Ultrastructure and cytochemistry of the mature spermatozoon of Khawia armeniaca (Cholodkovsky, 1915) (Caryophyllidea: Lytocestidae), a parasite of Capoeta capoeta sevangi (De Filippi, 1865) (Teleostei, Cyprinidae)
Mature spermatozoa of the Eucestoda show a high variability and provide important characters useful for phylogenetic studies (Justine, 1998, 2001; Levron et al., 2010). In general, they contain one or two axonemes, cortical microtubules, a nucleus, and various granules in the cytoplasm (Justine, 2003). Male gametes of the order Caryophyllidea are of special interest, as phylogenetic relationships within this group and its interrelationships with other tapeworms remain unclear (Waeschenbach et al., 2012; Caira & Littlewood, 2013).
Therefore, the present study aims to provide missing ultrastructural characters of the mature spermatozoa of K. armeniaca which are necessary for better understanding of the male gametes morphology within the genus Khawia, the order Caryophyllidea, and the Eucestoda.
Materials and Methods
Adult specimens of Khawia armeniaca (Cholodkovski, 1915) were collected from the intestine of Capoeta capoeta sevangi (De Filippi, 1865) (Pisces: Cyprinidae), from Lake Sevan, Armenia.
Transmission electron microscopy
The tapeworms were cut into small pieces, fixed immediately in ice cold 3 % glutaraldehyde in sodium cacodylate buffer (pH 7.4) for 3 – 8 h, followed by three changes of cacodylate buffer and postfixed in 1 % OsO4 for 1 h. The material was dehydrated in a graded alcohol series, acetone, and embedded in Araldite. The ultrathin sections (90 nm) were cut on diamond knife using a Leica Ultracut UCT ultramicrotome, placed on copper grids, and double-stained with uranyl acetate (30 min.) and lead citrate (20 min.). The grids were examined in a JEOL 1010 transmission electron microscope operated at 80 kV.
Cytochemistry
Cytochemical technique of Thiéry (1967) with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) was used for visualisation of glycogen in the mature spermatozooa. Ultrathin sections were placed on gold grids, treated in 1 % PA (20 – 25 min.), washed in destilled water, processed with 1 % TSC (40 min.), washed in 10 % acetic acid and destilled water, treated in 1 % SP (30 min.) and finally washed in destilled water. The grids were observed in JEOL 1010 transmission electron microscope.
Ethical Approval and/or Informed Consent
The research related to animals has been complied with all the relevant national regulations and institutional policies for the care and use of animals.
Results
A large number of cross- and longitudinal sections of the mature spermatozoa from vasa deferentia of Khawia armeniaca have been investigated in the present study.
Vas deferens
The vas deferens is situated between vasa eferentia and the ejaculatory duct of the cirrus pouch. The wall of convoluted vas deferens is formed by a thin syncytial epithelial layer, the luminal surface of which is lined by numerous sinuous lamellae and/or rare cilia (measuring up to 3.5 μm) (Fig. 1a).
Fig. 1
Cross sections of the mature spermatozoon of K. armeniaca: (a) vas deferens with five different regions of the spermatozoon (I-V); (b) region I presents one axoneme and six cortical microtubules; (c) region II shows cortical microtubules which are arranged in a semi-circle under the plasma membrane; (d-g) region III or nucleated region: small diameter of anterior part of the nucleus (d), which gradually increases (e), reaches a maximum in the middle part (f), and diminishes again more posteriorly (g); (h-j) region IV shows cortical microtubules which are arranged in two opposite rows (note one single microtubule in the middle between the two opposite rows) (h), rapidly reduced volume of cytoplasm and number of cortical microtubules (i, j); (k-l) region V with one axoneme which lost its central structure (k) and undergoes disorganization into doublets (l). Ax, axoneme; Arrowheads, attachment zones; BB, basal body of the cilium; CM, cortical microtubules; D, doublets; L, lamellae; N, nucleus. Scale bars: a = 1000 nm, b-h, l = 200 nm, i = 250 nm, j, k =125 nm.
Mature spermatozoon
Male gametes are long, filiform cells, tapered at both extremities, with one incorporated axoneme of the 9+‘1’ trepaxonematan structure, cortical microtubules and a nucleus which are situated parallel to the longitudinal axis of the spermatozoon, and granules of glycogen. Five different regions (I – V) with specific ultrastructural organisation can be recognized (Figs. 1, 2, 3).
Fig. 2
Granules of glycogen in the cytoplasm of the mature spermatozoon of K. armeniaca after application of Thiéry method (1967): (a) cross section of region II; (b) cross section of region III; (c) cross section of region IV; (d) longitudinal section of region II. Ax, axoneme; CM, cortical microtubules; G, glycogen; N, nucleus. Scale bars: a-c = 200 nm, d = 250 nm.
Fig. 3
Schematic reconstruction of the mature spermatozoon of K. armeniaca. I–V, five different regions of the mature spermatozoon; ASE, anterior spermatozoon extremity; Ax, axoneme; AZ, attachment zones; CC, central core; CM, cortical microtubules; G, glycogen; N, nucleus; PM, plasma membrane; PSE, posterior spermatozoon extremity.
Region I (Figs. 1a, b; 3I) corresponds to the anterior extremity of the spermatozoon. It contains one axoneme which is surrounded by a semiarc of up to 5 cortical microtubules (CM) located under the plasma membrane. The diameter of the spermatozoon is about 300 nm.
