Ultrastructure and motility pattern of the spermatozoa

of Aleochara curtula (Coleoptera, Staphylinidae)

Michael Wernera, Thomas Tscheulinb, Thomas Speckc, Dieter Zisslera, Klaus Peschkea,*

aInstitut fur Biologie I (Zoologie), Hauptstr. 1, D-79104 Freiburg i.Br., GermanybDepartment of Biology, Imperial College of Science, Technology and Medicine, Silwood Park, Ascot, Berkshire SL5 7PY, UK

cPlant Biomechanics Group, Botanischer Garten der Albert-Ludwigs-Universitat Freiburg, Schanzlestr. 1, D-79104 Freiburg, Germany

Received 4 July 2002; accepted 29 August 2002

Abstract

Ultrastructure and motility pattern of spermatozoa of the rove beetle Aleochara curtula were examined using electron and light

microscopic methods. The spermatozoon is about 100 mm long and filiform. The head piece comprises a 5 mm long triple layered acrosome

and 10 mm long nucleus. The flagellum consists of a 9þ9þ2 axoneme, two accessory bodies and two mitochondrial derivatives about equal

in size but of different shape in their cross sections. In both derivatives there are paracrystalline inclusions. The flagellum is attached to the

head by a 2 mm long centriole adjunct which is characterized by its electron dense material that forms a three layered folded lamellar

structure.

When liberated in buffer solution the sperm flagella assume a coiled hook-like form with the excentric stiff head protruding in front. The

spermatozoa are driven through the medium by a small helicoidal wave of high frequency superimposed to the bent flagella. The maximum

speed measured was 15.2 mm/s.

The sperm architecture of A. curtula is similar to that of other Aleochara species but differs in total length and dimensions of the

mitochondrial derivatives. For that reason Aleochara sperm can certainly prove useful to study the effect of the mitochondrial derivatives on

sperm motility. q 2002 Published by Elsevier Science Ltd.

Keywords: Insect spermatozoa; Sperm motility; Mitochondrial derivatives; Centriole adjunct

1. Introduction

Sperm ultrastructure in the vast insect order Coleoptera

has been investigated in many different families and was

found to match basically the classical pterygote type

(Jamieson, 1987; Jamieson et al., 1999), which is charac-

terized by the presence of a 9þ9þ2 axoneme, two

accessory bodies and two mitochondrial derivatives.

Variation in sperm architecture has been studied mostly

to resolve phylogenetic issues in beetles (Baccetti and

Daccordi, 1988; Burrini et al., 1988) as well as in other

insect species (Baccetti, 1972; Dallai, 1979; Carcupino et al.,

1995; Dallai and Afzelius, 1995; Jamieson, 1987). Obser-

vations and interpretations of sperm motility, however, are

scarce, in animals in general (Brokaw et al., 1970), and in

insects as well (Baccetti et al., 1973a,b; Phillips, 1974).

The 9þ9þ2 insect spermatozoa in motion generally

display a cylindrical-helicoidal type of wave (Baccetti,

1972). A much more complicated double wave motion

pattern has been described for the coleopteran Tenebrio

molitor (Baccetti et al., 1973a; Phillips, 1974), the

hemipteran Lygaeus (Phillips, 1974) and the phasmid

Bacillus rossius (Baccetti et al., 1973b). In this motion

pattern, according to Baccetti et al., (1973a), so called ‘large

waves’ which arise from behind the sperm head spreading

towards the posterior tail tip are superimposed by small

amplitude waves (‘small waves’) of higher frequency

spreading in the same direction. According to Baccetti and

Afzelius (1976), such small waves superimposed on large

waves are supposed to be restricted to occur in spermatozoa,

which possess two accessory bodies endowed with ATPase

activity. Besides the accessory bodies, however, the

mitochondrial derivatives have been considered to be

1467-8039/02/$ - see front matter q 2002 Published by Elsevier Science Ltd.

PII: S1 46 7 -8 03 9 (0 2) 00 0 46 -4

Arthropod Structure & Development 31 (2002) 243–254

www.elsevier.com/locate/asd

* Corresponding author. Tel.: þ 49(0) 761-203-2546; fax: þ 49(0) 761-

203-2546.

