Comparative Spermatology of Chelicerata: Review and Perspective Gerd ALBERTI Zoologischcs Institut I (Morphologie/Okologie), Universitat Heidelberg, Im Neuenheimer Feld 230, D-69120 Heidelberg, Germany. ABSTRACT Chelicerata have evolved separately from other arthropods since the Early Cambrian and thus provide many peculiarities aside of convergences realized within the framework of the arthropod concept. The present study describes the current knowledge on sperm cells of chelicerates. The most primitive types are found in the primarily marine taxa, in particular within the Xiphosura. Pantopoda have already quite derived sperm cells. In both groups, distinct character deviations are observable between spermatozoa of different taxa. These demonstrate the applicability of comparative spermatology for phylogenetic or systematic considerations. The most drastic modifications of the basic plan of spermatozoal structure are seen in the Arachnida. Representatives of all main taxa have now been studied, and their sperm cells are briefly discussed. In particular those of Araneae (spiders) and Acari (mites and ticks) have been extensively studied. The spider sperm cells are probably most interesting because of the various transport-forms such as encapsulated individual sperm cells (one cell per capsule: cleistospermia), capsules including several or numerous individual sperm cells (coenospermia and so called “spermatophores”), and, probably exceptional in the whole animal kingdom, capsules containing fused, syncytial sperm (synspermia). Acari all have aflagellate sperm of an extremely differing structure, which allowed the successful application of sperm ultrastructure for systematic purposes. In a subgroup of Acari it was possible to correlate alterations in the sperm structure with modifications in the genital systems and sexual behaviour. Thus, an evolutionary concept of sperm development in this taxon is suggested. RESUME Spermatologie comparee des Chelicerata: synthese et perspectives Les Chelicerata ont evolue ind£pendamment des autres arthropodes depuis le debut du Cambrien et done montrent de nombreuses particularity ainsi que des convergences r^alisees dans le cadre du concept des arthropodes. Ce travail d£crit l’etat actuel de nos connaissances sur les spermatozoides des Chelicerates. Les types les plus primitifs sont rencontres dans les taxons marins de manure primitive, en particular les Xiphosura. Les Pantopoda ont d6j& des spermatozoides tr£s derives. Dans les deux groupes, des deviations distinctes des caracteres sont observees entre les spermatozoides de differents taxons. Ceci demontre que la spermatologie comparee peut etre employee pour des considerations phylogenetiques ou systematiques. Les modifications les plus marquees du plan structural de base des spermatozoides sont rencontrees chez les Arachnida. Des representants de tous les taxons principaux ont maintenant ete etudies, et leurs spermatozoides sont rapidement commentes. En particulier, les spermatozoides des Araneae (araignees) et des Acari (acariens, tiques) ont ete etudies de maniere exhaustive. Les spermatozoides des araignees sont probablement les plus interessants du fait des formes de transport varies telles que les spermatozoides encapsuies individuellement (une cellule par capsule: cieistospermie), des capsules contenant quelques ou de nombreux spermatozoides (coenospermie ou pretendus “spermatophores”), et, probablement exceptionnelles au sein du Regne Animal, des capsules contenant des spermatozoides fusionnes ou syncytiaux (synspermie). Les Acariens ont tous des spermatozoides aflageltes de structures Alberti, G., 1995. — Comparative spermatology of Chelicerata: review and perspective. In: Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (eds), Advances in Spermatozoal Phylogeny and Taxonomy. Mem. Mus. natn. Hist, nat., 166 : 203-230. Paris ISBN : 2-85653-225-X. 204 G. ALBERTI : CHELICERATA extremement differentes, ce qui permet d'utiliser avec succes I’ultrastructure des spermatozoides pour la systematique. Dans un sous-groupe des Acariens il a ete possible de correler les alterations de la structure des spermatozoides avec des modifications du systcme genital et du comportcment sexuel. De ce fait, on suggere Vexistence d'un concept gvolutif du d£veloppement des spermatozoides dans ce taxon. Spermatozoa of Chelicerata are now well