Region II (Figs. 1a, c; 2a, d; 3II) exhibits an increase of both the volume of cytoplasm and the number of CM. The CM are arranged in one continuous row composed of up to 23 elements (Fig. 1c). Granules of glycogen (Figs. 2a, d), can be detected in the cytoplasm using Thiéry method (1967). One pair of attachment zones illustrates the points of fusion of the free flagellum with the median cytoplasmic process during spermiogenesis (Figs. 1c; 3II). The diameter of the spermatozoon reaches up to 460 nm.
Region III (Figs. 1a, d, e, f, g; 2b, 3III) corresponds to the middle part of the mature spermatozoon, containing the nucleus. The diameter of the nucleus is about 40 nm at the beginning (Fig. 1d), gradually enlarges (Fig. 1e) up to 500 nm in its middle region (Fig. 1f). Posteriorly, the nucles diameter diminishes again, having about 160 nm near its posterior extremity (Fig. 1g). The CM are arranged in a semicircle at first (Fig. 1d). Subsequently, an enlarged nucleus is approaching the plasma membrane of the spermatozoon, thus dividing originally continuous semicircle of CM in two opposite parts (Fig. 1e, f, g), each consisting of 12 – 15 elements. One pair of attachment zones can be recognized (Fig. 1f). Based on examination of cross sections, the maximum number of CM in the spermatozoon of K. armeniaca is 35 elements, which may occurr in the principal nucleated region of the male gamete. In addition, the cytoplasm of the spermatozoon contains scattered granules of glycogen (Fig. 2b). The diameter of the mature spermatozoon in region III is ranging from 480 nm (anterior and posterior parts) to 720 nm (middle part).
Region IV (Figs. 1a, i, j, 2c, 3IV) is the postnuclear part of the spermatozoon. The CM are arranged at first in two opposite rows, each having 9 elements, and one single microtubule is situated in the middle between the two opposite rows (Fig. 1h). More posteriorly, a strong reduction of the cytoplasm volume, and only a few CM (up to 4) are observed (Fig. 1i). Here, the diameter of the spermatozoon is diminished to 380 nm. At the end of Region IV only one axoneme is present (Fig. 1j).
Region V (Figs. 1a, k, l, 3V), or the posterior part of the spermatozoon, contains only one axoneme which undergoes major disorganization: the central core unit disappears at first (Fig. 1k), and is followed by further disorganization of peripheral doublets (Figs. 1l, 3V). The very posterior extremity of the K. armeniaca spermatozoon is around 140 nm in diameter.
Discussion
This study provides an evidence that ultrastructural architecture of the mature spermatozoon of Khawia armeniaca principally resembles that of other lytocestiids and caryophyllideans. Their male gametes exhibit one axoneme; cortical microtubules and nucleus which are situated parallel to the long axis of the spermatozoon (Bruňanská, 2010; Levron et al., 2010). Caryophyllidean tapeworms have the spermatozoon with 9+‘1’ axonemal structure which is typical for the Trepaxonemata (Ehlers, 1984). Their axoneme is composed of the central core unit interconnected with nine peripheral doublet microtubules which are associated with inner and outer dynein arms. Traditionally, it was believed that the central core unit contains the central electron-dense core, electron-lucent intermediate area, and peripheral electron-dense cortical sheath. In contrast, recent electron tomography observations have revealed the presence of the two tubular structures in the central axonemal electron-dense core of two lytocestiid caryophyllideans, Caryophyllaeides fennica and Khawia rossittensis (Matoušková et al., 2018, 2019). Future electron tomography studies are necessary to elucidate the structure of the central electron-dense core of the axonemes in the spermatozoa of K. armeniaca.
The parallel alignment of CM in a longitudinal axis occurrs in most Eucestoda (except for the Cyclophyllidea and Tetrabothriidea, which have spiralled CM), Digenea and Monogenea (Justine, 2001; Bruňanská 2010; Bakhoum et al.2017). Based on the number of axonemes (one or two), the parallel/spiralled pattern of CM, the parallel/spiralled pattern of the nucleus, and some other ultrastructural characters, seven types of the male gametes have been proposed for the Eucestoda (Levron et al., 2010). In cross sections, CM are distributed either into two fields located between the two axonemes (Types I and II) or in one field (Types III –VII). Type III spermatozoon exhibits ultrastructural organisation, which is specific exclusively for the Caryophyllidea. According classification of Levron et al. (2010) it includes one field of CM loosly scattered under the plasma membrane of the nucleated region of the spermatozoon in cross sections. In contrast, the present study reveals dual arrangement of CM, i.g. (i) in one field or a semicircle, and (ii) in two fields, or two opposite rows in regions III and IV of the spermatozoa in K. armeniaca. Whereas most caryophyllideans have the CM loosly scattered under the plasma membrane (Gamil, 2008; Bruňanská, 2009; Yoneva et al., 2011, 2012a, b; Bruňanská & Kostič, 2012; Matoušková et al., 2018; Bruňanská et al., 2019), the CM in K. armeniaca are tightly packed. A continuous field of tightly packed CM has been reported also in the spermatozoa of lytocestiid Monobothrioides chalmersius by Arafa and Hamada (2004). On the other hand, two opposite rows of CM have been reported in region II of the spermatozoa of lytocestiid Khawia rossittensis by Matoušková et al. (2019) and caryophyllaeid Glaridacris catostomi by Świderski and Mackiewicz (2002). However, up to date, the simultaneous occurrence of both types of arrangement of CM has never been reported in the spermatozoa of caryophyllidean species. Here, it is worth to note that CM are distributed into two fields situated between the two axonemes in the gyrocotyllidean and amphilinidean cestodes (Xylander 1989, Bruňanská et al. 2012) and in most digeneans (Bakhoum et al. 2017). However, the latter taxa differs from that of any Eucestoda by the presence of a mitochondrion in the spermatozoon.
