Recent Advances in Insect Embryology in Japan
Edited by H. Ando and K. Miya.
ISEBU Co. Ltd., Tsukuba 1985

1

Sperm Structure in the Praying Mantis,
Paratenodera aridifolia (Mantodea, Mantidae)

Yukio IWAIKAWA
and
Ken-ichi OGI

Synopsis
The sperm structure of the praying mantis, Paratenodera aridifolia (Mantodea, Mantidae) was studied
with light- and electron microscopy. The matured spermatozoon is a long filiform in shape, 2.2 mm inlength, and consists of the acrosome, nucleus, centriole adjunct, and flagellum composed of the axoneme
having the 9+9+2 pattern of microtubule arrangement, mitochondrial derivative, and accessory body. In
the seminal vesicle as well as in the seminal receptacle, 500 to 1200 sperms are bound to form a bundle
with dense material. The sperm bundle from the female sperm receptacle moved actively, whereas thosefrom the seminal vesicle moved only sluggishly.

Introduction
Many ultrastructural studies have been done on the spermatogenesis and the sperm
maturation in various animals (Yasuzumi, 1974). Detailed review on insect sperm was given
by Baccetti (1972) and Phillips (1970). The phylogeny and evolution of insects on the basisof sperm structures such as acrosome or flagellum have been discussed (Baccetti, 1979;
Dallai, 1979; Dallai et al., 1984; Friedlander, 1983; Grygier, 1982; Virkki, 1969).
Although more than five species of Mantidae are known from Japan, we have no
information on the gametes of these mantis. Thus, the ultrastructure of the mature sperm of
Paratenodera aridifolia was investigated to prove a basis for comparative studies on the
mantis sperm.

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Iwaikawa, Y. and K. Ogi

Materials and Methods
Materials
The matured males and females of the mantis, Paratenodera aridifolia Stoll(Mantodea, Mantidae) were collected in the vicinity of Nagoya Univeristy at September.
Spermatozoa isolated from a seminal vesicle and seminal receptacle were used as materials.
Light microscopy
1. Phase-contrast microscopy. Spermatozoa were isolated in some dropsof Hoyle's physiological saline and observed. 2. Fluorescence microscopy. Sperm cells were
stained with 0.01 % aCridinorange solution to clarify a nucleus and an acrosome. After
staining with the method described by Fujita and Fukuda (1975), cells were observed andphotog~aphed under Olympus BH-RFL microscope. The length of organelles was determined directly on a film.
Electron microscopy
1. Transmission electron microscope (TEM). Whole seminal vesicle
and seminal receptacle dissected in balanced saline were fixed in 2% glutaraldehyde buffered
to pH 7.2 with 0.2 M sodium cacodylate solution for 1 hr at room temperature. Then they
were reimmersed in a fresh fixative for 2 hr. After washing for 20 hr in several changes of thebuffer, specimens were post-fixed in 1% OS04 in the same buffer for 1 hr at 4°C. Dehydration was carried out in ethanol series and embedded in EPON. Thin sections were stainedwith uranyl acetate and lead citrate, and examined with a JEM T-8 electron microscope. 2.
Scanning electron microscope (SEM). Specimens were fixed in 2% glutaraldehyde bufferedin 0.2 M cacodylate. After rinsing, they were dehydrated through ethanol series, criticalpoint dried in CO 2 , and subsequently coated with gold and observed by JEM-T20 scanningelectron microscope.

