The flagellar apparatus of the golden algaSynura uvella: Four absolute orientations

Summary Flagellated vegetative cells of the colonial golden algaSynura uvella Ehr, were examined using serial sections. The two flagella are nearly parallel as they emerge from a flagellar pit near the apex of the cell. The photoreceptor is restricted to swellings on the flagella in the region where they pass through the apical pore in the scale case and the swellings are not associated with the cell membrane or an eyespot. A unique ring-like structure surrounds the axonemes of both flagella at a level just above the transitional helix. The basal bodies are interconnected by three striated, fibrous bands. Four short (<100 nm) microtubules lie between the basal bodies at their proximal ends. Two rhizoplasts extend down from the basal bodies and separate into numerous fine striated bands which lie over the nucleus. Three- and four-membered microtubular roots arise from the rhizoplasts and extend apically together. As the roots reach the cell anterior, the three-membered root bends and curves clockwise to form a large loop around the flagella; the four-membered root bends anticlockwise and terminates under the distal end of the three-membered root as it completes the loop. There are four absolute orientations, termed Types 1–4, in which the flagellar apparatus can occur. With each orientation type the positions of the Golgi body, nucleus, rhizoplasts, chloroplasts and microtubular roots change with respect to the flagella, basal bodies and photoreceptor. Two new basal bodies appear in pre-division cells, and three short microtubules appear in a dense substance adjacent to each new basal body. Based upon the positions of new pre-division basal bodies, a hypothesis is proposed to explain why there are four orientations and how they are maintained through successive cell divisions.     

Flagellar root contraction and nuclear movement during flagellar regeneration in Chlamydomonas reinhardtii

When Chlamydomonas cells are deflagellated by pH shock or mechanical shear the nucleus rapidly moves toward the flagellar basal apparatus at the anterior end of the cell. During flagellar regeneration the nucleus returns to a more central position within the cell. The nucleus is connected to the flagellar apparatus by a system of fibers, the flagellar roots (rhizoplasts), which undergo a dramatic contraction that coincides with anterior nuclear movement. A corresponding extension of the root system, back to its preshock configuration is observed as the nucleus retracts to a central position. Anterior displacement of the nucleus and flagellar root contraction require free calcium in the medium. Nuclear movement and flagellar root contraction and extension are not sensitive to inhibitors of protein synthesis (cycloheximide), or drugs that influence either microtubules (colchicine) or actin-based microfilaments (cytochalasin D). Detergent-extracted cell models contract and extend their flagellar roots and move their nuclei in response to alterations of free calcium levels in the medium. Cycles of nuclear movement in detergent-extracted models require ATP to potentiate the contractile mechanism for subsequent calcium-induced contraction. Flagellar root contraction and nuclear movement in Chlamydomonas may be causally related to signaling of induction of flagellar precursor genes or to the transport of flagellar precursors or their messages to sites of synthesis or assembly near the basal apparatus of the cell.     

Ultrastructure and membrane topography of special ciliary organelles in the ciliate Eufolliculina uhligi (Protozoa)

Summary In Eufolliculina uhligi and other folliculinid ciliates, a territory has been identified that differs ultrastructurally from other areas of the cell, and that is especially sensitive to mechanical stimuli. This territory is located around the anterior oral apparatus of the loricate trophont and posterior to the membranellar spiral of the swarmer. Each cilium in this territory is closely apposed to a small membrane-covered pin that is supported by transverse microtubules of the cilium. In front of the pin, the base of the cilium bulges out; the ciliary membrane is interconnected with the axoneme by filamentous material. Freeze-fractured cilia show a large rectangular particle array at the site of the basal swelling. Only scattered particles have been observed in the pin membrane. It is suggested that the cilium and the pin act as a unit, which has therefore been named the ciliumpin-complex. Comparison with ciliary organelles of unicellular and multicellular organisms indicates that, because of their polar organization, the complexes are involved in the transduction of oriented, presumably mechanical, stimuli.     