E-mail address: klaus.peschke@biologie.uni-freiburg.de (K. Peschke).

involved in insect sperm motility. They may impart stiffness

to the flagellar beat, affecting the form of the flagellar wave

(Phillips, 1974) and establish a particular motility pattern

specific to each insect species (Tokuyasu, 1974). Attempt-

ing to analyse the role of the mitochondrial derivatives in

sperm motility the rove beetle genus Aleochara

probably can prove useful, since variations in sperm

structure can be found mainly in total sperm length and

dimensions of the mitochondrial derivatives. Video

analyses of sperm movement in buffer solution of the

rove beetles Aleochara bilineata and A. tristis showed

that they move through the medium in a corkscrew-like

motion (Werner et al., 2000, 2001). A helical wave of high

frequency and low amplitude runs along the helically coiled

tail and propels the sperm through the medium, which is, on

first sight, similar to the double wave system described by

Baccetti et al. (1973a,b) for Tenebrio and Bacillus.

In this study, a detailed description of ultrastructure as well

as a hypothesis for the observed motility pattern is given for

spermatozoa of Aleochara curtula to provide additional

information for further comparative investigations of sperm

structure and function amongst different Aleochara species.

2. Materials and methods

2.1. Beetle culture and dissection

All beetles used in this survey were taken from our own

laboratory stock cultures, reared according to Fuldner

(1968) on blow fly pupae (Calliphora vicina ) as hosts for

the parasitoid beetle larvae. Females were kept individually

and males in groups of ten. To obtain spermatozoa from the

spermatheca, females were allowed to mate at least one day

before dissection. Spermathecae were dissected in the same

buffer solution that was used for further processing or sperm

observation (see later).

2.2. Sperm length measurements

Spermatozoa were liberated in a 0.1 M Phosphate

buffer (PB) containing 6 mM of the fluorochrome

diamidinophenylindole (DAPI), which stains DNA.

Combined fluorescence and phase contrast images

were taken with a Zeiss Axiocam mounted onto a Zeiss

Axioskop. Images were imported into image analysis

software (Optimas 6.51, Media Cybernetics). Acrosomes,

nuclei and sperm tails were measured by tracing these

elements with the cursor.

2.3. Transmission electron microscopy

Positive staining. Spermathecae from inseminated

females were cut into two or three parts and fixed

immediately in a 2% glutaraldehyde solution in 0.1 M PB

(pH 7.4) containing 1% tannic acid and 1.8% sucrose.

After rinsing in water several times the spermathecae were

block-stained in 1% uranyl acetate in distilled water

according to Afzelius (1988) and rinsed in water again.

Samples were dehydrated in a graded series of acetone and

embedded in Epon. Ultrathin sections were cut on a Reichert

OMU-3 ultramicrotome and stained with uranyl acetate and

lead citrate using a Leica Ultrostain. The sections were

examined in a Zeiss EM-9S2 and EM10 transmission

electron microscope operating at 60 and 80 kV, respectively.

Negative staining. Spermatozoa from the spermatheca

were liberated into a drop of distilled water by breaking or

puncturing the chitinized wall of the spermatheca. The

sperm suspension was then transferred onto slot grids

covered with formvar and negatively stained with 2%

phosphotungstate acid and 0.4% sucrose (pH 7.4).

2.4. Observations of sperm motility

For all live recordings of sperm we used a diphosphate

buffered saline (DPBS, pH 7.4) containing 52.02 mM NaCl,

39.70 mM KCl, 0.54 mM CaCl2, 1.18 mM MgCl2 £ 6H2O,

1.22 mM MgSO4 £ 7H2O, 1 mM glucose and 58.43 mM

succrose in 0.05 M PB. After dissection spermathecae were

placed in a drop of buffer solution on a slide and sperm were

liberated. Samples were examined in dark field mode using

a Zeiss Axioskop at 30 8C. The microscope stage was

equipped with a MINITUP HT 200 heating system for exact

temperature control. Sperm movements were recorded on an

S-VHS videotape with a co-recorded stop watch signal.

Single frames or sequences from this tape were digitalized

for further computer image analysis. Swimming velocity

was estimated using the ‘motion analysis’ macro of the

image analysis program. Using this tool, we followed the

trajectory of a spermatozoan head obtained from a

digitalized sequence with a frame interval of 0.5 s. The

relative coordinates of the sperm heads were recorded and

the straight line velocities were calculated. High-speed

recordings of sperm movement were made with a Red

Lake Motion Scope digital camera working at 250 or

500 frames/s. Digitalized sequences were read out from the

camera’s internal buffer and recorded directly on S-VHS

videotape at rates of 5 or 10 frames/s again with a co-

recorded stop watch signal. Flagella undulation frequencies

were measured from these recordings and calculated back to

their original recording speed.

3. Results

3.1. Sperm architecture

Morphology. Light microscopical examination of A.

curtula spermatozoa revealed them to be long and slender

cells with an overall length of 100.2 ^ 12.8 mm (�x ^ s:d:;

n ¼ 67). The headpiece, consisting of acrosome and

nucleus, is an elongated and stiff structure that is tapered

M. Werner et al. / Arthropod Structure & Development 31 (2002) 243–254244

off towards the tip. The lengths of acrosome and nucleus

were determined to be 4.9 ^ 0.2 mm (n ¼ 19) and

10.5 ^ 0.4 mm (n ¼ 158) respectively, such that the head-

piece comprises about 15% the of total sperm length.

3.2. Ultrastructure

A schematic diagram of the architecture of an A. curtula

spermatozoon as revealed by electron microscopy is given

in Fig. 1. The acrosome, which is situated apically to the

nucleus, is triple-layered: an axial rod is surrounded by an

acrosomal vesicle, which is itself covered by a layer of

extra-acrosomal material (Figs. 2 and 3). Cross sections

through the acrosome revealed a fibrous core within the

axial rod (Fig. 3). The basal part of the axial rod fits into an

invagination of the nucleus about 0.2 mm deep (Figs. 4

and 5). Though almost entirely filled with homogenous,

compact chromatin, the nucleus also presents small spots of

less electron dense areas containing electron dense granules

scattered within the chromatin mass (Figs. 6 and 7). The

base of the nucleus is bevelled at one side like the

mouthpiece of a whistle leaving space for the elements of

the centriole adjunct region (Figs. 8 and 11). The latter is

clearly recognizable by the presence of the electron dense

centriole adjunct material, which is folded forming a three

layered structure enclosing the apical axonemal elements

(Fig. 10). The tips of the axonemal microtubules are housed

at different depth whithin the centriole adjunct material and

surround a basical cylinder of about 60 nm in diameter. This

cylinder reaches up to the bevelled side of the nucleus and

protrudes slightly into a small nuclear invagination (Fig. 9).

Examination of consecutive cross-sections from anterior to

posterior reveal the axonemal accessory tubules to appear

first, accompanied by the anterior tip of the major

mitochondrial derivative (Figs. 12 and 13), and followed

by the doublets (Fig. 14). In more posterior sections the

radial spokes of the axoneme are found being in contact

with the basical cylinder (Fig. 15), which is ‘replaced’ by

the central tubules at about the level of the second

mitochondrial derivative’s tip (Fig. 16).

The whole axoneme exhibits the 9þ9þ2 arrangement of

microtubules (Fig. 17) typical of pterygote insect sperma-

tozoa. Each one of the outer accessory microtubules is

both interconnected to its adjacent microtubule and to

the neighbouring B-subtubule of the doublets by

electron dense intertubular material. In cross-sections,

intertubular material of the axoneme’s outside perimeter

can be seen in touch with the plasma membrane, which

in this area contours the axomene (Fig. 17). Half of the

cross sectional area of each B-subtubule is filled with

electron dense material whose staining is identical to

the content of the completely filled central singlet. The

second singlet microtubule is empty.

In the flagellum region of the spermatozoon, the

axoneme is accompanied by the two mitochondrial

derivatives nearly up to the posterior tail tip (Fig. 18).

Also flanking the axoneme there are two small accessory

bodies. Their bean-shaped cross sections measure

approximately 0.04 mm in length and 0.02 mm in width.

An accessory sheath is spread between both of them, com-

partmentalizing axoneme and mitochondrial derivatives

(Fig. 17)

As shown in the centriole adjunct region, the anterior tips

of the mitochondrial derivatives are located at different

levels. The major mitochondrial derivative, which reaches

closer to the nuclear base, is designated here as md1, the

smaller one as md2. Though both derivatives are approxi-

mately equal in cross-sectional size, they can easily be

distinguished by their cross-sectional shape and internal

arrangement. The md1 is more or less oval to reniform while

the md2 is pear-shaped. The largest diameter of either

derivative is about 0.3 mm. Within the derivatives, four

different regions can be distinguished (Fig. 17). (1) Most

remarkable is a circular electron lucent region occupying

most of the thicker part of the organelle. (2) A semi-circular

region at the abaxonemal side is less electron dense and

contains the mitochondrial cristae. (3) The portion of the

mitochondrial derivatives adjacent to the axoneme is taken

up by an unstructured electron dense region in which (4)

paracrystalline material is embedded. The latter fills almost

the whole pointed part of the md2 cross section, whereas in

the md1, there is always a considerable space between

paracrystalline material and the plasma membrane. Reg-

ularly spaced mitochondrial cristae can be seen clearly in

negative stained preparations (Fig. 20). The distance

between the cristae measures about 30 nm.

Towards the caudal end of the flagellum, the arrangement

of axonemal microtubules becomes less orderly and in serial

sections, they progressively disappear (Figs. 18 and 19).

Their tips are enclosed by a collar of electron dense material

that clings to the plasma membrane (Fig. 19). When sperm

are transferred into distilled water, the plasma membrane

bursts and the sperm assume a bow like structure held by

curled fibrils (Fig. 21). In negative contrast preparations (Fig.

22) the bow like structure is recognized as the axoneme and

the curled fibrils as the mitochondrial derivatives. Axoneme

and mitochondrial derivatives stick together as both in the

region of the centriole adjunct and the electron dense collar at

the posterior end of the flagellum.

3.3. Sperm motility

The tail of a motile A. curtula spermatozoon liberated in

buffer solution assumes a three-dimensional hook-like

structure that can be interpreted as an imperfect convolution

of a coil (Fig. 23). The diameter of this convolution is about

30 mm. The stiff sperm head is somewhat eccentric in regard

to the center of the helix. Along the tail there runs a small

helicoidal wave (Fig. 24) with an amplitude varying

between 0.8 and 2.1 mm (median ¼ 1.3 mm; n ¼ 114), and

a wavelength between 7.0 and 14.5 mm (median ¼ 9.9 mm;

n ¼ 114). The frequency ranged from 7.1 to 39.2 Hz

M. Werner et al. / Arthropod Structure & Development 31 (2002) 243–254 245

(median ¼ 19.2 Hz; n ¼ 84). This wave, originating behind

the sperm head and spreading towards the tail tip,

propagates the sperm through the medium (Fig. 25). The

trajectory is rectilinear and velocities measured varied

between 3.7 and 15.2 mm/s (median ¼ 8.4 mm/s; n ¼ 63).

During propagation the whole sperm rotates in a counter

clockwise direction in the course of which the eccentric

sperm head describes a helicoidal path (Fig. 23).

Fig. 1. Schematic diagram of an A. curtula spermatozoon. ab, accessory body; ar, axial rod; av, acrosomal vesicle; ax, axoneme; bc, basical cylinder; ca,

centriole adjunct; edc, electron dense collar; el, extra-acrosomal layer; md1, major mitochondrial derivative; md2, minor mitochondrial derivative; n, nucleus.

M. Werner et al. / Arthropod Structure & Development 31 (2002) 243–254246

4. Discussion

4.1. Sperm architecture

The spermatozoon of A. curtula exhibits all features

typical of pterygote insect sperm: it is filiform, has a

triple layered acrosomal complex, an elongated cylind-

rical nucleus, two mitochondrial derivatives, two

accessory bodies and a 9þ9þ2 axoneme (Jamieson,

1987). In the genus Aleochara, spermatozoan ultra-

structure has been investigated in A. tristis and A.

clavicornis (Werner et al., 2001) and, more intensively,

in A. bilineata (Werner et al., 1999). All three species

share the same sperm architecture, which is character-

ized by a prominent centriole adjunct region, two small

accessory bodies and two mitochondrial derivatives of

unequal size. At a length of 100 mm, the sperm of A.

curtula are shorter then those of the three other species.

Sperm length in A. bilineata was found to be 1000 mm

(Werner et al., 1999) and in A. tristis and A.

clavicornis, it was 400 and 500 mm, respectively

(Werner et al., 2001).

The triple layered structure of the acrosome is found in

almost all pterygote insects (Baccetti, 1972) and has been

described in Coleoptera for T. molitor (Baccetti et al.,

1973a), some Curculionoidea (Burrini et al., 1988), and

Chrysomelidae (Baccetti and Daccordi, 1988), the clerid

Divales bipustulatus (Mazzini, 1976), and the staphylinid A.

bilineata (Werner et al., 1999).

The nucleus of an A. curtula sperm is not completely

homogeneous, as has been described for the majority of

insect sperm (Baccetti, 1972), but it presents small electron

lucent areas filled with electron dense granules. Similar

structures were also found in the sperm nucleus of the

hymenopteran Bephratelloides pomorum (Lino-Neto et al.,

1999). The granules within this areas could possibly be

interpreted as polyribosomes as described by Baccetti et al.

(1973b) for the phasmid B. rossius, where polyribosomes

were found in an extranuclear space in the center of the

nucleus.

A centriole adjunct at the nucleus flagellum transition

region has been described for many insects (Cantacu-

zene, 1970). This structure has also been reported for

other coleopterans from the family Bathysciinae

Figs. 2–9. Sperm Head. Fig. 2: Negative contrast image of the anterior tip of the acrosome. The three layered structure is clearly visible in this preparation. Fig.

3: Cross section of the acrosome. The axial rod contains a fibrous inner core. Fig. 4: Longitudinal section of the acrosome-nucleus transition. The axial rod fits

into a nuclear invagination. Fig. 5: Cross section of the acrosome-nucleus transition. Fig. 6: Longitudinal section of the nucleus. Scattered within the condensed

chromatin mass there are less electron dense areas containing electron dense granules (arrows). Fig. 7: Cross section of the nucleus. Fig. 8: Negative contrast

preparation of the nucleus-flagellum transition region. The posterior end of the nucleus is bevelled like the mouthpiece of a whistle. Fig. 9: Cross section of the

nuclear base. The anterior tip of the basical cylinder extends into a nuclear invagination. bc, basical cylinder; ar, axial rod; av, acrosomal vesicle; el, extra-

acrosomal layer; md1, major mitochondrial derivative; n, nucleus.

M. Werner et al. / Arthropod Structure & Development 31 (2002) 243–254 247

(Juberthie-Jupeau et al., 1983), Bruchidae (Acantho-

scelides obtectus, Huignard, 1974), Dytiscidae (Dytiscus

marginalis and Colymbetes fuscus, Mackie and Walker,

1974; Acilius sulcatus, Werner, 1976), Lucanidae

(Aegus lavicollis, Kubo-Irie et al., 2000) and Staphyli-

nidae (A. bilineata, Werner et al., 1999). The structure

of the centriole adjunct in A. curtula is most peculiar. It

enfolds a complicated three-dimensional structure, in

which the anterior parts of the axoneme and the

mitochondrial derivatives are housed, quite similar to

the multilayered centriolar adjunct found in Locusta

migratoria (Cantacuzene, 1970) and in the grasshopper

Melanoplus differentialis (Lindsey and Biesele, 1974).

In contrast to A. curtula, the centriole adjunct of A.

bilineata is made of a compact mass of electron dense

material (Werner et al., 1999). In both species the

centriole adjunct extends along the initial segment of

the axoneme, whereas in the stag beetle Aegus

lavicollis, it covers the outside of the two mitochondrial

derivatives and extends almost half way along the

length of the whole flagellum (Kubo-Irie et al., 2000).

The authors suggested that the centriole adjunct is

Figs. 10–16. Centriole adjunct region. Fig. 10: Longitudinal section of the centriole adjunct region. The three layered electron dense material of the centriole

adjunct surrounds the basical cylinder. Figs. 11–16: Consecutive cross sections of the centriolar adjunct (all to the same scale). Fig. 11: The centriolar adjunct

extends into the space given free by the bevelled posterior top of the nucleus. Figs. 12 and 13: The folded three layered structure of the centriole adjunct

material encloses the anterior tips of some accessory tubules as well as the major mitochondrial derivative. Figs. 14 and 15: Further posterior, the axonemal

doublets (arrows) appear and came into contact with the basical cylinder via the radial spokes (arrowhead). Fig. 16: The centriole adjunct material is displaced

by the fully expressed axoneme and the second mitochondrial derivative. ab, accessory body; at, accessory tubule; ax, axoneme; bc, basical cylinder; ca,

centriole adjunct; md1, major mitochondrial derivative; md2, minor mitochondrial derivative; n, nucleus.

M. Werner et al. / Arthropod Structure & Development 31 (2002) 243–254248

involved in the movement of the flagellum in this

species. Baccetti (1972) also proposed a mechanical role

of this structure in the initiation of flagellar movements.

From the position of the centriole adjunct, Breland et al.

(1966), Phillips (1970) and Lindsey and Biesele (1974)

concluded that it serves to secure the flagellum to the

sperm head. Yasuzumi et al. (1970) supposed a nutritive

function for the developing axoneme during

spermiogenesis.

In pterygote insect flagella, the 9þ9þ2 axoneme is

usually flanked by two accessory bodies (Baccetti, 1972),

rarely by one (Werner, 1965) or three (Bawa and Kanwar,

1975). In the Coleoptera the accessory bodies are often of

considerable size relative to the axoneme. The largest ones

were found in the clerid D. bipustulatus (Mazzini, 1976). In

the Phasmatodea, the accessory bodies replace the mito-

chondrial derivatives during spermiogenesis, and they are

the most prominent organelles in the mature sperm

flagellum (Baccetti et al., 1973b). Some accessory bodies

showed ATPase and UTPase activity; they were considered

to maintain the large wave of the motile spermatozoa

(Baccetti et al., 1973a,b). However, in A. curtula, the

accessory bodies are very small and a mechanical role of

these organelles in sperm motility seems unlikely. The

same is true for A. bilineata, A. tristis and A. clavicornis

(Werner et al., 1999, 2001).

Although A. curtula sperm have two mitochondrial

derivatives of nearly equal cross sectional size, the major

Figs. 17–20. Flagellum region. Figs. 17–19. Cross sections of the flagellum (all to the same scale). Fig. 17: The mitochondrial derivatives are separated from

the axoneme by an accessory sheath that is spread between the accessory bodies. The mitochondrial derivatives show a central less electron dense region , the

region of the cristae , an unstructured electron dense matrix and the paracrystalline inclusion . Fig. 18: At the end of the tail the axonemal arrangement

of microtubules becomes disordered. Electron dense material can be seen between the accessory tubules and the plasma membrane (arrows). Fig. 19:

The plasma membrane at the very end of the flagellum is lined up with electron dense material which forms a collar-like structure around the remaining

microtubules. Fig. 20: Negative contrast image of the major mitochondrial derivative. The cristae are regularly spaced. Paracrystalline material is made of

longitudinal fibrils. ab, accessory body; as, accessory sheath; at, accessory tubule; ax, axoneme; cr, cristae; edc, electron dense collar; md1, major

mitochondrial derivative; md2, minor mitochondrial derivative; pc, paracrystalline material.

M. Werner et al. / Arthropod Structure & Development 31 (2002) 243–254 249

derivative (md1) can easily be distinguished by its cross

sectional shape and its position relative to centriole adjunct.

Flagellar structure varies somewhat between species in the

genus Aleochara, mainly in the dimensions of the major

mitochondrial derivative. In A. bilineata and A. tristis the

major mitochondrial derivative is considerable bigger than

the minor derivative and in A. clavicornis, it is excessively

enlarged (Werner et al., 2000, 2001).