Results
The matured spermatozoon of P. aridifolia is filiform in shape, which consists of aneedle-like head and a long movable flagellum. It measures approximately 2.2 mm in length(Fig. 1). The spermatozoa are deposited in the seminal vesicle and the seminal receptacle as
the sperm bundles (Fig. 2). SEM suggests that the sperms are bounded with each other at theheads to be spatulate but the flagella are not bound (Fig. 3). As shown in Fig. 2, the sperm
bundle taken out from the female spermatheca began a highly active helical movement
immediately after they were left in a saline solution. They continued to move for more than
24 hours in the saline solution. However, the sperm bundles from the seminal vesicles wereclustered firmly and they hardly moved even if they were separated mechanically into asingle bundle. It was difficult to distinguish the acrosome from the nucleus under aphase-contrast microscope. After staining with acridinorange the acrosome was seen in red
and the nucleus is green under a fluorescence microscope, and their length were determined
to be 25 .um and 63 .um respectively. Sperm tails were dyed only very weakly (Fig. 4).
TEM observation reveals that the mantis spermatozoon is thread-like in form, which
tapered toward both ends; it consists of the acrosome, elongated nucleus, centriole adjunct,and flagellum which is composed of the axoneme, mitochondrial derivatives and accessory
bodies. The acrosome is cone-shaped, packed with electron-dense material, situated at theanterior part of the nucleus and elongated inferiorly along one side of the organelle. The
striated bands with a periodicity of 8 nm are seen in the longitudinal section of the acrosome(Fig. 5). At the anterior part of the acrosome, the electron dense substances line to cause theSperm structure of mantis, Paratenodera

3thickening of the inner cell membrane (Fig. 6). Dense granules are seen to surround theacrosomal region like a cap. Therefore the acrosomes are embedded in these materials when
the sperm bundle was formed. The acrosome shows the moderate density and the internal
space may be found in an area slightly close to the distal end of the head (Fig. 7).
The nucleus occupies major part of the head and contains the most intensive substance.
The middle part of the nucleus is cylindrical and 360 nm in diameter. It extends taperingly
toward anterior and posterior ends. At the anterior end, the nucleus is lying along theacrosome, while at the posterior end, the nucleus is surrounded by the centriole adjunctterminates between two mitochondrial derivatives (Fig. 8). At a distallevel of the nuc.leus,
rod body, measured 430 nm in length, may be observed, but its function is unknown.
The flagellum is comprised three components, namely, the axoneme possessing the
microtubules in 9 + 9 + 2 arrangements, mitochondrial derivatives and accessory bodies (Fig.
9). Along the longitudinal axis of the spermatozoon these components may be observed in
the following order: 1. nine doublets and peripheral singlets of axoneme which occur in an
amorphous substance at the distallevel of the nucleus, and then, two central tubules arise
from the distal level of these structures (Fig. 8). 2. two mitochondrial derivatives containing
crystals which arise from the level of distal end of the amorphous substance beside theaxoneme, and 3. three accessory bodies shown as an aggregates of the dense granules foundat the most distal ends of the centriole adjunct and the nucleus. Two mitochondrial
derivatives and three accessory bodies disappear at the distal part of the tail and theaxoneme alone remains, and then it loses the regular arrangement of microtubules at theextreme end of the tail (Fig. 9D).
In this species, as shown in Figs. 6 and 7, about 500 - 1200 spermatozoa are bound with
one another to form a sperm bundle, which is often comet-shaped due to the long tails. The
direction of running of the axoneme is the same in almost all flagella and the sperms locatedin the central part of the packet shift their position backward.

Discussion
The spermatozoon of P. aridifolia is a long filamentous cell and its full length is 2.2 mm.
In general, it has been said that the length of sperm is 300 J.lm or more in many insects(Chapman, 1969). Although long sperms were found in Thermobia domestica (Bawa, 1964),
and in Pseudococcus obscurus (Ross and Robison, 1969), the sperm of the mantis isextremely long as observed in Drosophila (Cooper, 1950). In this mantis the spermatozoa aredeposited as a shape of a bundle in the seminal vesicle as well as in the seminal receptacle.
The bundles isolated from the spermatheca moved very actively in a saline solution, but
those from male did not move so actively. This suggests that the bundles may be activatedwith substances secreted from the female genital duct. This activated motion of the sperm
seems to be very similar to the sperm activation, "capacitation", that has been
demonstrated first in a mammalian sperm by Chang (1951) and Austin (1960). Shephered
et al. (1982) also reported in tick (Acari, Ixodoidea) that the sperm maturation accompaniedwith the structural changes corresponded to the capacitation.
The sperm bundle within a testis has been reported in various insects such as Chortophageviridifasciata (Payne, 1933), Locusta migratoria (Sz6ll6si, 1975), Leptocoris trivittatus(Jtaya et al., 1980), Pseudococcus obscurus (Nur, 1962; Ross and Robison, 1969),Parlatoria