MITOSIS AND CYTOKINESIS IN PLATYMONAS SUBCORDIFORMIS,A SCALY GREEN MONAD1

Scaly green monads are often placed in a separate class, Prasinophyceae, and have been considered to be among the most, primitive of green algae. Platymonas possesses rhizoplasts which resemble sarcomeric structures. At prophase, extranuclear spindle micro-tubules emanate from a granular region which appears to arise through dissolution or dispersion of the rhizoplasts. It is probable that the rhizoplasts are largely consumed during the formation of the spindle and only small fragments are left at metaphase. The rhizoplasts can be seen again at telophase but are short at this stage. The basal bodies are not at the spindle poles but remain at their interphase position. The interzonal spindle collapses early at telophase, and shortly thereafter cleavage microtubules appear. These microtubules extend from the region of the basal bodies to the posterior of the cell. The events of cell division are compared with these events in other green algae and in Ochromonas. The functional and phylogenetic significance of the observations is discussed.     

The cytology and ultrastructure of zoospores of Hydrurus foetidus (Chrysophyceae)

The unusual tetrahedral shape of Hydrurus foetidus (Vill.) Trev. zoospores is associated with a complex skeletal system of microtubules extending from a broad flagellar root (up to 19 microtubules) into each of three, pointed anterior processes. The posterior end, also pointed and supported by a separate set of microtubules, contains a single large chloroplast with a prominent posterior furrow containing mitochondrial elements. A large immersed pyrenoid is penetrated by paired thylakoids. There is no eyespot. Numerous large Golgi bodies occur immediately anterior to the nucleus and up to 5–6 contractile vacuoles lie near the cell surface at the anterior end. Two terminally inserted flagella extend from the cell surface, a long one serving for cell locomotion, and the other vestigial with an axonemal pattern of 9+0. The flagellar root system consists of: (1) a thin, striated rhizoplast extending from the basal body of the long flagellum and ramifying over the surface of a conspicuous, anteriorly directed, conical projection of the nucleus; (2) a broad microtubular root which emanates from near the basal body of the long flagellum and appears to function as a MTOC; (3) a compound root, consisting of a striated fiber and two associated microtubules, which runs alongside the basal body of the stubby flagellum before terminating at the cell surface; and (4) a short two-membered microtubular root, also associated with the basal body of the stubby flagellum. Other components of the flagellar apparatus include a large dense body near the proximal end of the basal body of the short flagellum, and a small, dense, core-like structure closely associated with one of its triplet fibers. The flagellar apparatus of H. foetidus is remarkably similar in ultrastructure to that of Chrysonebula holmesii Lund.     

THE FINE STRUCTURE OF MITOSIS AND CELL DIVISION IN THE CHRYSOPHYCEAN ALGA OCHROMONAS DANICA1

At the ultrastructural level, cell division in Ochromonas danica exhibits several unusual features. During interphase, the basal bodies of the 2 flagella replicate and the chloroplast divides by constriction between its 2 lobes. The rhizoplast, which is a fibrous striated root attached to the basal body of the long flagellum, extends under the Golgi body to the surface of the nucleus in interphase cells. During proprophase, the Golgi body replicates, apparently by division, and a daughter rhizoplast, appears. During prophase, the 2 pairs of flagellar basal bodies, each with their accompanying rhizoplast and Golgi body, begin to separate. Three or 4 flagella are already present at this stage. At the same time, there is a proliferation of microtubules outside the nuclear envelope. Gaps then appear in the nuclear envelope, admitting the microtubules into the nucleus, where they form a spindle. A unique feature of mitosis in O. danica is that the 2 rhizoplasts form the poles of the spindle, spindle microtubules inserting directly onto the rhizoplasts. Some of the spindle microtubules extend from pole to pole; others appear to attach to the chromosomes. Kinetochores, however, are not present. The nuclear envelope breaks down, except, in the regions adjacent, to the chloroplasts; chloroplast ER remains intact throughout mitosis. At late anaphase the chromosomes come to lie against part of the chloroplast ER. This segment of the chloroplast ER appears to be incorporated as part of the reforming nuclear envelope, thus reestablishing the characteristic nuclear envelope—chloroplast ER association of the interphase cell.     