In many insect species the mitochondrial derivatives

contain a paracrystalline center (Rosati et al., 1976)

consisting of the protein crystallomitin (Baccetti et al.,

1977). Cristae are found in a cortical zone only. Although

they show cytochrome-c oxidase activity (Bigliardi et al.,

1970; Baccetti et al., 1973a; Perotti, 1973; Bao et al., 1992;

Werner et al., 1999), sperm are cabable of obtaining their

energy by glycolysis alone (Baccetti et al., 1973b; Bloch

Qazi et al., 1998; Werner et al., 1999). The very complex

aggregation of spermatid mitochondria forming the neben-

kern and their transformation into two highly organized

mitochondrial derivatives (Andre, 1962; Pratt, 1968; Favard

and Andre, 1970) gave rise to various speculations about

their function within the mature spermatozoon. Besides

from their role in energy metabolism, mitochondrial

derivatives have been ascribed a mechanical function in

the modulation of axonemal movements (Phillips, 1974;

Tokuyasu, 1974). Specifically the crystalline content of the

derivatives was suggested to exhibit elastic properties

similar to that of the parergins in mammalian sperm

(Baccetti et al., 1977). Such mechanical effects are obvious

when the derivatives are of enormous size that they modify

the axonemal movements in such a way that, in artificial

media, even complete immobility results (D. bipustulatus,

Mazzini, 1976; A. clavicornis, Werner et al., 2001), though

locomotion in narrow ducts in the female genital tract may

still be enabled by the observed feeble axonemal motility.

4.2. Sperm motility pattern

Based on investigations on T. molitor (Baccetti et al.,

1973a) and B. rossius (Baccetti et al., 1973b), a complex

spermatozoa movement pattern which is characterized by

the simultaneous presence of two kinds of waves has been

described. A small amplitude wave of high frequency is

superimposed on a wave with higher amplitude and lower

frequency. These waves were termed as ‘small wave’ and

‘large wave’ by Baccetti et al. (1973a) and ‘minor wave’

and ‘major wave’ by Phillips (1974). According to Baccetti

et al. (1973a,b), the double wave system is a product of

two different flagellar components: the small wave is

propagated and maintained by the axoneme motor, and the

large wave by the ATPase and UTPase-rich accessory

bodies. However, Phillips (1974) described the same

motility pattern for the milkweed bug, Lygaeus, which

does not have any accessory bodies (Jamieson, 1987).

When liberated in buffer solution, the A. curtula

sperm tail assumes a helically coiled form which is

superimposed by a helical wave of high frequency and

low amplitude. The action of the latter propels the

sperm through the medium generating a corkscrew

motion of the sperm. At first sight, this motility pattern

resembles the two-wave system of movement described

for Tenebrio (Baccetti et al., 1973a). However, Baccetti

Figs. 21 and 22. Spermatozoa in distilled water. Fig. 21: Phase contrast image of a dissociated spermatozoon in distilled water. The bow-like structure

represents the axoneme the string-like structure both mitochondrial derivatives. Fig. 22: The same preparation in negative contrast. The curled mitochondrial

derivatives can be clearly distinguished from the axoneme. ax, axoneme; md, mitochondrial derivatives; md1, major mitochondrial derivative; md2, minor

mitochondrial derivative; sh, sperm head.

Figs. 23–25. A. curtula spermatozoon in movement. Fig. 23: Computer reconstruction of the three-dimensional bent sperm. The arrow indicates the rotation

during forward progression. Fig. 24: Single frame from a high speed recording showing the small amplitude, high frequency wave (arrow) superimposed to the

bent flagellum. Fig. 25: Sequence of a real time video recording showing a spermatozoon swimming in buffer solution. The frames are at 0.2 s intervals. fl,

flagellum; sh, sperm head. See also the electronic annex under www.elsevier.com for videoclips.

