Phylogenetic position of rhyacophiloid caddisflies (Insecta, Trichoptera): a spermatological analysis of Rhyacophilidae and Glossosomatidae

Most caddisflies (Insecta, Trichoptera) are classified into two suborders, Annulipalpia and Integripalpia. However, the use of the derived characters that are regularly applied in systematic and phylogenetic analyses of Trichoptera is insufficient to determine with certainty the position of the families belonging to Rhyacophiloidea, which are considered by different authors to be either Annulipalpia, or Integripalpia, or even a separate suborder. Rhyacophiloidea comprise four overall similar families: free-living Rhyacophilidae and Hydrobiosidae, saddle-case making Glossosomatidae, and purse-case making Hydroptilidae. It was previously found that Annulipalpia spermatozoa have aberrant axonemes while Integripalpia spermatozoa display the plesiomorph 9 + 2 axoneme. The present spermatological analysis of the families Rhyacophilidae and Glossosomatidae shows that both have spermatozoa with aberrant axonemes lacking the two central microtubules found in the typical axoneme of insect spermatozoa. This is an apomorphic character shared with the superfamily Hydropsychoidea, indicating that from this point of view, Rhyacophiloidae are more closely related to Annulipalpia than to Integripalpia.     

Sperm structure of Trichoptera. III. Hydropsychidae,polycentropodidae and philopotamidae (Annulipalpia)

Spermatozoa from 9 species belonging to 3 families of trichopteran suborder Annulipalpia (Philopotamidae : Philopotamus ludificatus, P. montanus, Wormaldia occipitalis, W. copiosa; Polycentropodidae: Plectrocnemia geniculata, Polycentropus mortoni, P. irroratus, Cyrnus trimaculatus; Hydropsychidae: Hydropsyche pellucidula) were studied by light, scanning, and transmission electron microscopy. The absence of axonemal dynein arms and a consequent sperm immotility are characteristic of the species studied. The presence of 7, rather than 2, central microtubules is shared by Polycentropodidae and some Philopotamidae. A greater variability in the sperm axoneme was found within Philopotamidae than in the other families. The species of the genus Wormaldia have an axial vesicle rather than microtubules and their axonemes have a 9 + 9 + 0 or 13 + 13 + 0 pattern. Hydropsychidae have spermatozoa provided with numerous finger-like appendages containing microtubular doublets. The progressive loss of sperm flagellum and motility within the group is discussed.     

Spermatozoon ultrastructure and phylogenetic relationships in the monogeneans (platyhelminthes)

Justine J.-L., Lambert A. and Mattei X. 1985. Spermatozoon ultrastructure and phylogenetic relationships in the monogeneans (Platyhelminthes). International Journal for Parasitology15: 601–608. New observations reported in this study together with bibliographical data allow comparisons of spermatozoon ultrastructure in 28 genera of monogeneans, belonging to 19 families. The authors propose to compare and classify monogenean spermatozoa using two simple ultrastructural characteristics: (a) the number of axonemes, 1 or 2, (b) the presence or absence of cortical microtubules. These traits make it possible to group monogenean spermatozoa in four patterns. Pattern 1 (2 axonemes plus microtubules) is characteristic of the polyopisthocotyleans (9 families). The three other patterns are found in the monopisthocotyleans. Pattern 2 (2 axonemes without microtubules) is found in the Capsalidae and Dionchidae, which seem closely related, and also in the Udonellidae, Gyrodactylidae and Euzetrema. Pattern 3 (1 axoneme plus 1 altered axoneme plus microtubules) is found in the Monocotylidae and Loimoidae. Pattern 4 (1 axoneme without microtubules) is found in the Amphibdellatidae, Ancyrocephalidae, Calceostomatidae and Diplectanidae. A phylogeny of the monogeneans is drawn from the data of comparative spermatology; this scheme coincides in many points with the phylogeny of Lambert (1980) which was based on the study of chaetotaxy and ciliated cells of the oncomiracidium.     

Axoneme-specific beta-tubulin specialization: a conserved C-terminal motif specifies the central pair.