The location of the maximum number of CM would be an interesting ultrastructural criterion, especially in connection with their presumed role in spermatozoan movements. The maximum number of CM in the spermatozoa within the order Caryophyllidea is not constant (Tab. 1). It occurs in the anterior parts of the spermatozoa most frequently, and varies between 10 and 30 in the Caryophyllaeidae, or 15 – 40 in the Lytocestidae. At the intraspecific level of Khawia, the highest maxim number of the CM (35) has been reported in K. armeniaca (present study), whereas K. rossittensis exhibits maximum 22 CM (Matoušková et al., 2019) and K. sinensis only 15 elements (Bruňanská, 2009).
Glycogen is the major carbohydrate storage form in animals and represents the major energy store of the spermatozoa in the Eucestoda (Euzet et al., 1981). The presence of glycogen was detected using Thiéry method (1967) in the spermatozoa of the Caryophyllidea (Bruňanská, 2009; Yoneva et al., 2011; Bruňanská & Kostič, 2012; Matoušková et al., 2018; Bruňanská et al., 2019; present study), and in male gametes of other cestodes, e.g. Diphyllobothriidea, Bothriocephalidea, Haplobothriidea, Diphyllidea, Trypanorhyncha, Tetraphyllidea, Proteocephalidea, and Tetrabothriidea (Levron et al., 2010). In these cestodes, glycogen is localised in the sperm cytoplasm and it was never found in the sperm axonemes.
The spermatozoa travel on their route from the site of the origin towards the cirrus pouch through various canals of the male reproductive system, including vas deferens. The basic structure of the vasa deferentia of K. armeniaca resembles that in caryophyllidean Atractolytocestus huronensis (Bruňanská et al., 2011), gyrocotylideans or amphilinideans (Rohde & Watson, 1986; Xylander, 1989). Interestingly, vasa deferentia of other Caryophyllidea, Spathebothriidea, Diphyllobothriidea or Proteocephalidea have no cilia (Davydov et al., 1994; Korneva & Davydov, 2001; Poddubnaya, 2002; Poddubnaya et al., 2005).
Cross sections of the mature spermatozoon of K. armeniaca: (a) vas deferens with five different regions of the spermatozoon (I-V); (b) region I presents one axoneme and six cortical microtubules; (c) region II shows cortical microtubules which are arranged in a semi-circle under the plasma membrane; (d-g) region III or nucleated region: small diameter of anterior part of the nucleus (d), which gradually increases (e), reaches a maximum in the middle part (f), and diminishes again more posteriorly (g); (h-j) region IV shows cortical microtubules which are arranged in two opposite rows (note one single microtubule in the middle between the two opposite rows) (h), rapidly reduced volume of cytoplasm and number of cortical microtubules (i, j); (k-l) region V with one axoneme which lost its central structure (k) and undergoes disorganization into doublets (l). Ax, axoneme; Arrowheads, attachment zones; BB, basal body of the cilium; CM, cortical microtubules; D, doublets; L, lamellae; N, nucleus. Scale bars: a = 1000 nm, b-h, l = 200 nm, i = 250 nm, j, k =125 nm.
Fig. 2
Granules of glycogen in the cytoplasm of the mature spermatozoon of K. armeniaca after application of Thiéry method (1967): (a) cross section of region II; (b) cross section of region III; (c) cross section of region IV; (d) longitudinal section of region II. Ax, axoneme; CM, cortical microtubules; G, glycogen; N, nucleus. Scale bars: a-c = 200 nm, d = 250 nm.
Fig. 3
Schematic reconstruction of the mature spermatozoon of K. armeniaca. I–V, five different regions of the mature spermatozoon; ASE, anterior spermatozoon extremity; Ax, axoneme; AZ, attachment zones; CC, central core; CM, cortical microtubules; G, glycogen; N, nucleus; PM, plasma membrane; PSE, posterior spermatozoon extremity.
Variation of the maximum number of cortical microtubules (CM) in the spermatozoa of the Caryophyllidea.