Cell morphology and formal description of Ergobibamus cyprinoides n. g., n. sp., another Carpediemonas-like relative of diplomonads

About 20 new isolates of Carpediemonas-like organisms (CLOs) have been reported since 2006. Small subunit rRNA gene phylogenies divide CLOs into six major clades: four contain described exemplars (i.e. Carpediemonas, Dysnectes, Hicanonectes, and Kipferlia), but two include only undescribed organisms. Here we describe a representative of one of these latter clades as Ergobibamus cyprinoides n. g., n. sp., and catalogue its ultrastructure. Ergobibamus cyprinoides is a bean-shaped biflagellated cell, 7-11.5 μm long, with a conspicuous groove. Instead of classical mitochondria there are cristae-lacking rounded organelles 300-400 nm in diameter. The posterior flagellum has a broad ventral vane and small dorsal vane. There are normally four basal bodies, two non-flagellated. There is one anterior root (AR), containing six microtubules. The posterior flagellar apparatus follows the "typical excavate" pattern of a splitting right root supported by fibres "I,"B," and "A," a "composite" fibre, a singlet root, and a left root (LR) with a "C" fibre. The B fibre originates against the LR--a synapomorphy of the taxon Fornicata--supporting the assignation of Ergobibamus to Fornicata, along with diplomonads, retortamonads, and other CLOs. Distinctive features of E. cyprinoides include the complexity of the AR, which is intermediate between Hicanonectes, and Carpediemonas and Dysnectes, and a dorsal extension of the C fibre.     

FLAGELLAR ROOTS AND THE RESERVOIR CYTOSKELETON OF COLACIUM LIBELLAE (EUGLENOPHYCEAE)1

The three flagellar roots of Colacium Ehrenberg give rise to the three microtublar bands of the reservoir cytoskeleton. The dorsal root (DR) originates at the basal body (bb1) of the emergent flagellum. It is initiated on the left side of the cell, runs toward the right side under the posterior end of the reservoir and thence anteriorly in a spiral path over the dorsal surface of the reservoir until it terminates on the left side of the eyespot. Along its length, it appears to initiate a dorsal band (DB) which forms the major dorsal portion of the reservoir cytoskeleton—the dorsal microtubules (DMT). Two roots originate at the basal body (bb2) of the non-emergent flagellum. The ventral root (VR) runs up the left side of the cell and initiates the band of microtubules which forms part of the presumptive vestigial cytopharynx. Therefore, it forms the reinforcing microtubules (MTR) of Colacium. The intermediate root (IR) forms the para-reservoir microtubules (PMT). Flagellar root correlation with the reservoir cytoskeletal bands strengthens their homologies with the bodonid bands and further supports the hypothesis that the euglenoids are derived from the kinetoplastid flagellates.     

CELL DIVISION IN THE FILAMENTOUS PLEURASTRUM AND ITS COMPARISON WITH THE UNICELLULAR PLATYMONAS (CHLOROPHYCEAE)1

At prophase in Pleurastrum, extranuclear spindle microtubules develop from the region of centrioles, which lie lateral to the nucleus midway between the future sites of the metaphase spindle poles. The microtubules then move laterally to overarch the nucleus and finally become incorporated into the spindle. The centrioles do not migrate and therefore lie in the same plane as the chromosomes at metaphase. At telophase, 2, more different systems of microtubules develop from the vicinity of the centrioles—a phycoplast and extensive arrays of microtubules that ensheath the daughter nuclei. Cell division in the filamentous Pleurastrum is compared to that in the green flagellate, Platymonas. The similarities between cell division in the 2 algae are interpreted as evidence: (i) that rhizoplasts (which in Platymonas resemble myofibrils) are somehow homologous to microtubules; and, (ii) that cell division in Pleurastrum differs from cell division in other examined filamentous chlorophycean genera because Pleurastrum has an independent evolutionary origin from a monad with Platymonas-like characteristics.     

A light and electron microscopical study of the green flagellateSpermatozopsis similis spec. nova

The green flagellateSpermatozopsis similis spec. nova has been studied in culture by light and electron microscopy. The flagellate bears two flagella, is naked and has a characteristic crescent and spirally twisted cell shape. The two flagella are of subequal length, each with a prominent hair-point. Each cell contains two contractile vacuoles, a single chloroplast with an anterior eyespot but lacking a pyrenoid, an anteriorly located nucleus, a single dictyosome associated with the posterior end of the nucleus, a single mitochondrion posterior to the nucleus and associated with a small microbody, some conspicuous vacuoles, and a greater number of secondary cytoskeletal microtubules which probably are responsible for maintaining the peculiar shape of this species. SinceS. similis in culture is only biflagellate, it cannot be accommodated within the quadriflagellate, but otherwise very similar speciesS. exsultans. Spermatozopsis similis is compared with other green flagellates and is shown to share common ultrastructural characters withChlamydomonas-type green algae.     