Axonemes are ancient organelles that mediate motility of cilia and flagella in animals, plants, and protists. The long evolutionary conservation of axoneme architecture, a cylinder of nine doublet microtubules surrounding a central pair of singlet microtubules, suggests all motile axonemes may share common assembly mechanisms. Consistent with this, alpha- and beta-tubulins utilized in motile axonemes fall among the most conserved tubulin sequences [1, 2], and the beta-tubulins contain a sequence motif at the same position in the carboxyl terminus [3]. Axoneme doublet microtubules are initiated from the corresponding triplet microtubules of the basal body [4], but the large macromolecular "central apparatus" that includes the central pair microtubules and associated structures [5] is a specialization unique to motile axonemes. In Drosophila spermatogenesis, basal bodies and axonemes utilize the same alpha-tubulin but different beta-tubulins [6--13]. beta 1 is utilized for the centriole/basal body, and beta 2 is utilized for the motile sperm tail axoneme. beta 2 contains the motile axoneme-specific sequence motif, but beta 1 does not [3]. Here, we show that the "axoneme motif" specifies the central pair. beta 1 can provide partial function for axoneme assembly but cannot make the central microtubules [14]. Introducing the axoneme motif into the beta 1 carboxyl terminus, a two amino acid change, conferred upon beta 1 the ability to assemble 9 + 2 axonemes. This finding explains the conservation of the axoneme-specific sequence motif through 1.5 billion years of evolution.     

Immunocytochemical study of tubulin in the 9+‘1’ sperm axoneme of a monogenean (Platyhelminthes), Pseudodactylogyrus sp.

The spermatozoon of the monopisthocotylean monogenean Pseudodactylogyrus sp. (a gill parasite of eels) has a single axoneme showing a 9+‘1’ pattern, a nucleus and a mitochondrion, but has no cortical microtubules. This species thus provides a very simple model for the study of tubulin in the 9+‘1’ axonemes of the Platyhelminthes, in contrast with digenean sperm which have a more complex spermatozoon with two such axonemes and cortical microtubules. Indirect immunofluorescence labelling of tubulin shows that the elongating spermatids, initially lying in all directions in the early stages, are arranged as parallel elements in further stages. The number of spermatids in an isogenic group could also be precisely counted and equals 32. Nuclear labelling with fluorescent dyes shows that the nuclei, first located in the common mass of the spermatids, later elongate and migrate into the growing spermatids, and that the nucleus is located in the central part of the mature spermatozoon, with the two extremities devoid of nucleus. Labelling with antibodies directed against acetylated, tyrosinated, and polyglutamylated tubulin gave positive results, thus indicating that these post-translational modifications of tubulin are present in the axoneme of spermatids and spermatozoa of monopisthocotylean monogeneans.     

Ultrastructure of the spermatozoon of Bothriocotyle sp. (Cestoda: Bothriocephalidea), a parasite of Schedophilus velaini (Sauvage, 1879) (Perciformes: Centrolophidae) in Senegal

The mature spermatozoon of Bothriocotyle sp. is filiform and tapered at both extremities. It possesses 2 axonemes of unequal length, showing the 9 + "1" pattern of Trepaxonemata. The anterior extremity exhibits a crest-like body. Thereafter, the crest-like body disappears, and the first axoneme is surrounded by a ring of cortical microtubules (about 27 units) that persist until the appearance of the second axoneme. This ring of cortical microtubules is characteristic only for species of Bothriocephalidea and represents a very useful phylogenetic character. The spermatozoon cytoplasm is slightly electron-dense and contains numerous electron-dense granules of glycogen in several regions. The anterior and posterior extremities of the spermatozoon lack cortical microtubules. The posterior extremity of the spermatozoon of Bothriocotyle sp. possesses a nucleus and a disorganized axoneme, which also characterizes spermatozoa of the Echinophallidae studied to date.     

The pattern of microtubules in the axonemes of Gymnosphaera albida sassaki: evidence for 13 protofilaments.