Arafa, S.Z., Hamada, S.F. (2004): Spermatogenesis and sperm ultrastructure of the caryophyllidean cestode, Monobothrioides chalmersius (Woodland, 1924) Hunter, 1930. Egypt. J. Zool., 43: 49 – 70ArafaS.Z.HamadaS.F.2004Spermatogenesis and sperm ultrastructure of the caryophyllidean cestode, Monobothrioides chalmersius (Woodland, 1924) Hunter, 1930Egypt. J. Zool434970Search in Google Scholar
Bakhoum, A.J., Miquel, J., Ndiaye, P.I., Justine, J.L., Falchi, A., BÂ, C.T., Marchand, B., Quilichini, Y. (2017): Advances in spermatological characters in the Digenea: review and proposal of spermatozoa models and their phylogenetic importance. Adv. Parasitol., 98: 111 – 165. DOI: 10.1016/bs.apar.2017.04.001BakhoumA.J.MiquelJ.NdiayeP.I.JustineJ.L.FalchiA.BÂC.T.MarchandB.QuilichiniY.2017Advances in spermatological characters in the Digenea: review and proposal of spermatozoa models and their phylogenetic importanceAdv. Parasitol9811116510.1016/bs.apar.2017.04.00128942768Open DOISearch in Google Scholar
Bruňanská, M. (2009): Spermatological characters of the caryophyllidean cestode Khawia sinensis Hsü, 1935, a carp parasite. Parasitol. Res., 105: 1603 – 1610. DOI: 10.1007/s00436-009-1599-2BruňanskáM.2009Spermatological characters of the caryophyllidean cestode Khawia sinensis Hsü, 1935, a carp parasiteParasitol. Res1051603161010.1007/s00436-009-1599-219727822Open DOISearch in Google Scholar
Bruňanská, M. (2010): Recent insights into spermatozoa development and ultrastructure in the Eucestoda. In: Lejeune, T., Delvaux, P. (Eds) Human spermatozoa: maturation, capacitation and abnormalities. Nova Science Publishers, Inc., New York, pp. 327 – 354BruňanskáM.2010Recent insights into spermatozoa development and ultrastructure in the EucestodaLejeuneT.DelvauxP.Human spermatozoa: maturation, capacitation and abnormalitiesNova Science Publishers, IncNew York327354Search in Google Scholar
Bruňanská, M., Kostič, B. (2012): Revisiting caryophyllidean type of spermiogenesis in the Eucestoda based on spermatozoon differentiation and ultrastructure of Caryophyllaeus laticeps (Pallas, 1781). Parasitol. Res., 110: 141 – 149. DOI: 10.1007/s00436-0112463-8BruňanskáM.KostičB.2012Revisiting caryophyllidean type of spermiogenesis in the Eucestoda based on spermatozoon differentiation and ultrastructure of Caryophyllaeus laticeps (Pallas, 1781)Parasitol. Res11014114910.1007/s00436-0112463-8Open DOISearch in Google Scholar
Bruňanská, M., Matey, V., Nebesářová, J. (2012b): Ultrastructure of the spermatozoon of the diphyllobothriidean cestode Cephalochlamys namaquensis (Cohn, 1906). Parasitol. Res., 111: 1037 – 1043. DOI: 10.1007/s00436-012-2928-4BruňanskáM.MateyV.NebesářováJ.2012bUltrastructure of the spermatozoon of the diphyllobothriidean cestode Cephalochlamys namaquensis (Cohn, 1906)Parasitol. Res1111037104310.1007/s00436-012-2928-422576853Open DOISearch in Google Scholar
Bruňanská, M., Matoušková, M., Nebesářová, J., Mackiewicz, J.S., Poddubnaya L.G. (2019): First ultrastructural and cytochemical data on the spermatozoon and its differentiation in progenetic and adult Archigetes sieboldi Leuckart, 1878 (Cestoda, Caryophyllidea, Caryophyllaeidae). Parasitol. Res., 118: 1205 – 1214. DOI: 10.1007/s00436-019-06276-zBruňanskáM.MatouškováM.NebesářováJ.MackiewiczJ.S.PoddubnayaL.G.2019First ultrastructural and cytochemical data on the spermatozoon and its differentiation in progenetic and adult Archigetes sieboldi Leuckart, 1878 (Cestoda, Caryophyllidea, Caryophyllaeidae)Parasitol. Res1181205121410.1007/s00436-019-06276-z30847613Open DOISearch in Google Scholar
Bruňanská, M., Nebesářová, J., Oros, M. (2011): Ultrastructural aspects of spermatogenesis, testes, and vas deferens in the parthenogenetic tapeworm Atractolytocestus huronensis Anthony, 1958 (Cestoda: Caryophyllidea), a carp parasite from Slovakia. Parasitol. Res., 108: 61 – 68. DOI: 10.1007/s00436-010-2038-0BruňanskáM.NebesářováJ.OrosM.2011Ultrastructural aspects of spermatogenesis, testes, and vas deferens in the parthenogenetic tapeworm Atractolytocestus huronensis Anthony, 1958 (Cestoda: Caryophyllidea), a carp parasite from SlovakiaParasitol. Res108616810.1007/s00436-010-2038-020838810Open DOISearch in Google Scholar
