of fibres.
1961. The fine
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.
21:623-630.
Zur Anatomie des Insektenhodens.
Zool. Anz.
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 thesilkworm.
Agric. Imp. Univ. Japan.
2:125-157.
Verson, E.
1889.
Zur Spermatogenese.
Zool. Anz.
Bull. Coll.
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
*
05a j
.
CSS
04 4 a j .
NU
RL
.
04 a j
04
a j ,
,
MS
RL
MS-
■ 044 a j .
6
AFCMT-
027 a
-AFC
j .
7
-MS
CPF-
.
022 a j
FDIMFARCF-
021 a j
Q 3 aj
10
J9
o
0-26 M
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
EXAM INATION A N D THESIS REPORT
Candidate:
G o n g -t s eh Ch en
Major Field:
Entomology
Title of Thesis:
S pe r m a t o g e n e s i s and su bm ic ro sc op ic structure of s p er m at o zo a
H e l i o th i s v irescens (L ep id optera:Noctuidae)
M ajor
ifessor and C hairm an
Dean of the G raduate School
EXAM INING COMMITTEE:
Date of Examination:
May
15.
1969
in