Andalucia (n. gen.)--the deepest branch within jakobids (Jakobida; Excavata), based on morphological and molecular study of a new flagellate from soil

A new heterotrophic flagellate (Andalucia godoyi n. gen. n. sp.) is described from soil. Earlier preliminary 18S rRNA analyses had indicated a relationship with the phylogenetically difficult-to-place jakobid Jakoba incarcerata. Andalucia godoyi is a small (3-5 mum) biflagellated cell with a ventral feeding groove. It has tubular mitochondrial cristae. There are two major microtubular roots (R1, R2) and a singlet root associated with basal body 1 (posterior). The microtubular root R1 is associated with non-microtubular fibres "I,"B," and "A," and divides in two parts, while R2 is associated with a "C" fibre. These structures support the anterior portion of the groove. Several features of A. godoyi are characteristic of jakobids: (i) there is a single dorsal vane on flagellum 2; (ii) the C fibre has the jakobid multilaminate substructure; (iii) the dorsal fan of microtubules originates in very close association with basal body 2; and (iv) there is no "R4" microtubular root associated with basal body 2. Morphological analyses incorporating the A. godoyi data strongly support the monophyly of all jakobids. Our 18S rRNA phylogenies place A. godoyi and J. incarcerata as a strong clade, which falls separately from other jakobids. Statistical tests do not reject jakobid monophyly, but a specific relationship between Jakoba libera and J. incarcerata and/or A. godoyi is rejected. Therefore, we have established a new genus Andalucia n. gen. with the type species Andalucia godoyi n. sp., and transfer Jakoba incarcerata to Andalucia as Andalucia incarcerata n. comb.     

Triggering and regulatory mechanisms of ciliary motion in the ctenophore Bolinopsis. III. Electric excitability and inherent contractility of ciliated cells]

Rhythmical electrostimulation is able to produce in a tissue strip of Bolinopsis series of beats of combs-plates. However, the beat frequency, within one series, is only poorly controlled by electrical stimuli. Isolated comb-plate cells with a cilium cut off maintain rhythmical contractions stimulated both mechanically and electrically. A lot of microtubules has been found in the apical region of these cells. It is supposed that it is these microtubules, probably together with the associated root filaments, that are responsible for cell motility. Mutual mechanical stimulation of ciliary cells in these motions is, presumably, a signal providing intratissue propagation of metachronal wave.     

Molecular phylogeny of Cercomonadidae and kinetid patterns of Cercomonas and Eocercomonas gen. nov. (Cercomonadida, Cercozoa)

Cercomonads are among the most abundant and widespread zooflagellates in soil and freshwater. We cultured 22 strains and report their complete 18S rRNA sequences and light microscopic morphology. Phylogenetic analysis of 51 Cercomonas rRNA genes shows in each previously identified major clade (A, B) two very robust, highly divergent, multi-species subclades (A1, A2; B1, B2). We studied kinetid ultrastructure of five clade A representatives by serial sections. All have two closely associated left ventral posterior microtubular roots, an anterior dorsal root, a microtubule-nucleating left anterior root, and a cone of microtubules passing to the nucleus. Anterior centrioles (=basal bodies, kinetosomes) of A1 have cartwheels; the posterior centriole does not, suggesting it is older, and implying flagellar transformation similar to other bikonts. Strain C-80 (subclade A2) differs greatly, having a dorsal posterior microtubule band, but lacking the A1-specific fibrillar striated root, nuclear extension to the centrioles, centriolar diaphragm, extrusomes; both mature centrioles lack cartwheels. For clade A2 we establish Eocercomonas gen. n., with type Eocercomonas ramosa sp. n., and for clade B1 Paracercomonas gen. n. (type Paracercomonas marina sp. n.). We establish Paracercomonas ekelundi sp. n. for culture SCCAP C1 and propose a Cercomonas longicauda neotype and Cercomonas (=Neocercomonas) jutlandica comb. n. and Paracercomonas (=Cercomonas) metabolica comb. n.     