The axonemes of Gymnosphaera albida radiate from a central axoplast. Their proximal ends form a shell around the axoplast. Within the shell each axoneme is enveloped by a fibrillar sheath and the microtubules are interconnected by electron-dense linkages, which sometimes appear to be double. In nearly transverse sections the microtubules and their linkages form hexagons of 2 irregular types arranged in alternating rows. The shapes of the hexagons vary from one axoneme to the next. The variation is caused largely by the inclination of the axonemes to the line of sight, but also by distortion occurring during the preparation, observation and photography of the sections. Calculations show that, of a number of likely basic patterns (as would be seen in strictly transverse section), only one is compatible with measurements made on 9 of the axonemes. This involves only one type of hexagon oriented in 2 directions to form a 'parquet-floor' pattern. The hexagon is bilaterally symmetrical and its 6 microtubules all have the same set of angles between their linkages, namely an unpaired angle of 138 degrees 28' and paired angles of 110 degrees 46'. Because these angles are in the ratio of 5:4:4, it is deduced that the microtubules have 13 protofilaments forming their walls. Morphogenetically the lateral growth of the pattern is governed by 2 rules: (1) there must be one, and only one, direction of 2-step zig-zagging of the linkages, and (2) linkages forming opposite sides of a hexagon must be in parallel.     

Bending motion of Chlamydomonas axonemes after extrusion of central- pair microtubules

Relatively little is known about the functions of central-pair microtubules (Tamm, S. L., and G. A. Horridge, 1970, Proc. Roy. Soc. Lond. B, 175: 219-233; Omoto, C. K., and C. Kung, 1979, Nature (Lond.). 279:532-534) and radial spokes (Warner, F. D., and P. Satir, 1974, J. Cell Biol., 63:35-63), although a sliding microtubule mechanism has been established for the flagellar movement (Summers, K. E., and I. R. Gibbons, 1971, Proc. Natl. Acad. Sci. USA., 68:3092-3096). In the present report, an attempt was made to determine the functions of central-pair microtubules in flagellar motility. Central-pair microtubules were found to extrude from the tips of elastase-digested axonemes of demembranated Chlamydomonas flagella after the addition of ATP. The length of the extruded central-pair microtubules was approximately 70-100% that of the axoneme. After extrusion, axonemes continued to swim slowly backwards in the reactivation medium, with a trailing central pair attached like a tail to the flagellar tip. During bending movement of the axonemes, partially extruded central pairs rotated counterclockwise about the axoneme axis, as viewed from the distal end (Kamiya, R., 1982, Cell Motil. [Suppl.]:169-173). Axonemes swam backwards with a symmetric waveform and a beat frequency of approximately 10 Hz in the reactivation medium containing 10(-9)-10(-4) M Ca ions. Even at a lower Ca++ concentration, no ciliary-type swimming was noted on the axonemes.     

Axoneme-dependent tubulin modifications in singlet microtubules of the Drosophila sperm tail

Drosophila melanogaster sperm tubulins are posttranslationally glutamylated and glycylated. We show here that axonemes are the substrate for these tubulin C-terminal modifications. Axoneme architecture is required, but full length, motile axonemes are not necessary. Tubulin glutamylation occurs during or shortly after assembly into the axoneme; only glutamylated tubulins are glycylated. Tubulins in other testis microtubules are not modified. Only a small subset of total Drosophila sperm axoneme tubulins have these modifications. Biochemical fractionation of Drosophila sperm showed that central pair and accessory microtubules have the majority of poly-modified tubulins, whereas doublet microtubules have only small amounts of mono- and oligo-modified tubulins. Glutamylation patterns for different beta-tubulins experimentally assembled into axonemes were consistent with utilization of modification sites corresponding to those identified in other organisms, but surrounding sequence context was also important. We compared tubulin modifications in the 9 + 9 + 2 insect sperm tail axonemes of Drosophila with the canonical 9 + 2 axonemes of sperm of the sea urchin Lytichinus pictus and the 9 + 0 motile sperm axonemes of the eel Anguilla japonica. In contrast to Drosophila sperm, L. pictus sperm have equivalent levels of modified tubulins in both doublet and central pair microtubule fractions, whereas the doublets of A. japonica sperm exhibit little glutamylation but extensive glycylation. Tubulin C-terminal modifications are a prevalent feature of motile axonemes, but there is no conserved pattern for placement or amount of these     