known from the comparative point of view. Several authors have included spermatological characters in their considerations on phylogenetic systematics of Chelicerata [117, 136, 137], comparative spermatology in general [33], on comparative spermatology as a tool in phylogenetic systematics [138], on arthropod phylogeny [32] or on insect spermatology [75], However, since not earlier than 1987 (Schizomida) [21] sperm cells of all main taxa had been described at least from one representative species, only an incomplete basis was available to these authors. Moreover, the many remarkable peculiarities shown by chelicerate spermatozoa require a separate study. This may stimulate further investigators to focus on spermatology of this interesting, diverse, and ancient group of arthropods, which has evolved separate from the other arthropods since the Early Cambrian [36, By far, the greatest structural diversity of spermatozoa is shown among the terrestrial and most species-rich Arachnida, which thus will dominate the following review. OBSERVATIONS AND DISCUSSION General aspects Spermatozoa ol Chelicerata represent the main categories of sperm structure distinguished today [32, 33, 35, 66, 74, 75], The primitive sperm among chelicerates is only found in horse-shoe crabs (Xiphosura) [18, 29, 56, 124] and is characteristic of those taxa exhibiting aquatic fertilization (aquasperm) [sensu 74], This type is classically characterized by a spherical head containing the acrosomal complex and nucleus with condensed chromatin, a middle piece containing a few relatively large mitochondria, and an elongate sperm tail representing the flagellum. The distal centriole is connected by an elaborate anchoring complex to the plasmalemma. It is already distinctly modified, however, in the Xiphosura [1, 18] (Fig. 1). The acrosomal complex is remarkable because of its very long acrosomal filament (perforatorium) which runs through the nucleus and is coiled around it. The chromatin is not as condensed as is usually the case in mature spermatozoa. The number of rather small mitochondria is quite high and a distinct middle piece is not always recognizable since the mitochondria may be scattered throughout the cytoplasm surrounding the nucleus in the spherical head region [18], There is also no prominent anchoring complex [18, 56] and the axoneme of the flagellum may lack the central tubules (in the Indowest-Pacific species of Tachypleus and Carcinoscorpius) [18, 147], The modified (derived) sperm (filiform-flagellate, biflagellate, or aflagellate to mention some obvious deviations from the primitive type) is characteristic of taxa exhibiting a modified fertilization. It is found thus in those aquatic (marine) taxa which have developed internal fertilization or are at least close to it and in terrestrial animals which depend on internal eitilization. Thus, in Chelicerata modified sperm cells are found in the marine Pantopoda and in Arachnida, which are primarily terrestrial animals. In Pantopoda (Pycnogomda), the sperm of Nymphon species are still close to the primitive type (big. 1). However, an acrosomal complex is lacking and the nucleus is elongated. It is surrounded by longitudinally arranged microtubules which persist in the mature sperm cell. Similar to xiphosurans, chromatin condensation is only weak. The proximal centriole is reduced. !?e 'S/n * f§CUUm m which considerable inter- and intraspecific variation in the axonemal pattern (9+0 to 18+0) occurs [48, 49, 52], The sperm cells of Nymphon are motile. On the contiary, the elongate, immotile spermatozoa of Pycnogonum littorale are highly modified and Source: ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY 205 contain numerous longitudinally arranged microtubules (more than 1000) forming complex patterns. Aside of folded membranes (nuclear derivative?) found in the central part of the cell no further organelles were seen [49]. Fig. 1. — Diagrams of sperm of the primary marine cheli cerates, Xiphosura and Pantopoda. a: Tachypleus gigas (Xiphosura) sperm are still close to the primitive type of sperm (aquasperm). The spermatozoon possesses a spherical head, a pronounced acrosomal complex, and a long flagellum. Note characters which deviate from the primitive sperm: very long acrosomal filament penetrating'the nucleus and coiling around it, indistinct middle piece. In contrast to