Bruňanská, M., Poddubnaya, L.G. (2006): Spermiogenesis in the caryophyllidean cestode Khawia armeniaca (Cholodkovski, 1915). Parasitol. Res., 99: 449 – 454. DOI: 10.1007/s00436-006-0155-6BruňanskáM.PoddubnayaL.G.2006Spermiogenesis in the caryophyllidean cestode Khawia armeniaca (Cholodkovski, 1915)Parasitol. Res9944945410.1007/s00436-006-0155-616602020Open DOISearch in Google Scholar
Bruňanská, M., Poddubnaya, L.G. (2010): Spermatological characters of the spathebothriidean tapeworm Didymobothrium rudolphii (Monticelli, 1890). Parasitol. Res., 106: 1435 – 1442. DOI: 10.1007/s00436-010-1822-1BruňanskáM.PoddubnayaL.G.2010Spermatological characters of the spathebothriidean tapeworm Didymobothrium rudolphii (Monticelli, 1890)Parasitol. Res1061435144210.1007/s00436-010-1822-120352452Open DOISearch in Google Scholar
Bruňanská, M., Poddubnaya, L.G., Xylander, W.E.R. (2012a): Spermatozoon cytoarchitecture of Amphilina foliacea (Platyhelminthes, Amphilinidea). Parasitol. Res., 111: 2063 – 2069. DOI: 10.1007/s00436-012-3053-0BruňanskáM.PoddubnayaL.G.XylanderW.E.R.2012aSpermatozoon cytoarchitecture of Amphilina foliacea (Platyhelminthes, Amphilinidea)Parasitol. Res1112063206910.1007/s00436-012-3053-022932939Open DOISearch in Google Scholar
Bruňanská, M., Scholz, T., Dezfuli, B.S., Poddubnaya, L.G. (2006): Spermiogenesis and sperm ultrastructure of Cyathocephalus truncatus (Pallas, 1781) Kessler, 1868 (Cestoda: Spathebothriidea). J. Parasitol., 92: 884 – 892. DOI: 10.1645/GE-718R1.1BruňanskáM.ScholzT.DezfuliB.S.PoddubnayaL.G.2006Spermiogenesis and sperm ultrastructure of Cyathocephalus truncatus (Pallas, 1781) Kessler, 1868 (Cestoda: Spathebothriidea)J. Parasitol9288489210.1645/GE-718R1.117152927Open DOISearch in Google Scholar
Caira, J.N., Littlewood, D.T.J. (2013): Worms, platyhelminthes. In Levin, S.A. (Ed) Encyclopedia of Biodiversity, vol. 7, 2nd edn. Waltham, MA: Academic Press, pp. 863 – 899CairaJ.N.LittlewoodD.T.J.2013Worms, platyhelminthesLevinS.A.EdEncyclopedia of Biodiversity, vol. 7, 2nd ednWaltham, MAAcademic Press86389910.1016/B978-0-12-384719-5.00166-0Search in Google Scholar
Davydov, V.G., Poddubnaya, L.G., Kolesnikova, G.A. (1994): Ultrastructure of genital system ducts of Caryophyllaeus laticeps (Cestoda, Caryophyllidea). Parazitologiya, 28: 501 – 509 (In Russian)DavydovV.G.PoddubnayaL.G.KolesnikovaG.A.1994Ultrastructure of genital system ducts of Caryophyllaeus laticeps (Cestoda, Caryophyllidea)Parazitologiya28501509(In Russian)Search in Google Scholar
Ehlers, U. (1984): Phylogenetishes System der Platyhelminthes [Phylogenetic System of the Platyhelminthes]. Verh. Naturwiss. Ver. Hamb. 27: 291 – 294 (In German)EhlersU.1984Phylogenetishes System der Platyhelminthes [Phylogenetic System of the Platyhelminthes]Verh. Naturwiss. Ver. Hamb27291294In GermanSearch in Google Scholar
Euzet, L., Świderski, Z., Mokhtar-Maamouri, F. (1981): Ultrastructure comparée du spermatozoïde des cestodes. Relations avec la phylogenèse [The comparative ultrastructure of the spermatozoa in cestodes and its phylogenetic implications]. Ann. Parasitol. Hum. Comp., 56: 247 – 259. DOI: 10.1051/parasite/1981563247 (In French)EuzetL.ŚwiderskiZ.Mokhtar-MaamouriF.1981Ultrastructure comparée du spermatozoïde des cestodes. Relations avec la phylogenèse [The comparative ultrastructure of the spermatozoa in cestodes and its phylogenetic implications]Ann. Parasitol. Hum. Comp5624725910.1051/parasite/1981563247In FrenchOpen DOISearch in Google Scholar
Gamil, I.S. (2008): Ultrastructural studies of the spermatogenesis and spermiogenesis of the caryophyllidean cestode Wenyonia virilis (Woodland,1923). Parasitol. Res., 103: 777 – 785. DOI: 10.1007/s00436-008-1040-2GamilI.S.2008Ultrastructural studies of the spermatogenesis and spermiogenesis of the caryophyllidean cestode Wenyonia virilis (Woodland,1923)Parasitol. Res10377778510.1007/s00436-008-1040-218568447Open DOISearch in Google Scholar
Justine, J.-L. (1998): Spermatozoa as phylogenetic characters for the Eucestoda. J. Parasitol., 84: 385 – 408. DOI: 10.2307/3284502JustineJ.-L.1998Spermatozoa as phylogenetic characters for the EucestodaJ. Parasitol8438540810.2307/3284502Open DOISearch in Google Scholar