Flagellar Axonemes with 10 Microtubular Doublets in Spermatozoa from Gall-midges (Diptera,Cecidomyiidae)

Abstract The spermatozoa of some gall-midges (Cecidomyiidae, Lestremiinae), belonging to the tribe Micromyini, were seen to have an axoneme that consists of 10, instead of nine, microtubular doublets surrounding a central cylinder. In some related species within the same tribe the axoneme was found to contain a similar cylinder but to have nine doublets, as in typical flagella, or to have nine doublets and no central structure. These three types of axonemes can be given the shorthand designations “10+cyl”,“9+cyl”, and “9+0”. The tribe Lestremiini is characterized by a giant axoneme having 150 doublets in two rows reversely oriented. Other characteristics of examined spermatozoa are the electron density of the B-tubules of the axoneme, a feature shared by all members of the subfamily Lestremiinae, and the presence of a prominent cytoplasmic droplet containing numerous, regularly spaced microtubules, which is shared by all Micromyidi. These axonemal models are discussed from a phylogenetic point of view.     

Direct measurement of inter-doublet elasticity in flagellar axonemes.

The outer doublet microtubules in ciliary and flagellar axonemes are presumed to be connected with each other by elastic links called the inter-doublet links or the nexin links, but it is not known whether there actually are such elastic links. In this study, to detect the elasticity of the putative inter-doublet links, shear force was applied to Chlamydomonas axonemes with a fine glass needle and the longitudinal elasticity was determined from the deflection of the needle. Wild-type axonemes underwent a high-frequency, nanometer-scale vibration in the presence of ATP. When longitudinal shear force was applied, the average position of the needle tip attached to the axoneme moved linearly with the force applied, yielding an estimate of spring constant of 2.0 (S.D.: 0.8) pN/nm for 1 microm of axoneme. This value did not change in the presence of vanadate, i.e., when dynein does not form strong cross bridges. In contrast, it was at least five times larger when ATP was absent, i.e., when dynein forms strong cross bridges. The measured elasticity did not significantly differ in various mutant axonemes lacking the central-pair microtubules, a subset of inner-arm dynein, outer-arm dynein, or the radial spokes, although it was somewhat smaller in the latter two mutants. It was also observed that the shear displacement in an axoneme in the presence of ATP often took place in a stepwise manner. This suggests that the inter-doublet links can reversibly detach from and reattach to the outer doublets in a cooperative manner. This study thus provides the first direct measure of the elasticity of inter-doublet links and also demonstrates its dynamic nature.     

The Homology of Cortical Microtubules in Platyhelminth Spermatozoa: a Comparative Immunocytochemical Study of Acetylated Tubulin

Spermatozoa of certain acoels, a group of primitive Platyhelminthes, and spermatozoa of the most derived Platyhelminthes, the Cercomeridea (parasitic Platyhelminthes), show a general morphological resemblance in that they are long filiform cells with two incorporated axonemes and longitudinal cortical microtubules. A possible way to test the homology of these cortical microtubules in the different groups is to analyze the presence/absence of post-translational modifications of tubulin. An indirect immunofluorescence study showed that the doublet microtubules of the sperm axonemes are labelled by an anti acetylated-alpha-tubulin antibody in all groups, irrespective of the axoneme pattern (9 + 0, 9 + 2 and non-trepaxonematan 9 + “1” in various acoels, and trepaxonematan 9 + “1” of the temnocephalid Troglocaridicola sp., the digenean Echinostoma caproni and the monopisthocotylean monogenean Pseudodactylogyrus sp.). Significant differences are found in the sperm cortical microtubules: they are acetylated in the acoel Actinoposthia beklemischevi, but not in the digenean E. caproni and the temnocephalid Troglocaridicola sp. These results suggest that the sperm cortical microtubules of the acoels are not homologous with the morphologically similar elements found in the higher Platyhelminthes.     

A light and electron microscopic study of the flagellate-to-ameba conversion in the MyxomyceteStemonitis pallida

Summary The flagellate-to-ameba conversion process of the MyxomyceteStemonitis pallida was investigated with Nomarski optics and electron microscopy. The flagellate has two flagella, a long and a short one. When the water film containing the flagellates becomes very thin, they retract their flagella, usually the short one first and then the long one. The short flagellum is retracted by only one method, in which the sheath membrane of the flagellum fuses with the cell membrane, consequently causing the axoneme to be absorbed into the cytoplasm. Retraction of the long flagellum can be divided into four types. In all cases, fusion of the sheath membrane and the cell membrane takes place. The retracted axoneme of the long flagellum sometimes beats convulsively for about 10 minutes after retraction, and after 10–15 minutes it became indistinguishable as it was detached from the blepharoplast.Analysis of thin sections shows that the retracted axonemes disintegrate in the following squence: B-tubules, A-tubules, spokes, central microtubules. In almost all cells the degradation begins immediately after retraction and is completed within 90 minutes. Only on rare occasions, structures which seem to have been derived from retracted axonemes are observed in the ameba about 90 minutes after conversion. The basal bodies and cytoplasmic microtubules are a little more stable than the retracted axonemes. Some basal bodies of the short flagellum, whose C-tubules are affected, are present in the amebae more than 90 minutes after conversion. Cytoplasmic microtubules decrease in number and become shorter in the amebae after about 24 hours, when newly formed regions filled with flocculent material appear.     

X-ray diffraction recording from single axonemes of eukaryotic flagella

We report the first X-ray diffraction patterns recorded from single axonemes of eukaryotic flagella with a diameter of only <0.2 μm, by using the technique of cryomicrodiffraction. A spermatozoon isolated from the testis of a fruit fly, Drosophila melanogaster, either intact or demembranated, was mounted straight in a glass capillary, quickly frozen and its 800-μm segment was irradiated end-on with intense synchrotron radiation X-ray microbeams (diameter, ~2 μm) at 74 K. Well-defined diffraction patterns were recorded, consisting of a large number of isolated reflection spots, extending up to 1/5 nm(-1). These reflections showed a tendency to peak every 20°, i.e., the patterns had features of an 18-fold rotational symmetry as expected from the 9-fold rotational symmetry of axonemal structure. This means that the axonemes remain untwisted, even after the manual mounting procedure. The diffraction patterns were compared with the results of model calculations based on a published electron micrograph of the Drosophila axoneme. The comparison provided information about the native state of axoneme, including estimates of axonemal diameter, interdoublet spacing, and masses of axonemal components relative to those of microtubules (e.g., radial spokes, dynein arms, and proteins associated with accessory singlet microtubules). When combined with the genetic resource of Drosophila, the technique presented here will serve as a powerful tool for studying the structure-function relationship of eukaryotic flagella in general.     

Ultrastructure de la spermiogenèse et du spermatozoïde de Loimosina wikoni et affinités phylétiques des Loimoidae (Plathelminthes, Monogenea, Monopisthocotylea)

In Loimosina, during spermiogenesis, the zone of differentiation of the spermatid contains two centrioles continued as two 9 +‘1’axonemes. One of the axonemes lengthens and will become the principal axoneme of the spermatozoon. The other axoneme is as long as the first one at the beginning of spermiogenesis, but is shorter in the mature sperm cell. The spermatozoon consists of several regions: (a) at the anterior end, the centriolar derivative of the principal axoneme; (b) a short region which shows cortical microtubules coinciding with external ornamentations; (c) a long uniflagellate region, with mitochondrion; (d) a biflagellate region containing the anterior slender part of the nucleus; (e) the posterior part of the nucleus, with no accompanying cytoplasmic organelles. Spermiogenesis and sperm structure in Loimosina differ from what is known in all other described monogeneans, excepting the monocotylid Heterocofyle to which they show close resemblances. However, the alteration of the second axoneme is more complete in Heterocofyle than in Loimosina. Comparative study of spermiogenesis and sperm ultrastructure thus shows an interesting coincidence with classical phylogenies of the monogeneans, in which the families Loimoidae and Monocotylidae are closely related. Chez Loimosina, pendant la spermiogenèse, la zone de différenciation de la spermatide contient deux centrioles prolongés par deux axonemes de type 9 +‘1′. L'un des axonemes s'allonge et deviendra l'axonème principal du spermatozoïde mûr. Le deuxième axontme est aussi long que le premier au début de la spermiogenèse, mais il est plus court dans le spermatozoïde mûr. Le spermatozoïde comprend: (a) à l'avant, le dérivé centriolaire de l'axonème principal; (b) une courte région contenant quelques microtubules corticaw longitudinaux coincidant avec des omementations extramembranaires; (c) une longue région miflagellée avec mitochondrie; (d) une région biflagellée contenant La partie antérieure effilée du noyau; (e) la région postérieure du noyau, sans organites cytoplasmiques accompagnateurs. La spermiogenèse et la structure du spermatozoïde de Loimosina sont différentes de ce qui est connu chez tous les autres Monogènes décrits, excepté le Monocotylidae Heterocofyle auquel elles ressemblent beaucoup. Toutefois, l'altération du deuxième axonème est moins complète chez Loimosina que chez Heterocotyle. L'étude comparée des spermatozoïdes et des spermiogenèses montre une bonne coincidence avec les phylogenèses classiques, dans lesquelles les familles Loimoidae et Monocotylidae sont proches.     

Differentiation between dynamic instability and end-to-end annealing models for length changes of steady-state microtubules

Short microtubules can be formed by shearing a sample at polymerization steady state of microtubules formed by glycerol-induced assembly of pure tubulin dimer. Such short microtubules show a rapid increase in mean length. The rate of this increase is too fast to be accounted for by statistical redistribution of subunits between microtubules. We propose that the fast length changes are a result of the end-to-end annealing of microtubules demonstrated by Rothwell et al. (Rothwell, S. W., Grasser, W. A., and Murphy, D. B. (1986) J. Cell Biol. 102, 619-627). This proposal has been tested by measuring the rate of annealing of free microtubules to Tetrahymena axonemes under conditions identical to those used for the lengthening of sheared microtubules. That free microtubules anneal to axonemal microtubules is indicated by the following observations. Axonemes elongate at both ends in the presence of steady state microtubules, as predicted for a symmetrical annealing process; under conditions where the microtubule number concentration is greater than that for axonemes, the initial rate of axoneme elongation is more rapid with a low concentration of long microtubules at steady state than with a high number concentration of short microtubules at steady state. These observations are inconsistent with the predictions of a model based on microtubule dynamic instability (Mitchison, T., and Kirschner, M. (1984) Nature 312, 237-242). The annealing rate observed with axonemes can account for the rate of elongation of sheared steady state microtubules.     

Spermiogenesis and spermatozoon ultrastructure of Nicolla wisniewskii (Digenea: Opecoelidae), an intestinal parasite of brown trout Salmo trutta (Pisces: Teleostei)

Spermiogenesis and ultrastructure of spermatozoon of Nicolla wisniewskii (Digenea, Opecoelidae), an intestinal parasite of Salmo trutta, were studied by electron microscopy. Spermiogenesis follows the general pattern found in the Digenea. It begins with the formation of a differentiation zone, including striated rootlets associated with 2 centrioles and an intercentriolar body. The flagella undergo a rotation of greater than 90 degrees. Then, their fusion with the median cytoplasmic process is proximodistal and asynchronous. A peculiarity was observed before the fusion of flagella, i.e., the attachment zones joined as 2 pairs by an electron-dense bridge. The mature spermatozoon is characterized by 2 axonemes, cortical microtubules, a nucleus, 2 mitochondria, external ornamentation, and spinelike bodies. At the posterior end of flagella, the spermatozoon is also characterized by the presence of a central element of the axoneme and without the 9 microtubule doublets. These results were compared with those of the other digeneans and, in particular, with other species of Opecoelidae. It appears that the number of cortical microtubules and their localization in the spermatozoon may be an interesting feature of their phylogeny.     

Sperm structure of Trichoptera. I. Integripalpia : Limnephiloidea

Spermal ultrastructure in 16 caddisflies (Trichoptera) belonging to the suborder Integripalpia, superfamily Limnephiloidea, was examined in a search for apomorphic and plesiomorphic features. In all species examined, the sperm tail axoneme was of the 9 + 9 + 2 + type, the axonemal doublets lacked outer dynein arms but had inner ones, and the cell membrane was scalloped with a prominent glycocalyx. The number of protofilaments in the accessory tubules depended on the phylogenetic position: 18 in the family Leptoceridae, 19 in Limnephilidae, Goeridae and Odontoceridae (with a reduction in the number distally), and 20 in Sericostomatidae. Spermatozoa in Leptoccridae are further characterized by the 2 central microtubules being flattened and eccentric and not being surrounded by a central sheath. Spermatozoa of Sericostomatidae have an accessory body and a helicoidal array of the sperm tail.     

Spermiogenesis and sperm ultrastructure in the monogenean parasite Acanthocotyle lobianchi

The ultrastructural events of spermiogenesis and the ultrastructure of the mature spermatozoon of an acanthocotylid monogenean, Acanthocotyle lobianchi, are described. The early zone of differentiation (ZD) contains two roughly perpendicular centrioles which become parallel and produce two free flagella, although these later become incorporated into the same body of cytoplasm. No cortical microtubules were found supporting the ZD at any stage of spermiogenesis. Much of the length of the thread-like sperm contains two axonemes of the 9 + '1' pattern together with a nuclear and mitochondrial profile but the 'posterior' region is occupied only by a single axoneme and the nucleus. A laterally situated electron-lucent vesicle with specialization of the adjacent surface membrane is found in the 'anterior' region of the sperm. The phylogenetic implications of these observations are discussed.     

Ultrastructural and immunocytochemical investigation of acoel sperms with 9 + 1 axoneme structure: new sperm characters for unraveling phylogeny in Acoela

Acoel sperm characters proved useful in deciphering acoel taxonomy. The phylogenetic value of sperm characters in closely related sub-groups or in a monophyletic taxon has not yet been assessed. We have investigated sperm ultrastructure in seven members of the monophyletic taxon Childia sensu (Tekle et al. J Zool Sys Evol Res 43(1):72–90, 2005) and in their closest relatives, the Mecynostomidae (four taxa). All members of Childia examined show little variation in their sperm ultrastructure. The common characters of Childia taxa are: 9 + 1 axoneme structure, the presence of six distal cytoplasmic microtubules in the absence of axial or cortical ones, long nucleus and extensive nucleus–flagella overlap. We have identified a new set of cytoplasmic microtubules lying in the centriolar end of the sperm cell, distal microtubules. The origin and phylogenetic significance of this character is discussed. The types and arrangement of cytoplasmic granules could be used as phylogenetic characters at a low taxonomic level. A loose membrane amorphous core type of granule was found to be a synapomorphy for the following clade within the taxon Childia: C. crassum + C. groenlandica + C. vivipara + C. brachyposthium + C. macroposthium. Sausage shaped granules are plesiomorphic among the taxa examined. The rest of the granule characters were found to be homoplasious. Sperm ultrastructural characters have again proven their concordance with molecular phylogeny. The only morphological synapomorphies known for the sister taxa Childia–Mecynostomidae, in the molecular phylogeny, are characters derived from sperm ultrastructure: distal microtubules arranged in two groups of three microtubules each and a 9 + 1 axoneme structure. The spermatozoa of Childia and Mecynostomidae show 9 + 1 axoneme configuration, seemingly similar to the 9 + ‘1’ axoneme pattern of the Platyhelminthes—Trepaxonemata. Using electron-microscope immunocytochemistry, we have demonstrated that, unlike the central cylinder of trepaxonematans, the central cylinder of the 9 + 1 axonemal pattern in acoels is immunoreactive to tubulin and contains a single central microtubule. Therefore, the 9 + 1 patterns in acoels and trepaxonematans are homoplasious.