Limulus polyphemus, this species has a flagellum with 9x2+0 axoneme [18]. b: Nymphon sp. (Pantopoda). The sperm is further derived. It lacks an acrosomal complex, the head is elongated, the middle piece is indistinct, there is only one centriole [49, 52]. AF, acrosomal filament; AV. acrosomal vacuole; N, nucleus. In Arachnida the main subtypes of modified sperm cells are distributed through the taxa as follows: filiform-flagellate (Scorpiones), coiled-flagellate (Pseudoscorpiones, Uropygi, Amblypygi, Araneae, Ricinulei), and aflagellate spermatozoa (Opiliones, Palpigradi, Solifugae, Acari) [4-8, 12, 13, 29, 21, 32, 33]. However, this classification gives only a rough impression of the diversity found within Arachnida: any conventional structure of sperm cells may be varied. Fig. 2 gives an impression of the various types of sperm depicting an example of each of the major taxa. The extraordinary diversity may be first simply expressed by variations in magnitude. Sperm cells of Arachnida range between less than 2 |im (Nicoletiella lutealAcari-Actinotrichida; Siro r/wr/cor/wT/Opiliones-Cyphophthalmi) [5, ALBERTI, unpublished] to 1000 pm in the argasid tick Omithodorus tholozani (capacitated sperm cell; see below) [63], 206 G. ALBERTI : CHELICERATA The individual sperm cell may be of a very complex structure (e.g. Pseudoscorpiones; certain Acari-Anactinotrichida; Fig. 2) or may be rather simple (e.g. Solifugae; certain Acari- Actinotrichida) [5, 7] (Fig. 2). In the spider mite Tetranychus urticae (Acari-Actinotrichida), spermatozoa merely are comprised of a chromatin body, some cytoplasm, and a plasmalemma with tubular indentations [22], However, primarily arachnid spermatozoa possess the same set of organelles which characterizes the ground plan of the metazoan sperm cell [33-35] (see above). BACCETTI [32, 33] several times stressed the major evolutionary alterations in the arachnids: coiling of sperm cells and loss of flagellum. In the following, modifications of conventional structures will be described at first, and secondly, new types of organization will be considered. Acrosomal complex The acrosomal complex typically is composed of an acrosomal vesicle (vacuole) and an acrosomal filament (perforatorium) (Fig. 3). The space between acrosomal vacuole and plasmalemma is occupied by a more or less distinct substance termed preacrosomal (also extraacrosomal or periacrosomal) material. The acrosomal filament is part of the subacrosomal material containing actin filaments which are highly ordered [124], Another component appears as an amorphous substance and is termed an intermediate substance. This acrosomal complex is primarily present in all arachnid orders, probably with the only exception of Palpigradi, in which no acrosomal filament is found (however, only one species has been observed until now) [4], Sometimes these conventional structures achieve a peculiar organization. In contrast to certain other scorpions in which the acrosomal complex is at the tip of the elongate nucleus, in the sperm of Buthus occitanus the very small acrosomal vacuole is located aside of the helical anterior part of the nucleus. The subacrosomal material (more strictly the intermediate substance) surrounds the whole tip of the nucleus [8], comparable to the subacrosomal cone found in certain Onychophora [74, 75]. A peculiar situation is observable in the opilionid Nemastoma lugubre [ALBERTI, unpublished] where subacrosomal material spreads between nuclear envelope and plasmalemma! Thus it apparently connects the latter to the nucleus and moulds the cell parallel to chromatin condensation and nuclear shaping [see also 81], At the first glance, the situation found in certain Acari-Anactinotrichida (namely in the vacuolated type of sperm) [6, 9, 13] is quite similar as in the opilionid just mentioned. Here the acrosomal vacuole is developed into a flat cisterna growing from a central acrosomal plate [6, 41,42], This flat cisterna is connected to the plasmalemma by preacrosomal material and the shape of the cell obviously is influenced by this (Fig. 8e). The preacrosomal material often contains filaments which are probably stabilizing the anterior region of the cell during the acrosomal reaction [124]. Such filaments were found in several Araneae [24, 26, 109] and in the micro whip scorpion Schizomus palaciosi [21] (Fig. 3b). In the latter