Justine, J.-L. (2001): Spermatozoa as phylogenetic characters for the Platyhelminthes. In: Littlewood D.T.J., Bray R.A. (Eds) Interrelationships of the Platyhelminthes. Taylor and Francis, London, pp. 231 – 238JustineJ.-L.2001Spermatozoa as phylogenetic characters for the PlatyhelminthesLittlewoodD.T.J.BrayR.A.Interrelationships of the PlatyhelminthesTaylor and FrancisLondon231238Search in Google Scholar
Justine, J.-L. (2003): Ultrastructure des spermatozoïdes et phylogénie des Neodermata [Ultrastructure of the spermatozoa and phylogeny of Neodermata]. In Combes, C., Jourdane, J. (Eds) Taxonomie, Ecologie et Evolution des Métazoaires Parasites [Taxonomy, Ecology and Evolution of Metazoan Parasites]. Tome I, PUP, Perpignan, France, pp. 359 – 380 (In French)JustineJ.-L.2003Ultrastructure des spermatozoïdes et phylogénie des Neodermata [Ultrastructure of the spermatozoa and phylogeny of Neodermata]CombesC.JourdaneJ.Taxonomie, Ecologie et Evolution des Métazoaires Parasites [Taxonomy, Ecology and Evolution of Metazoan Parasites]. Tome IPUP, PerpignanFrance359380In FrenchSearch in Google Scholar
Korneva, Z.V., Davydov, V.G. (2001): Ultrastructure of the male reproductive system in three proteocephalidean cestodes. Zool. Zh., 80: 921 – 928 (In Russian)KornevaZ.V.DavydovV.G.2001Ultrastructure of the male reproductive system in three proteocephalidean cestodesZool. Zh80921928(In Russian)Search in Google Scholar
Levron, C., Bruňanská, M., Kuchta, R., Freeman, M., Scholz, T. (2006b): Spermatozoon ultrastructure of the pseudophyllidean cestode Paraechinophallus japonicus, a parasite of deep-sea fish Psenopsis anomala (Perciformes, Centrolophidae). Parasitol. Res., 100: 115 –121. DOI: 10.1007/s00436-006-0224-xLevronC.BruňanskáM.KuchtaR.FreemanM.ScholzT.2006bSpermatozoon ultrastructure of the pseudophyllidean cestode Paraechinophallus japonicus, a parasite of deep-sea fish Psenopsis anomala (Perciformes, Centrolophidae)Parasitol. Res10011512110.1007/s00436-006-0224-x16835797Open DOISearch in Google Scholar
Levron, C., Bruňanská, M., Poddubnaya, L.G. (2006a): Spermatological characters of the pseudophyllidean cestode Bothriocephalus scorpii (Müller, 1776). Parasitol. Int., 55: 113 –120. DOI: 10.1016/j.parint.2005.11.055LevronC.BruňanskáM.PoddubnayaL.G.2006aSpermatological characters of the pseudophyllidean cestode Bothriocephalus scorpii (Müller, 1776)Parasitol. Int5511312010.1016/j.parint.2005.11.05516387529Open DOISearch in Google Scholar
Levron, C., Bruňanská, M., Poddubnaya, L.G. (2006c): Spermatological characters in Diphyllobothrium latum (Cestoda, Pseudophyllidea). J. Morphol., 267: 1110 – 1119. DOI: 10.1002/jmor.10460LevronC.BruňanskáM.PoddubnayaL.G.2006cSpermatological characters in Diphyllobothrium latum (Cestoda, Pseudophyllidea)J. Morphol2671110111910.1002/jmor.1046016752406Open DOISearch in Google Scholar
Levron, C., Miquel, J., Oros, M., Scholz, T. (2010): Spermatozoa of tapeworms (Platyhelminthes, Eucestoda): advances in ultrastructural and phylogenetic studies. Biol. Rev., 85: 523 – 543. DOI: 10.1111/j.1469-185X.2009.00114.xLevronC.MiquelJ.OrosM.ScholzT.2010Spermatozoa of tapeworms (Platyhelminthes, Eucestoda): advances in ultrastructural and phylogenetic studiesBiol. Rev8552354310.1111/j.1469-185X.2009.00114.x20015312Open DOISearch in Google Scholar
Levron, C., Sitko, J., Scholz, T. (2009): Spermiogenesis and spermatozoon of the tapeworm Ligula intestinalis (Diphyllobothriidae): phylogenetic implications. J. Parasitol., 95: 1 – 9. DOI: 10.1645/GE-1646.1LevronC.SitkoJ.ScholzT.2009Spermiogenesis and spermatozoon of the tapeworm Ligula intestinalis (Diphyllobothriidae): phylogenetic implicationsJ. Parasitol951910.1645/GE-1646.118576887Open DOISearch in Google Scholar
Levron, C., Yoneva, A., Kalbe, M. (2013): Spermatological characters in the diphyllobothriidean Schistocephalus solidus (Cestoda). Acta. Zool., 94: 240 – 247. DOI: 10.1111/j.1463-6395.2011.00549.xLevronC.YonevaA.KalbeM.2013Spermatological characters in the diphyllobothriidean Schistocephalus solidus (Cestoda)Acta. Zool9424024710.1111/j.1463-6395.2011.00549.xOpen DOISearch in Google Scholar