Thoracic output of crayfish giant fibres

Summary The thoracic homologue of the abdominal segmental giant neurone of crayfish Pacifastacus leniusculus is identified and described. It has a small cell body located in the anterior ventro-lateral quadrant of the ganglion and a large neuropil arborization, with dendrites aligned along the tracts of the giant fibres. The SG axon exits the ganglion within the major root which innervates the leg, usually in the anterior region of this root. Within 1–2 mm of the ganglion the axon terminates in a mass of fine branches, apparently randomly located within the base of the root.The SG receives suprathreshold input from the ipsilateral MG and LG fibres through rectifying electrical synapses. It makes output to FF motor neurones, also through electrical synapses. The SG also makes output to at least one corollary discharge interneurone. The SG receives depolarizing inhibitory synaptic potentials which can prevent its activation by the GFs. Some but not all of these synaptic potentials are common to similar potentials occurring in a large leg promotor motor neurone.Abbreviations AC anterior connective - GF giant fibre - IPSP inhibitory post-synaptic potential - LG lateral giant fibre - MG medial giant fibre - MoG motor giant neurone - PC posterior connective - PMM promotor motor neurone - r1 first root - r3 third root - rAD anterior distal root - rPD posterior distal root - rPM promotor muscle root - SG segmental giant neurone     

Fine structure of the dorsal bristle complex and pellicle of Euplotes

The fine structure of the dorsal bristle complex and pellicle of non-developing Euplotes eurystomus is described in detail by scanning and transmission electron microscopy. The bristle-pit unit is a highly differentiated complex of organelles. The bristle complex is composed of a pair of kinetosomes (basal bodies) joined by a connective. The anterior kinetosome bears the bristle cilium, which contains a polarized network of particles (“lasiosomes”). The posterior kinetosome bears a very short, knob-like “condylocilium,” and has an associated striated fiber. Accessory ribbons of microtubules are also associated with the kinetosome couplets. Parasomal sacs, a septum connecting the bristle cilium to the anterior wall of the pit, core granules of the kinetosomes, and large membranous ampules are described. The organization of the bristle complex bears many similarities to the somatic ciliature of other ciliates. The pellicle of Euplotes is composed of a continucus outer cell membrane subtended by membranous alveoli, which contain a “fibrous mat.” Two sheets of subpellicular microtubules (longitudinal and transverse) are located just beneath the alveoli. The “epiplasm” seen in some other ciliates is apparently absent in Euplotes. The texture of the cell surface is a pattern of folds or rugae composed of the outer cell membrane and the upper membrane of the alveolus. The pattern of rugae probably defines the “silverline-system” of light microscopy.     

On Core Jakobids and Excavate Taxa: The Ultrastructure of Jakoba incarcerata

The cellular organisation of the 'excavate' flagellate Jakoba incarcerata Bernard, Simpson and Patterson 2000 is described. Cells have one nucleus and dictyosome. The putative mitochondria lack cristae. Two flagella (anterior and posterior) insert anterior to the feeding groove. The posterior flagellum bears a dorsal vane. An 'anterior' microtubular root arises against the anterior basal body. Two main microtubular roots, left and right, and a singlet 'root' arise around the posterior basal body and support the groove. Non-microtubular fibres termed 'A', 'B', 'I', and 'composite' associate with the right root. A multilaminar 'C' fibre associates with the left root. The cytoskeleton of J. incarcerata indicates a common ancestry with other excavate taxa (i.e. diplomonads, retortamonads, heteroloboseids, 'core jakobids', Malawimonas, Carpediemonas, and Trimastix). Overall, J. incarcerata is most similar to (other) core jakobids, namely Jakoba libera, Reclinomonas, and Histiona. We regard J. incarcerata as a core jakobid and identify the group by the synapomorphy 'vanes restricted to dorsal side of the posterior flagellum'. The anterior root and position of the B fibre (and presence of dense inclusions in the cartwheels and a conscpicuous singlet root-associated fibre) in J. incarcerata are novel for core jakobids and argue for close relationships with Trimastix and/or Heterolobosea. The C fibre is similar in substructure to the costal fibre of parabasalids and it is possible that the structures are homologous.     