an intricate paracrosomal lattice structure appears in the late stages of spermiogenesis as a transient structure (Figs 3d, 5). Regarding the various shapes of the acrosomal vacuole one extraordinary example was already demonstrated from the Acari-Anactinotrichida (Figs 2, 8a, c, e). Another well-known example is that of pseudoscorpions. In this group the acrosomal vacuole continues into a spiral band which surrounds the elongate smooth nucleus [40, 133] (Fig. 2). A peculiar sickle-shaped acrosomal vacuole was observed in Ricinulei [20] (Fig. 3c). Finally it may be mentioned that certain taxa of Opiliones and Acari have lost the acrosomal filament (as Prokoenenia wheeled, Palpigradi) [4] or are completely devoid of an acrosomal complex. Sometimes this occurs in the same genus: Siro rubens (Opiliones) with all acrosomal components) [80], Siro duricorius without acrosomal complex [ALBERTI, unpublished]. ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY 207 Nucleus Whereas the corkscrew appearance of the nuclear region in Pseudoscorpiones is derived from the peculiar spiral band (acrosomal vacuole), the nuclei of certain scorpions and of Uropygi themselves exhibit a helical, in Amblypygi and basically also in Araneae a corkscrew shape (with sharp edges) [8, 24, 40, 76, 103, 107, 126, .133, 134] (Fig. 5). In the latter and also in the uropygid Mastigoproctus giganteus, the acrosomal filament spirals around the periphery of the nucleus enclosed in a nuclear canal [24, 103, 107] (Figs 3e, 4, 5). In Amblypygi and Araneae there is a tendency toward asymmetry of the nucleus which extends beyond the implantation fossa into a postcentriolar nuclear elongation [12, 26]. This asymmetry may be extremely developed in spiders of the genus Tetragnatha (Tetragnathidae) [12], in which the basis of the axoneme comes close to the acrosomal vacuole. On the other hand, the nucleus of Pholcus phalangioides (Pholcidae; Araneae) is nearly of radial symmetry, which is a derived characteristic as can be concluded by comparison with related taxa [24] (Fig. 5). EURYPELMA CRYPTOCELLUS k Ricinulei Araneae CHEIRIDIUM Pseudoscorpiones SIRO Opiliones DRURUS jrpiones SCHIZOMUS Uropygi sf TARANTULA Amblypygi // CYTA Acari Actinotrichida PROKOENENIA Palpigradi OPILIOACARUS EUSIMONIA Acari Anactinotrichida Solifugae Fig. 2. — Synoptic view of spermatozoa of the terrestrial Arachnida (representatives of some taxa have secondarily invaded limnic and marine habitats). The spermatozoon of the scorpion is drawn in reduced length (full length: 275 pm). Spermatozoa of Siro duricorius and Cheiridium museorum [original], others [4-7, 9, 21, 26. 76, 77]. Opilioacarus: now Neocarus. Scale bar: 5 pm. A comparable tendency as in Tetragnatha, probably even more peculiar, is observable in the ricinuleid Cryptocellus boneti [20]. In this species the axoneme is directly connected with the acrosomal vacuole, a situation - to the author's knowledge - not found anywhere else. However, the nucleus is not asymmetrical in contrast to Tetragnatha, though of extraordinary shape. The anterior part is a thin tube containing the acrosomal filament (Fig. 3c). 208 G. ALBERTI : CHELICERATA Even though these are only few examples, it is evident, that the nuclei may exhibit a considerable variety of shapes. In the flagellate spermatozoa, whether filiform or coiled, it is basically a large elongate organelle (Scorpiones, Pseudoscorpiones, Uropygi, Amblypygi, Araneae) [8, 20, 21, 76, 77, 103, 107, 126, 133, 134], which may even extend beyond the posterior end of the flagellum (Cryptocellus boneti) [20]. It is of interest, also from the viewpoint of functional spermatology, that a manchette of microtubules is observed only in some of these spermatozoa, namely in those of Uropygi, Amblypygi, Araneae and Ricinulei [20, 24, 26, 76, 126]. No manchette is found in Scorpiones and Pseudoscorpiones [40, 72, 77, 106, 133]. At this point it is not intended to continue the discussion on the possible function of a manchette of microtubules in nuclear shaping [see 21, 58, 127 for references]. It may only be mentioned that the microtubules exhibit alterations in their arrangement parallel to nuclear condensation in Schizomus palaciosi [21], They follow exactly the sometimes aberrant configurations of the spermatid nuclei (e.g. in Tetragnatha) [12], The manchette microtubules disappear at the