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Iwaikawa, Y. and K. Ogi

Fig. 1 Phase-contrast micrograph of living spermatozoon in seminal receptacle of Paratenodera aridifolia. Needle-like head seen upper middle. x 190.
Fig. 2 Phase-contrast micrograph of living sperm bundle dissociated from seminal receptacle.
Helical movement of flagella may be seen clearly. x 190.
Fig. 3 SEM micrograph of sperm bundle in seminal vesicle. x 350.
Fig. 4 Fluorescence micrograph of sperm bundle stained with acridinorange. Most brightpart corresponds to acrosome followed by nucleus and fuzzy tails. x 360.

Sperm structure of mantis, Paratenodera

5Fig. 5 A. Longitudinal section showing acrosome and nucleus. Cell membrane narrows
slightly at the part of end of nucleus (small arrow) and bears no dense substances.
x 35,000. B. Enlargement of a part showing striations (arrow heads). x 100,000.
A acrosome (arrow head, acrosomal space), Nu nucleus.
Fig. 6 A. Cross section of sperm bundle showing acrosomal region. 500 cells are boundedwith dense material. x 9,000. B. Cross section of sperm bundle at its anterior partshowing the tip of acrosome and dense material (*) around cells. x 35,000.

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Iwaikawa, Y. and K. Ogi

Fig. 7 A. Cross section of sperm bundle at its posterior part shown in Fig. 6. At the centerof bundle various levels of sperms from acrosome to nucleus may be seen.
Numerous nuclei are seen in periphery. In this bundle, sperm number in 1200. x
6,000. B. Cross sections of sperms distributing at the center of bundle. A
acrosome (arrow head, acrosomal space), Nu nucleus, (*) dense material. x 35,000.

Sperm structure of mantis, Paratenodera

Fig. 8 A. Longitudinal section showing the distal part of head connected to tail withcentriole adjunct. Arrow indicates the terminal of centriole adjunct. x 35,000. B.
Longitudinal section showing sperm cut at another plane of cells illustrated in A.
Peripheral singlet (arrow) expands through amorphous substance (*). x 35,000. C.
Cross section showing various levels of head of the sperms. Numbers (I ~ 7)
indicate levels shifting in position anterior to posterior. x 35,000.
Ax axoneme, Ce centriole adjunct, Nu nucleus, R rod body.

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Iwaikawa, Y. and K. Ogi

Fig. 9 A. Longitudinal section of middle part of flagella . x 35,000. B. Cross section of
middle part of flagella. x 35,000. C. Cross section of flagellum showing 9 + 9 + 2
arrangement of microtubules. x 80,000. D. Cross sections at the level close to the
distal end of tail (arrow and arrow heads). x 35,000. Ac accessory body, Ax
axoneme, M mitochondrial derivatives.