Ultrastructural Observations on the Spermatozoon,Oocyte and Fertilization Process in Gonapodasmius,a Gonochoristic Trematode (Trematoda: Digenea: Didymozoidae)

Observations were performed in the uterus of a female Gonapodasmius sp., a gonochoristic didymozoid Trematode. The oocyte is a round cell 6 μm in diameter, which shows a ‘nucleolus-like cytoplasmic body’ and cortical granules. The spermatozoon is filiform, mobile and about 50 μm long. There is no acrosome. The anterior tip of the spermatozoon contains two centrioles made up of singlets and cortical microtubules with associated glycocalyx. The centrioles are continued as two axonemes of the classical 9 + ‘1’ pattern of flatworms, accompanied by a mitochondrion and a short row of cortical longitudinal microtubules. It is the posterior part of the sperm cell which contains the nucleus. At the outset of fertilization, the anterior part of the spermatozoon coils around the oocyte and penetrates it by lateral fusion. The posterior region of the spermatozoon, with the nucleus, is the last part to enter the oocyte, after passing through a perforation in the forming eggshell. The whole spermatozoon thus penetrates the female cell.     

Zoospore ultrastructure in species ofTrebouxia andPseudotrebouxia (Chlorophyta)

Zoospore ultrastructure (incl. flagellar apparatus) has been investigated in three species ofTrebouxia (T. glomerata, T. erici, T. pyriformis) and one species ofPseudotrebouxia (P. impressa) using an absolute configuration analysis. Zoospores in all taxa studied are nearly identical in ultrastructure and exhibit a very distinctive disposition of cell organelles: cells are naked, biflagellate and considerably flattened along the plane of flagellar beat, the single contractile vacuole is located anteriorly in the ventral region of the cell, the nucleus is anteriorly to centrally located in the dorsal region of the cell. A single dictyosome is located close to the anterior, ventral edge of the nucleus. The chloroplast occupies a posterior position in the cell and usually has an anterior profile in the left region of the cell. There are two branched mitochondria per cell or a single mitochondrial reticulum with profiles anterior to the nucleus (in the dorsal region of the cell), and posterior to the nucleus. In zoospores ofTrebouxia spp. the posterior mitochondrial profile is associated with a microbody, inP. impressa zoospores the anterior mitochondrial profiles are associated with a microbody. The zoospores contain a distinctive system of three ER-cisternae: one system links to both basal bodies and extends to the nucleus, the other two systems subtend the plasmamembrane on the left and right broad cell surfaces and extend to the posterior region of the cell. The flagellar apparatus is structurally identical to that previously described for zoospores ofFriedmannia israelensis and exhibits basal body displacement by one basal body diameter into the 11/5 o'clock direction, a non-striated distal connecting fiber, a cruciate microtubular root system lacking system I fibers and presence of a single system II fiber which connects the basal bodies with the nucleus and runs parallel to one of the ER-strands. The left flagellar roots (X-roots) are subtended by a complex set of amorphous and striated material that connects each left root with both basal bodies.—This study demonstrates the close systematic relationship between the phycobiontsTrebouxia andPseudotrebouxia and the generaFriedmannia, Pleurastrum, andMicrothamnion and supports recent classification schemes which place all these taxa into a single order separate from otherChlorophyta. Dedicated to Prof. DrElisabeth Tschermak-Woess on the occasion of her 70th birthday.     

Ultrastructure of spermiogenesis and spermatozoa in Marchalina hellenica (Gennadius) (Hemiptera: Sternorrhyncha,Marchalinidae)

The spermiogenesis, the sperm structure and the sperm motility of Marchalina hellenica (Gennadius) were examined. In the early spermiogenesis a centriolar apparatus was identified, but this structure is not involved in the production of the sperm flagellum. As in other Coccoidea, the flagellar axoneme originates by the activity of the thickened tip of the numerous microtubules surrounding the nuclear anterior region close to the periphery of the cell. This region pushes against a narrow cytoplasmic layer, giving rise to a papilla. In this region a novel structure, consisting of a regular network of thin filaments, arranged orthogonally to the bundle of microtubules, is visible. The sperm flagellum consists of a series of about 260 microtubules, regularly arranged in rings around the axial nucleus. This latter extends in the middle part of the sperm length. As usual in scale insects, sperm form a bundle, which in M. hellenica is composed of 64 sperm cells, surrounded by somatic cyst cells. The sperm bundle has an helicoidal array, with a cap of dense material at its apex, lending the anterior and the posterior region of the sperm bundle with a different structural organization. This difference is responsible of the different speed gradient observed in the helical wave propagating along the sperm bundle.