end of spermiogenesis (see the pantopod Nymphon, however). Another characteristic which largely varies within the Arachnida is the implantation fossa, a posterior region of the nucleus which usually contains the centrioles or their derivatives. The fossa may be a shallow, funnel-shaped indentation as in scorpions [8, 77], Uropygi/Thelyphonida [107], Amblypygi [76, 126] and Pseudoscorpiones [40, 134]. On the contrary, in Uropygi/Schizomida and many spiders the implantation fossa is deep, sometimes making the sperm almost a hollow tube (e.g. Schizomus palaciosi-Schizomida, Pholcus phalangioides-Araneae) [21, 24] (Figs 4, 5). Often dense material occupies the lumen of the fossa. This material may be homogeneous, granular (e.g. Pholcus) or may contain filaments or microtubules [92]. Within aflagellate spermatozoa an implantation fossa is observable only in opilionids. This is connected, in the genus Siro, with the transient appearance of a flagellum during spermiogenesis and is only a very shallow depression of the nuclear surface [80]. Nevertheless, in other opilionids a deep and broad invagination which contains the centrioles may occur [78, 81, 125]. In the aflagellate spermatozoa elongate, bulky, spherical, disc-, and bowl-shaped nuclei may be encountered (Fig. 2). It appears as if, after giving up the “basic plan” in early times, nearly any possibility of altering the shape of nucleus and cell has been realized. What is remarkable in particular with regard to the nucleus is the disappearance of the nuclear envelope in Fig. 3. Details of spermatozoa of various Cheliceraia. a: Acrosomal region of Tachypleus gigas (Xiphosura). Note acrosomal filament originating in the centre of a slight posterior indentation of the acrosomal vacuole and showing regularly arranged subfibres (actin) and nucleus with only loosely arranged chromatin [18], x 53 000. b: Late spermatid of Filisiaia insidiatrix (Araneae). The cap-like acrosomal vacuole is sectioned transversely and the acrosomal filament composed of subfibres is shown. The acrosomal vacuole is surrounded by thin filaments. Note manchette microtubules [24]. x 49 500. c: Spermatid of Cryptocellus boneti (Ricinulei). The small, sickle shaped acrosomal vacuole is sectioned transversely (large arrowhead). It is surrounded by dense streaks. Note axoneme originating immediately behind the acrosomal vacuole (the nucleus lies parallel to the axoneme and is not shown in this figure). At left two of the dense plates which later ensheath the spermatozoon are seen (small arrowheads) (compare Figs. 2 and 5) [20]. x 28 500. d: Spermatid of Schizomus palaciosi (Uropygi-Schizomida). The basis of the long acrosomal vacuole, which is deeply indented from behind, rests on the nucleus. The acrosomal filament is rather thick and shows the same pattern of subfibres as in Tachypleus gigas (compare a). Note the paracrosomal lattice structure, which is only present during a short period in spermiogenesis [21], x 63 000. e: Part of synspermium, i.e. the product of four fused spermatids, of Segestria senoculata (Araneae) showing basis of one axoneme and transverse sections through the coils of all four axonemes. Note 9x2 +3 axonemal pattern (synapomorphy of Megoperculata: Uropygi, Amblypygi and Araneae) and the absence of membranes separating the axonemes. what indicates the complete fusion between the four spermatids. A secretion sheath surrounds the synspermium [24], AF. acrosomal filament; AV, acrosomal vacuole; N, nucleus; PAL paracrosomal lattice structure; SH. sheath of secretion. Source: MNHN, Paris ADVANCES IN SPERMATOZOA!, PHYLOGENY AND TAXONOMY 209 Source: MNHN, Paris 210 G. ALBERTI : CHEL1CERATA Solifugae and Acari-Actinotrichida at the end of spermiogenesis. In these taxa the acrosomal filament penetrates through the chromatin body and thus directly contacts the cytoplasmic matrix [6, 7] (Figs 6d. 8d). A similar situation is to the author's knowledge found only in Xiphosura [18,56] (Fig. 1). Midpiece Since the work of ANDRE [28] the differentiation of the middle piece and chondriome in scorpions is known in detail. This is an example of extensive reorganization of mitochondria into elongate tubular structures (compare Fig. 2). No comparable elongation apparently occurs in Amblypygi and the early derived spider Heptathela kimurai (Mesothelae) [103] in which numerous mitochondria are arranged