Sperm structure of mantis, Paratenodera

9oleae (Robison, 1966), Bombyx mori ,Katsuno, 1978), Papilio xuthus (Numata and Hidaka,1980), Drosophila (Liebrich et al:, 1982), Popillia japonica (Anderson, 1950), and Apis
cerana indica (Bawa and Narwaha, 1975). Approximately 500 - 1200 spermatozoa make abundle in P. aridifolia. FIQwever whether these abundant cells have been derived from asingle spermatogonium or not may not be known definitely till the spermatogenesis isanalyzed. In Odonata, Omura (1953) found that a single follicle of the testis contained 500- 2000. sperms, which were formed after a single spermatogonium repeated cell division 11
tiimes in each cyst. Robison (1966) has shown that the sperm bundle in the scale insect,Parlatoria oleae, containing 16 sperms in encysted form, was derived from a single
spermatogonium. According to Bawa and Marwaha (1975), the sperm bundle of thehoneybee, Apis cerana indica, is corn prized on an average of 72 sperms. Each sperm bundle
has a hyaline cap at its anterior region. Szollosi (1982) has described on the formation of the
sperm bundle in locusts, in which acrosomes of the spermatids become tightly linked by acap of glycoprotein in the cyst during spermiogenesis. In the sperm bundles of P. aridifolia,
the acrosome is coated with electron dense material and bounded with each other (Figs. 5,6). It is very likely that this substance may contain some kind of protein, because the bundle
may be released by a treatment with trypsin (Iwaikawa, unpublished). Bawa and Marwaha(1975) pointed out that, in the honeybee, the hyaline cap disappeared and the sperms wereno longer seen in bundle as the sperms entered into the seminal vesicle. In this mantis, on theother hand, the sperm bundles were preserved not only in the seminal vesicle but also in theseminal receptacle immediately after oviposition. These results suggest that the cap
substance may be different among the insects and male mantes may be able to transfer many
sperms safely into the female genital duct in the form of the bundle.
It is well known that the ultrastructures of insect sperms are different from species tospecies (Baccetti, 1972; Phillips, 1970). TEM observation revealed that the matured spermsof P. aridifolia tapered toward both anterior and posterior ends. The head region of sperm of
this insect consists of the acrosome, nucleus, and centriole adjunct. The acrosome iscone-shaped, within which an internal space may be found. The rod-like structure previously
known from the sperms of the cockroach, Phycnoscelus indicus (Shahaney et al., 1972) was
not found in P. aridifolia. The posterior part extends laterally along the nucleus. This
resembles with that of the sperm in the orthopteran, Conocephalus saltator (Cruz-Landim
and Ferreira, 1977). It is clear that a periodic striation is seen in the acrosome sectioned
longitudinally (Fig. 5). We assume that this repeated pattern is characteristic of this mantis
acrosome. Electron-dense material is accumulated over the inner cell membrane at moreanterior part of the acrosome. This dense layer may correspond to an extraacrosomallayerof triple-layered acrosomal complex exhibited by Baccetti (1972). The nucleus is a solid
homogeneous and the most intensive densely packed organelle as observed in other insect
sperms. The distal part of the nucleus is extensively surrounded by the centriole adjunct, as
occurred in various other species. In P. aridifolia the centriole adjunct may connect thenucleus to the flagellum which has the axoneme arranged in 9 + 9 + 2 pattern ofmicrotubules. In various animals including mammals, it has been shown that the centrioles ofthe sperms which have nine triplets exist near the nucleus (yasuzumi, 1974). In the insects,
however, Phillips (1970) stated that no centriole exists in the sperms of many species. We
also failed to verify nine triplets beneath the nucleus in the matured sperms of P. aridifolia.
In this species only a mass of an amorphous material, 300 nm in length, may be found,instead of the centriole and an origin of nine doublets and peripheral singlets of flagellar

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Iwaikawa, Y. and K.Ogi

axon. Two mitochondrial derivatives were seen to extend along the axon till the distal end of
the tail. This extension resembles closely to that of the cockroach sperm (Shahaney et al.,1972) except that three accessory bodies lie along the axoneme (see also Baccetti, 1972).
Microtubules found in the axons of all sperms in the bundles are arranged in such a way thatthey are pointing the same direction (Fig. 9B). This might be correlated with thesynchronous helical movement of the sperm flagella in the bundle.
In a preliminary study using another mantis, Statilia maculata Thunberg, a positive
reaction for acridinorange staining was detected at the base of the flagellum, as occurred in
the acrosome. This suggests that the ultrastructural variations of the sperms might be present
in another species of mantes, which will be usefull to discuss the sperm structures in future.

Acknowledgments
We wish to thank Dr. M. Takaoka, Professor Emeritus, Nagoya University, for his criticalreading and suggestions in preparing the manuscript.

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Author's address:

Dr. Y. Iwaikawa and Prof. K. Ogi,
Department of Biology,
Faculty of General Education,
Nagoya University,
Chikusa-ku, Nagoya 464, Japan