Marigo, A.M., Świderski, Z., BÂ, C.T., Miquel, J. (2011): Spermiogenesis and ultrastructure of the spermatozoon of the trypanorhynch cestode Aporhynchus menezesi (Aporhynchidae), a parasite of the velvet belly lanternshark Etmopterus spinax (Elasmobranchii: Etmopteridae). Folia Parasitol., 58: 69 – 78. DOI: 10.14411/fp.2011.007MarigoA.M.ŚwiderskiZ.BÂC.T.MiquelJ.2011Spermiogenesis and ultrastructure of the spermatozoon of the trypanorhynch cestode Aporhynchus menezesi (Aporhynchidae), a parasite of the velvet belly lanternshark Etmopterus spinax (Elasmobranchii: Etmopteridae)Folia Parasitol58697810.14411/fp.2011.007Open DOISearch in Google Scholar
Matoušková, M., Bílý, T., Bruňanská, M., Mackiewicz, J.S., Nebesářová, J. (2018): Ultrastructure, cytochemistry and electron tomography analysis of Caryophyllaeides fennica (Schneider, 1902) (Cestoda: Lytocestidae) reveals novel spermatology characteristics in the Eucestoda. Parasitol. Res., 117: 3091 – 3102. DOI: 10.1007/s00436-018-6001-9MatouškováM.BílýT.BruňanskáM.MackiewiczJ.S.NebesářováJ.2018Ultrastructure, cytochemistry and electron tomography analysis of Caryophyllaeides fennica (Schneider, 1902) (Cestoda: Lytocestidae) reveals novel spermatology characteristics in the EucestodaParasitol. Res1173091310210.1007/s00436-018-6001-9Open DOISearch in Google Scholar
Matoušková, M., Bílý, T., Bruňanská, M., Oros, M., Kostič, B., Nebesářová, J. (2019): New data on spermiogenesis and trepaxonematan axoneme in basal tapeworms (Cestoda, Caryophyllidea, Lytocestidae) parasitizing cyprinid fishes. Sci. Rep., 9: 1 – 11. DOI: 10.1038/s41598-019-49312-9MatouškováM.BílýT.BruňanskáM.OrosM.KostičB.NebesářováJ.2019New data on spermiogenesis and trepaxonematan axoneme in basal tapeworms (Cestoda, Caryophyllidea, Lytocestidae) parasitizing cyprinid fishesSci. Rep911110.1038/s41598-019-49312-9Open DOISearch in Google Scholar
Miquel, J., Świderski, Z., Neifar, L., Eira, C. (2007): Ultrastructure of the spermatozoon of Parachristianella trygonis Dollfus, 1946 (Trypanorhyncha, Eutetrarhynchidae). J. Parasitol., 93: 1296 – 1302. DOI: 10.1645/GE-1193.1MiquelJ.ŚwiderskiZ.NeifarL.EiraC.2007Ultrastructure of the spermatozoon of Parachristianella trygonis Dollfus, 1946 (Trypanorhyncha, Eutetrarhynchidae)J. Parasitol931296130210.1645/GE-1193.1Open DOISearch in Google Scholar
Miquel, J., Świderski, Z. (2006): Ultrastructure of the spermatozoon of Dollfusiella spinulifera (Beveridge and Jones, 2000) Beveridge, Neifar and Euzet, 2004 (Trypanorhyncha, Eutetrarhynchidae). Parasitol. Res., 99: 37 – 44. DOI: 10.1007/s00436-005-0094-7MiquelJ.ŚwiderskiZ.2006Ultrastructure of the spermatozoon of Dollfusiella spinulifera (Beveridge and Jones, 2000) Beveridge, Neifar and Euzet, 2004 (Trypanorhyncha, Eutetrarhynchidae)Parasitol. Res99374410.1007/s00436-005-0094-7Open DOISearch in Google Scholar
Poddubnaya, L.G. (2002): Ultrastructure of the male reproductive ducts in Diphyllobothrium latum (Cestoda, Pseudophyllidea). Zool. Zh., 81: 394 – 405 (In Russian)PoddubnayaL.G.2002Ultrastructure of the male reproductive ducts in Diphyllobothrium latum (Cestoda, Pseudophyllidea)Zool. Zh81394405(In Russian)Search in Google Scholar
Poddubnaya, L.G., Mackiewicz, J.S., Bruňanská, M., Dezfuli, B.S. (2005): Fine structure of the male reproductive ducts, vagina and seminal receptacle of Cyathocephalus truncatus (Cestoda: Spathebothriidea). Folia Parasitol., 52: 241 – 250. DOI: 10.14411/fp.2005.032PoddubnayaL.G.MackiewiczJ.S.BruňanskáM.DezfuliB.S.2005Fine structure of the male reproductive ducts, vagina and seminal receptacle of Cyathocephalus truncatus (Cestoda: Spathebothriidea)Folia Parasitol5224125010.14411/fp.2005.032Open DOISearch in Google Scholar
Rohde, K., Watson, N. (1986): Ultrastructure of spermiogenesis and sperm of Austramphilina elongata (Platyhelminthes, Amphilinidea). J. Submicrosc. Cytol., 18: 361 – 374RohdeK.WatsonN.1986Ultrastructure of spermiogenesis and sperm of Austramphilina elongata (Platyhelminthes, Amphilinidea)J. Submicrosc. Cytol18361374Search in Google Scholar
Scholz, T., Brabec, J., Kráľová-Hromadová, I., Oros, M., BazsalovicsovÁ E., Ermolenko, A., HanzelovÁ, V. (2011): Revision of Khawia spp. (Cestoda: Caryophyllidea), parasites of cyprinid fish, including a key to their identification and molecular phylogeny. Folia Parasitol., 58: 197 – 223ScholzT.BrabecJ.Kráľová-HromadováI.OrosM.BazsalovicsovÁE.ErmolenkoA.HanzelovÁV.2011Revision of Khawia spp. (Cestoda: Caryophyllidea), parasites of cyprinid fish, including a key to their identification and molecular phylogenyFolia Parasitol5819722310.14411/fp.2011.020Search in Google Scholar
Scholz, T., Oros, M. (2017): Caryophyllidea van Beneden in Carus, 1863. In Caira, J. N., Jensen, K. (Eds) Planetary Biodiversity Inventory (2008 – 2017): Tapeworms from Vertebrate Bowels of the Earth. University of Kansas, Natural History Museum, Special Publication No. 25, Lawrence, KS, USA, pp. 47 – 64ScholzT.OrosM.2017Caryophyllidea van Beneden in Carus, 1863CairaJ. N.JensenK.Planetary Biodiversity Inventory (2008 – 2017): Tapeworms from Vertebrate Bowels of the EarthUniversity of Kansas, Natural History Museum, Special Publication No. 25Lawrence, KS, USA4764Search in Google Scholar
Świderski, Z., Mackiewicz, J.S. (2002): Ultrastructure of spermatogenesis and spermatozoa of the caryophyllidean cestode Glaridacris catostomi Cooper, 1920. Acta Parasitol., 47: 83 – 104. DOI: 10.2478/s11686-008-0013-zŚwiderskiZ.MackiewiczJ.S.2002Ultrastructure of spermatogenesis and spermatozoa of the caryophyllidean cestode Glaridacris catostomi Cooper, 1920Acta Parasitol478310410.2478/s11686-008-0013-zOpen DOISearch in Google Scholar
Thiéry, J.P. (1967): Mise en évidence des polysaccharides sur coupes fines en microscopie électronique [The localisation of polysaccharides in fine sections using electron microscopy]. J. Microsc. (Paris), 6: 987 – 1018 (In French)ThiéryJ.P.1967Mise en évidence des polysaccharides sur coupes fines en microscopie électronique [The localisation of polysaccharides in fine sections using electron microscopy]J. Microsc. (Paris)69871018In FrenchSearch in Google Scholar
Waeschenbach, A., Webster, B.L., Littlewood, D.T.J. (2012): Adding resolution to ordinal level relationships of tapeworms (Platyhelminthes: Cestoda) with large fragments of mtDNA. Mol. Phylogenet. Evol., 63: 834 – 847. DOI: 10.1016/j.ympev.2012.02.020WaeschenbachA.WebsterB.L.LittlewoodD.T.J.2012Adding resolution to ordinal level relationships of tapeworms (Platyhelminthes: Cestoda) with large fragments of mtDNAMol. Phylogenet. Evol6383484710.1016/j.ympev.2012.02.020Open DOISearch in Google Scholar
Xylander, W.E.R. (1989): Ultrastructural studies on the reproductive system of Gyrocotylidea and Amphilinidea (Cestoda): spermatogenesis, spermatozoa, testes and vas deferens of Gyrocotyle. Int. J. Parasitol., 19: 897 – 905. DOI: 10.1016/00207519(89)90117-3XylanderW.E.R.1989Ultrastructural studies on the reproductive system of Gyrocotylidea and Amphilinidea (Cestoda): spermatogenesis, spermatozoa, testes and vas deferens of GyrocotyleInt. J. Parasitol1989790510.1016/00207519(89)90117-3Open DOISearch in Google Scholar
Yoneva, A., Levron, C., Ash, A., Scholz, T. (2012b): Spermatological characters of monozoic tapeworms (Cestoda: Caryophyllidea), including first data on a species from the Indomalayan catfish. J. Parasitol., 98: 423 – 430. DOI: 10.1645/GE-2794.1YonevaA.LevronC.AshA.ScholzT.2012bSpermatological characters of monozoic tapeworms (Cestoda: Caryophyllidea), including first data on a species from the Indomalayan catfishJ. Parasitol9842343010.1645/GE-2794.122010629Open DOISearch in Google Scholar
Yoneva, A., Levron, C., Oros, M., Orosová, M., Scholz, T. (2011): Ultrastructure of spermiogenesis and mature spermatozoon of Breviscolex orientalis (Cestoda: Caryophyllidea). Parasitol. Res., 108: 997 – 1005. DOI: 10.1007/s00436-010-2144-zYonevaA.LevronC.OrosM.OrosováM.ScholzT.2011Ultrastructure of spermiogenesis and mature spermatozoon of Breviscolex orientalis (Cestoda: Caryophyllidea)Parasitol. Res108997100510.1007/s00436-010-2144-z21085990Open DOISearch in Google Scholar
Yoneva, A., Levron, C., Oros, M., Orosová, M., Scholz, T. (2012a): Spermiogenesis and spermatozoon ultrastructure of Hunterella nodulosa (Cestoda: Caryophyllidea), a monozoic parasite of suckers (Catostomidae) in North America. Folia Parasitol., 59: 179 – 186. DOI: 10.14411/fp.2012.025YonevaA.LevronC.OrosM.OrosováM.ScholzT.2012aSpermiogenesis and spermatozoon ultrastructure of Hunterella nodulosa (Cestoda: Caryophyllidea), a monozoic parasite of suckers (Catostomidae) in North AmericaFolia Parasitol5917918610.14411/fp.2012.02523136798Open DOISearch in Google Scholar