helically. Though quite different, in these three taxa a typical middle piece is established with mitochondria surrounding the flagellum or the axoneme respectively (Figs 4,5). In pseudoscorpions a different situation is found. The mitochondria are very elongate, thin tubular structures which are attached to the basis of the axoneme but they are otherwise situated in the cytoplasm of the coiled spermatozoon (see below) independently from the axoneme, where they establish a mitochondrial ring [40, 125] (Fig. 2). Contrary to scorpions, in pseudoscorpions the mitochondria are attached to the bases of the flagellum by an intricate structure (compare Fig. 2). In Uropygi no middle piece is encountered [21, 107, & PHILLIPS, 1986, pers. com.] (Fig. 5) as is the case in Ricinulei [20]. In Araneae, beside the primitive condition shown in Heptathela, mitochondria can be located without special arrangement (e.g. orthognath spiders, several labidognath spiders) [24, 26] or can be completely absent [93, 104] in the mature spermatozoa. Sometimes mitochondria pass through a “primitive” position in spermiogenesis being for some time located at the posterior end of the nucleus close to the basis of the flagellum (Schizomus palaciosi, several Araneae) [12, 21] (Fig. 5: Schizomus). In the aflagellate spermatozoa mitochondria are found in various positions sometimes “embedded” in the nucleus or chromatin body respectively (in the opilionid genus Siro and certain Acari- Actinotrichida) [6, 27, 64, 80, 142] (Fig. 7). Spermatozoa of some taxa may be devoid of mitochondria (e.g. Tetranychus w/T/cae/Acari-Actinotrichida) [22]. Axoneme The flagellum or axoneme respectively, if present, starts with two centrioles (Pantopoda are different in this respect; see above) arranged coaxially, an arrangement also found in the Xiphosura [18, 56] (Fig. 1). Thus this derived characteristic may be regarded as a synapomorphy of the chelicerates [136, 137], Sometimes, however, the centrioles are arranged nearly orthogonally as in the mature spermatozoa of bird spiders [26]. Spermiogenesis reveals that this position is a derived arrangement in these spiders, probably caused by the coiling process, and follows a phase in which the centrioles are in tandem position. Even the original orthogonal arrangement is observable in the early stages of spermatogenesis [26]. One of the most interesting results from the viewpoint of comparative spermatology in Arachnology was the discovery of the 9x2 +3 axoneme in Araneae [103, 112], Uropygi [21, 107] and Amblypygi [76, 126] demonstrating the close relationship of these taxa with a spermatological criterion (Fig. 3e). However, even this apparently very stable characteristic (compared with the variability found e.g. in scorpions) [8, 72, 77] is altered in certain spiders (Linyphiidae) in which a 9x2 +0 axoneme occurs [12], Thus this axoneme is by convergence similar to that of the xiphosuran genera Tachypleus and Carcinoscorpius [18, 147] and several scorpions [72], Sometimes dense material surrounds the distal centriole thus probably establishing an anchoring complex (Pholcus phalangioides, Dysdera sp., Segestria senoculata; Araneae) [24] (Figs 3e, 6b). In spiders the number of tubules constituting the centrioles may be reduced and/or Source: ADVANCES IN SPERMATOZOA!. PHYLOGENY AND TAXONOMY 211 dense fibres may be attached to the base of the axoneme. In Cryptocellus boneti (Ricinulei) the proximal centriole disappears during late spermiogenesis [20]. The only case where an aflagellate spermatozoon reveals relicts of an axoneme is represented by the cyphophthalmid Siro [80], In these tiny opilionids a flagellate stage is observable during spermiogenesis. Fig. 4. — Different shapes of nuclei of spermatids of spiders and their relation to acrosomal complex and axoneme. a: Heptathela kimurai (Liphistiidae, Mesothelae). b: Filisiata insidiairix (Filistatidae. Opisthothelae). c: Tetragnatha montana (Tetragnathidae, Opisthothelae). d: Pholcus phalangioides (Pholcidae, Opisthothelae). The Filislata-lype is the most common in spiders and may be close to the plesiomorphic type of Opisthothelae A peculiarity of scorpions and pseudoscorpions is the flagellar tunnel surrounding the axoneme and separating it from the middle piece mitochondria (in scorpions) and from the cytoplasmic matrix respectively (of the coiled cell in the