New data on spermiogenesis and trepaxonematan axoneme in basal tapeworms (Cestoda, Caryophyllidea, Lytocestidae) parasitizing cyprinid fishes

Monozoic caryophyllidean cestodes, intestinal parasites of cyprinid fishes, represent a group of tapeworms with an unclear evolutionary history. As spermatology may provide phylogenetically important data, the spermiogenesis and ultrastructure of the mature spermatozoon have been investigated using an integrative approach combining transmission electron microscopy, cytochemistry and electron tomography in Khawia rossittensis (Szidat, 1937). The process of spermatid formation is accompanied by the presence of ultrastructural characters not described in traditional models of spermiogenesis, e.g., apical electron-dense material, the two striated roots situated unusually opposite each other, branching of typical striated roots, an intercentriolar body comprising five electron-dense and four electron-lucent layers, rotation of both free flagella and flagellar buds to the median cytoplasmic process at 90°, and a complete proximodistal fusion. The synchronous rotation of both flagellar buds and growing free flagella is an evolutionarily linked pattern favouring the hypothesis that the Caryophyllidea are not ancestral but are secondarily derived from polyzoic forms. Electron tomography analysis has revealed a unique feature of two helicoidal tubular structures in the central electron-dense core of the axoneme of mature spermatozoon. These data provide new insights into the architecture of the 9 + ‘1’ axoneme, which is shared by male gametes of all trepaxonematan Platyhelminthes.

using electron tomography analysis in one-axoneme spermatozoa of the evolutionarily more derived tapeworm Nippotaenia mogurndae 17 . These interesting findings call for further detailed electron tomography studies of other caryophyllidean cestodes and cestode groups to elucidate the basic sperm structure of type 9 + '1' in the Trepaxonemata.
Therefore, the spermatological characteristics of Khawia rossittensis, another lytocestid tapeworm and parasite of Carassius gibelio, have been examined to shed more light on the cytodifferentiation and fine structural architecture of the male gametes of caryophyllidean cestodes.

Results
The very early stage of spermiogenesis in Khawia rossittensis is marked by the formation of the zone of differentiation (Fig. 1A,B). This area contains cortical microtubules, apical dense material and two centrioles associated with striated roots, which are oriented tangential to the apex of the nucleus (Figs 1A and 2a). Atypically, the striated roots are situated opposite of each other (Figs 1C and 2b). In advanced stages of spermiogenesis, a typical position of striated roots can be observed (Figs 1D,F and 2c 1 ,d 1 ). A remarkable feature is represented by the intercentriolar body, which comprises five electron-dense and four electron-lucent layers (Fig. 1D inset, 2b). The electron-dense plates are of different thicknesses: the thickest plate is the central plate, the less thick plates are the two peripheral plates, and the thinnest plates are the inner dense plates, which can be detected at higher magnification (Figs 1D inset, 2b). The branching of typical striated roots, where one typical root is branched into two or three arms, is another bizarre spermatology characteristic of K. rossittensis (Figs 1E,G and 2c 2 ,d 2 ). The formation of free flagella and flagellar buds is accompanied by their rotation towards the prolonging median cytoplasmic process (MCP) (Figs 1D,F,G and 2c 1 -d 2 ). In the advanced stages of spermiogenesis, the two arching membranes are formed under the plasma membrane, and the nucleus migrates into the MCP (Figs 1D,E,F and 2c 1 -e). The free flagellum approaches (Figs 1G,I and 2d 1 ,d 2 ) and merges with the MCP during proximodistal fusion (Figs 1H-J and 2e). The attachment zones are present as two small islands of electron-dense material on the inner surface of the plasma membrane of the MCP (Figs 1I and 2d 1 ,d 2 ). At the end of spermiogenesis, the ring of arching membranes is constricted, and the spermatids are detached from the residual cytoplasm (Fig. 2e).
The mature spermatozoa of K. rossittensis are filiform cells tapered at both ends. These cells contain a single axoneme of trepaxonematan type 9 + '1' , cortical microtubules, cytoplasm with granules of glycogen and a nucleus. Five different regions with typical cytoarchitecture were recognized in ultrastructural studies of the male gametes of K. rossittensis.
Region I (Figs 3A and 4I), or the anterior part of the spermatozoon, contains one typical axoneme surrounded by a semi-arc of five cortical microtubules situated under the plasma membrane.
Region II (Figs 3B-E and 4II) exhibits an enlarged volume of cytoplasm and an increasing number of cortical microtubules (7)(8)(9)(10)(11). The cytoplasm is electron lucent in conventional ultrathin longitudinal and cross sections (Fig. 3B,C). The application of the Thiéry method (1967) 18 enabled the detection of electron-dense granules of glycogen (Fig. 3D,E). One pair of attachment zones corresponds to the points of fusion of the free flagellum with the median cytoplasmic process during spermiogenesis (Figs 3B,F,G,I,J and 4II,III,IV).
Region III (Figs 3F-I and 4III) is a nucleated part of the spermatozoon. Anteriorly, the nucleus has a small diameter and is located near the axoneme (Fig. 3F). In the middle part of region III, the nucleus occupies the largest area of the cytoplasm, and the number of cortical microtubules increases up to 22 (Fig. 3G,H). The size of the nucleus and the number of cortical microtubules decrease posteriorly (Fig. 3I). Glycogen granules are situated in the peripheral cytoplasm of the spermatozoon (Fig. 3H).
Region IV (Figs 3J and 4IV) is characterized by a reduction in the cytoplasmic volume and the number of cortical microtubules (up to 3).
Region V (Figs 3K and 4V) or posterior part of the spermatozoon shows the axoneme without its central structure, i.e., nine peripheral doublets, which are continuously transforming into singlets.
Electron tomography investigations of one-axoneme mature spermatozoon of K. rossittensis with the 9 + '1' trepaxonematan structure were based on the analysis of a tomogram virtual slices with increasing Z-position ( Fig. 5A-D). Our results showed that the central core unit (CU) is interconnected with nine peripheral axonemal doublets (D) by regularly spaced spokes (S). The central core unit consists of a central electron-dense core (EC) and electron-lucent intermediate area (IA), which is peripherally surrounded by an electron-dense cortical sheath (CS). The EC has a nearly elliptical shape and contains two tubular structures that are visible in all tomogram virtual slices as a white central electron-lucent area surrounded by a dark electron-dense border (white arrowhead in Fig. 5). The diameter of each tubular structure is 14.4 ± 1.4 nm (measured black-to-black border). The

Discussion
Our observations on spermiogenesis in Khawia rossittensis revealed interesting ultrastructural features not described previously in traditional models of sperm formation in the Caryophyllidea 19,20 , e.g., apical electron-dense material and striated roots situated unusually in opposite direction in early stages, and rotation of both free flagella and flagellar buds. A branching of the striated root has not been described in the Caryophyllidea. A unique tubular doublet was detected using electron tomography in the central axonemal core of the spermatozoon with 9 + '1' trepaxonematan structure in the present study.
The atypical arrangement of the two striated roots in opposite direction during the early stages of spermiogenesis as described in K. rossittensis in the present study has been reported in the lytocestid Caryophyllaeides fennica 11 , the caryophyllaeid Caryophyllaeus laticeps 25 , and the diphyllobothriidean Diphyllobothrium latum 29 . www.nature.com/scientificreports www.nature.com/scientificreports/ These spermatology findings may support the hypothesis based on molecular analysis that Caryophyllidea and Diphyllobothriidea are sister groups 40 . Another remarkable and unusual feature of Eucestoda is the branching of typical striated roots, where one typical root is branched into two or three arms. However, further observations are needed to elucidate the occurrence and/or importance of this unusual architecture of striated roots in cestodes. www.nature.com/scientificreports www.nature.com/scientificreports/ The intercentriolar body (ICB), a phylogenetically important character, is considered to be a plesiomorphic feature of the Eucestoda 5,6 . Its composition may be closely related to the evolution of cestodes. The remarkable variability of this cell structure has been recorded in caryophyllidean cestodes of the family Lytocestidae. Here, the ICB is either absent, namely, in Monobothrioides chalmersius 10 and Atractolytocestus huronensis 13 , or present and may consist of a variable number of electron dense layers, i.e., one in K. armeniaca 12 , three in K. sinensis 13 and L. indicus 14 , or five in K. rossittensis (present study). Whereas the ICB of most caryophyllideans contains three electron-dense layers [21][22][23][24][25] , five electron-dense layers have been detected in Archigetes sieboldi 41 and in K. rossittensis (present study). The latter pattern has also been reported in some diphyllobothriideans 29,30 and trypanorhynchs 42 .
The rotation of both free flagella and flagellar buds to the median cytoplasmic process at 90° has not been described in traditional models of the spermiogenesis of the Caryophyllidea 10,19,20,22,43 . Subsequently, this feature was observed in some lytocestids 8,[11][12][13] , caryophyllaeids 21,25,41 , capingentids 23 and some evolutionarily higher non-cyclophillidean cestodes with one axoneme spermatozoa, i.e., tetraphyllideans 44 , tetrabothriideans 45 , or nippotaeniids 17 . The flagellar bud may represent a strongly reduced flagellum developed during the spermiogenesis of cestodes with two-axoneme spermatozoa, and consequently, the rotation of both free flagella and flagellar buds may indicate a derived stage of spermiogenesis in the Caryophyllidea 17 .
Glycogen is a branched polymer of glucose residues that presumably serves as an energy reserve for the motility of spermatozoa and other activities essential to the fertilization of ova 46 . An investigation of glycogen using a cytochemical method by Thiéry 18 revealed the presence of glycogen granules in the cytoplasm of prenuclear,   www.nature.com/scientificreports www.nature.com/scientificreports/ nuclear, and postnuclear regions in the mature spermatozoa of K. rossittensis. Similar results were reported in two other caryophyllideans, C. laticeps 25 and C. fennica 11 . It should be noted, however, that intraaxonemal glycogen has not been detected in the mature spermatozoa of any caryophyllideans or Platyhelminthes with the 9 + '1' trepaxonematan structure.
Our electron tomography analysis revealed unique two tubular structures in the centre of the electron-dense core of the 9 + '1' trepaxonematan axoneme in the spermatozoon in K. rossittensis. A similar structure was described in the lytocestid C. fennica for the first time 15 . Virtual tomogram slices of both lytocestid cestodes showed the two central tubular structures, which may resemble two free parallel central singlet microtubules of the 9 + 2 axoneme of cilia and flagella, as described by Fawcett and Porter 47 for the first time. On the other hand, the helical appearance of central doublet microtubules in cestodes (Fig. 6) does not support any hypothesis about the homology of these structures. Notably, the 9 + 2 axonemes are supposed to be almost ubiquitous structures, which are evolutionarily conserved and evolved in early eukaryotes nearly a billion years ago 48 . Our recent electron tomography findings have introduced a new model for the spermatozoon axoneme with a 9 + '1' trepaxonematan structure, which, until recently, has been described exclusively in lytocestid caryophyllideans (Fig. 7).
The helicoidal nature of the central tubular doublet in K. rossittensis resembles the spiral shape of the central electron-dense core within the central cylinder of the single axoneme of the spermatozoon in nippotaeniid cestodes 17 . A helical substructure is supposed to be a more fundamental configuration than the derived protofibrilar substructure 49 .
The present study provides an original dataset of the novel spermatology features of lytocestid caryophyllidean tapeworms. The unique architecture of the 9 + '1' axoneme of mature spermatozoa, as revealed by electron tomography, has been reported in only two lytocestids. Therefore, further studies of the spermatozoa of the tapeworms sensu stricto (Eucestoda) are urgently needed for the effective use of these new characters for the clarification of systematic and phylogenetic questions within the Platyhelminthes.

Materials and Methods
parasite sampling. Live adult specimens of Khawia rossittensis (Szidat, 1937) were obtained from the intestine of prussian carp Carassius gibelio (Bloch, 1782) (Cypriniformes, Cyprinidae) captured from the Tisa River, Southeastern Slovakia. The fish were dissected using the standard methods described by Ergens and Lom 50 . All applicable institutional, national and international guidelines for the care and use of animals were followed and approved by the Animal Care and Use Committee of the Institute of Parasitology, Slovak Academy of Sciences, Košice (Slovakia). Tapeworms were cooled in a 0.9% NaCl solution and immediately processed for ultrastructural studies in the following way. electron microscopy. The worms were cut into small pieces, fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 10 days at 4 °C, washed with 0.1 M sodium cacodylate buffer, postfixed in 1% OsO 4 in 0.1 M cacodylate buffer for 2 hours at 4 °C, dehydrated in a graded alcohol series and embedded in Spurr epoxy resin. Semithin sections were cut with glass knives on an LKB Bromma 8880 ultramicrotome, stained with methylene blue and examined under a light microscope for the localization of the testes and vas deferens. Ultrathin sections were cut with a diamond knife on a Leica Ultracut UCT ultramicrotome, placed on copper grids and double stained with uranyl acetate and lead citrate. The grids were examined on a JEOL 1010 transmission electron microscope operating at 80 kV. cytochemistry. Visualization of glycogen granules in male gametes was carried out using the Thiéry method 18 , the periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) technique, using the following protocol: ultrathin sections were placed on gold grids, treated in 1% PA (20-25 minutes), washed with distilled water, processed in 1% TSC (40 minutes), washed with 10% acetic acid and distilled water, treated in 1% SP (30 minutes), and finally washed with distilled water and dried. Gold grids were examined on a JEOL 1010 transmission electron microscope operating at 80 kV. www.nature.com/scientificreports www.nature.com/scientificreports/ electron tomography. Ultrathin sections of 100 nm thickness were placed on 300 mesh copper grids (SPI), and staining was performed using alcoholic uranyl acetate and lead citrate both for 15 minutes. Au nanoparticles 10 nm conjugated with protein-A (BBI) as fiducial markers were placed on both sides and the surfaces were covered by carbon. We performed electron tomography as a dual axis in the range ±70° with a tilt step of 0.8° by means of a JEOL 2100F transmission electron microscope (TEM) equipped with a motorized tilt stage and a Orius SC 1000 Gatan camera automatically controlled by SerialEM software 51 . Tomogram reconstruction and model segmentation were performed with the IMOD software package 52 . The reconstructed tomogram with an isotropic voxel size of 0.9 nm was filtered for noise reduction by a 3D median filter and stored as an MRC file 53 . The contrast of virtual tomogram slices was enhanced by using an ImageJ plugin Enhanced Local Contrast (CLAHE) (imagej.net/Enhanced_Local_Contrast_(CLAHE)).

Data Availability
The datasets generated and/or analysed during the current study are available from the corresponding authors on reasonable request.