M. Werner et al. / Arthropod Structure & Development 31 (2002) 243–254250

M. Werner et al. / Arthropod Structure & Development 31 (2002) 243–254 251

et al. (1973a) assume that the large helical coil is an

actively propelled wave evoked by the accessory bodies,

and it does indeed look like such a wave in the light

microscopic image, even in A. curtula. Nonetheless, we

propose that the actual sperm movement is caused by

the high frequency wave alone, whereas the coiling of

the flagellum is not a wave, but a static structure that

results from an interplay of static forces of axoneme,

mitochondrial derivatives and plasma membrane. When

sperm of A. curtula are kept in distilled water, they

burst and the flagellar filaments assume a bow and

string-like form. The same structure was found in

spermatozoa of the chrysomelid Lilioceris lilii (Furieri,

1977). Furieri interpreted the bow as the mitochondrial

derivatives and the string as the axoneme. However, our

negative contrast preparations of A. curtula sperm show

that the reverse is actually the case. The curvature of

the bow-like axoneme is maintained by the two curled

mitochondrial derivatives (the ‘strings’ in Fig. 21),

which are attached to the initial segment of the

axoneme via the centriole adjunct and to the posterior

end of the axoneme via the electron dense collar. In

intact sperm the functional unit composed of these

organelles is tightly packed inside the plasma mem-

brane. If one assumes that the mitochondrial derivatives

have elastic properties, as suggested by Baccetti et al.

(1977) for the paracrystalline inclusions, then this

configuration can lead to the observed three-dimensional

bending of the sperm tails in A. curtula spermatozoa,

which represents the ‘large wave’ in the interpretation

of Baccetti et al. (1973a). The small-amplitude high-

frequency wave which is driven by the axoneme is

identical to the ‘small wave’ of Baccetti et al. (1973a).

Spermatozoa of A. bilineata (Werner et al., 2000), A.

tristis and A. clavicornis (Werner et al., 2001) all show

a motility pattern similar to that of A. curtula, although

the exact shape of the three-dimensional flagellar

structure varies, partially due to the different flagellar

length. A. curtula has the shortest flagellum, which is

too short for a complete convolution of the helix. A.

clavicornis sperm are distinguished from those of the

other species by their complete immobility in buffer

solution. Since the sperm of all these species share the

same general architecture, but are distinguished by the

overall length and the dimensions of the major

mitochondrial derivative, the genus Aleochara may

prove highly valuable for the study of the biomechani-

cal role of these organelles in sperm motility.

In A. curtula, the passage of sperm from the male to the

female storage organ, the spermatheca, is accomplished by

means of a spermatophore that is transmitted into the female

genital chamber. Probably due to osmotic processes the

spermatophore grows a tube through the spermathecal duct

into the spermatheca, where the tube swells up to form a

balloon in which the spermatozoa are flushed in (Gack and

Peschke, 1994; Forster et al., 1998). Based on these

findings, it appears that sperm motility does not contribute

to spermathecal filling in A. curtula. However, sperm

motility may be required for the transfer of sperm from the

storage organ to the fertilization site, about which there is no

information to date. Active movement of sperm seems to

play a minor role in the transport of sperm into the

spermatheca in the Diptera and is probably assisted by

female processes (Linley and Simmons, 1981; Arthur et al.,

1998; Simmons et al., 1999). In honey bees, however, the

activity of spermatozoa themselves is critical to their

migration to the spermatheca as shown by Collins (2000)

in instrumentally inseminated queens. Therefore, the issue

of functionality of the observed sperm motility in insects

remains contentious (Leopold, 1976).

It is important to note that most studies of insect sperm

motility have been carried out in artificial environments

where the sperm may show a relaxed shape that differs

considerably from their shape inside the male or female

genital tracts. According to Linley and Simmons (1981), it

would be useful to study the effects of mechanical

restriction on sperm motility in the context of movements

along narrows ducts.

Acknowledgements

The authors are indebted to S. Collatz (1941–1997) for

her competent assistance with electron microscopy, Dr

W. Federle (Wurzburg) for his introduction and help in the

high speed recordings and C. Traiser (Tubingen) for

creating the computer reconstruction. Dr A.-K. Eggert

(Illinois State University) kindly checked on our English.

Thanks also to Prof R. Hartmann for help with the digital

images. This study was supported by a grant of the Deutsche

Forschungsgemeinschaft (PE 231/12-1).

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