pseudoscorpions) (Fig. 2). Whereas in the scorpions the flagellar tunnel comprises a distinct extracellular space, this is completely absent in pseudoscorpions owing to the close apposition of the membranes, which in early stages had delimited it. In scorpions and in some pseudoscorpions a dense cylinder surrounds the flagellar 212 G. ALBERTI : CHEL1CERATA tunnel. A flagellar tunnel is also observable during spermiogenesis of Uropygi, Amblypygi, Araneae and Ricinulei but disappears later [20, 21, 76, 107, 125]. Thus the axoneme is not surrounded by its genuine membrane in the mature spermatozoa of these taxa (Figs 3e, 6a, b). It is important to note that the flagellar tunnel separates the flagellum from the mitochondrial sheath (in scorpions) or mitochondrial ring (in pseudoscorpions). In Amblypygi and the early derived spider Heptathela, in which a middle piece is still existent, the flagellar tunnel disappears before the mitochondria occupy their definite position (Fig. 5). A flagellar tunnel appears also in connection with the transient flagellum of Siro. It is retained after the axonemal tubules have been withdrawn as a peculiar “crypt” containing numerous microvilli [80, ALBERTI, unpublished]. The flagellar tunnel may be derived from the cytoplasmic collar around the flagellar basis in xiphosuran sperm. No such structure occurs in Pantopoda (Fig. 1). In addition to the diversity achieved in arachnids by variations of the basic sperm components “new” structures are introduced into this cell type resulting in new structural and functional possibilities and extraordinary peculiarities. These are dealt with in the following. Vesiculation and vacuoles Arachnid spermatozoa are very often rich in vesicles and cisternae, especially those belonging to the coiled-flagellate type. These vesicles - at least partly a result of the coiling process during which surface membranes are likely internalized - may be arranged very regularly under the plasmalemma (Uropygi/Thelyphonida, Amblypygi) [76, 107, 126] (Figs 2, 5). In Araneae and Ricinulei such vesicles and cisternae often contain dense material forming “dense streaks” (Fig. 3c). In certain Araneae vesicles fuse to large vacuoles [24]. In addition to these structures further vesicles are derived by an extended activity of the Golgi apparatus. In Ricinulei dark plates are formed early in spermatogenesis and contribute to an intracellular capsule enclosing the central components of the mature spermatozoon (Figs 2, 3c) [20]. In the palpigrade Prokoenenia xvheeleri a huge vacuole dominates the large cell, which was previously thought to be a spermatophore (Fig. 2). The spermatozoa of certain Acari/Anactinotrichida develop during a very complex spermatogenesis a conspicuous vacuole bordered by a complex periphery bearing numerous “cellular processes” [5, 42, 63, 131] (Figs 2, 7, 8a,c). Perhaps the pouches of the ribbon sperm of related mites are derived from the vesicular precursors of the large vacuole characterizing the vacuolate type (Figs 8b) [5, 9, 13, 139-141], Transport forms - sperm aggregates Another aspect which was newly (or independently from non-arachnids) achieved during the evolution of spermatozoa of Arachnida is the establishment of secondary events. These transform elongate spermatozoa into the already mentioned coiled spermatozoa (Figs 2, 6a, b). In Uropygi and Amblypygi the coiling process starts from the posterior (flagellar) part of the cell resulting in a spherical mature spermatozoon. It is of interest to note that this coiling process is incomplete in the early derived spider Heptathela kimurai (Mesothelae) [89, 103], Only the flagellum and posterior part of the nucleus are involved. In the bird spiders (orthognath spiders) the coiling is more complete, leaving only the acrosomal region projecting slightly from the otherwise more or less spherical cell [26]. Finally, in (most) labidognath spiders the whole cell including the acrosomal region is completely coiled or spherical again [12, 15, 24, 39, 92, 93, 104, 109].. If it is assumed that the spherical shape is the plesiomorphic state within the “Pedipalpi”-Araneae, the spiders demonstrate, probably, a nice example of a round about way in the evolution of their sperm cells. In Araneae the coiled cell is surrounded by an extracellular secretion sheath in different ways (Figs 3e, 6b and see below). Source: