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J. Parasitol., 93(3), 2007, pp. 545–552� American Society of Parasitologists 2007

MESOCESTOIDES LINEATUS (GOEZE, 1782) (MESOCESTOIDIDAE):NEW DATA ON SPERM ULTRASTRUCTURE

Jordi Miquel, Catarina Eira, Zdzisław Swiderski*†, and David Bruce Conn‡Laboratori de Parasitologia, Departament de Microbiologia i Parasitologia Sanitaries, Facultat de Farmacia, Universitat de Barcelona,Av. Joan XXIII, sn, 08028 Barcelona, Spain. e-mail: jordimiquel@ub.edu

ABSTRACT: Spermiogenesis and the ultrastructural characters of the spermatozoon of Mesocestoides lineatus are described bymeans of transmission electron microscopy, including cytochemical analysis for glycogen. Materials were obtained from a goldenhamster (Mesocricetus auratus) after experimental infection with tetrathyridia metacestodes obtained from naturally infectedlizards (Anolis carolinensis) from Louisiana. Spermiogenesis in M. lineatus is characterized by the orthogonal growth of a freeflagellum, a flagellar rotation, and a proximodistal fusion. The zone of differentiation contains 2 centrioles associated with striatedrootlets and a reduced intercentriolar body. The mature spermatozoon of M. lineatus lacks a mitochondrion, and it is characterizedby the presence of (1) a single, spiraled, crested body 150 nm thick; (2) a single axoneme of the 9�‘1’ pattern of trepaxonematanPlatyhelminthes; (3) a parallel and reduced row of submembranous cortical microtubules; (4) a spiraled cordon of glycogengranules; and (5) a spiraled nucleus encircling the axoneme.

Mesocestoididae Perrier, 1897, comprises only 2 genera, Me-socestoides Vaillant, 1863, and Mesogyna Voge, 1952; speciesin the family are distinguished by the presence or absence ofthe paruterine organ. According to several researchers (seeRausch, 1994), the discrimination of higher taxa based on thischaracter is inconsistent, and consequently, the inclusion of the2 genera in the same family is controversial. Moreover, theinclusion of Mesocestoididae in Cyclophyllidea constitutes an-other contentious issue (see Rausch, 1994). The representativesof that family differ from the rest of cyclophyllideans in severalmorpho-anatomical aspects, such as the median ventral locationof the genital atrium in both Mesocestoides and Mesogyna gen-era and the bipartite vitelline gland in species of Mesocestoides.The presumed 3-host life cycles of Mesocestoides spp. differfrom all the cyclophyllideans. Supplementary molecular and ul-trastructural data (Mariaux, 1998; Miquel et al., 1999) also con-tribute to the complexity of the present systematic status of thisfamily. With respect to the ultrastructural data, spermiogenesisand the spermatozoon of Mesocestoides litteratus (Miquel etal., 1999) revealed the presence of striated rootlets, an inter-centriolar body, flagellar rotation during spermiogenesis, andthe reduced row of parallel cortical microtubules in the maturespermatozoon, among other characters. These spermatologicalcharacters contrast with those exhibited by the rest of cyclo-phyllideans (Justine, 1998, 2001; Miquel et al., 1999).

Here, we present the ultrastructural results on spermiogenesisand the mature spermatozoon of Mesocestoides lineatus to cor-roborate the observations made in the previous study of thecongeneric M. litteratus and thus increase the ultrastructuraldata for the future establishment of a general pattern of spermorganization in the Mesocestoididae.

MATERIALS AND METHODSLive tetrathyridia metacestode stages of Mesocestoides lineatus (Goeze,

1782) were obtained from the body cavity of naturally infected lizards,Anolis carolinensis, from Louisiana (see Conn and Etges, 1984). Tetra-

Received 21 July 2006; revised 4 October 2006; accepted 27 Novem-ber 2006.

* W. Stefanski Institute of Parasitology, Polish Academy of Sciences,Warsaw, Poland.

† Department of General Biology and Parasitology, Warsaw MedicalUniversity, Warsaw, Poland.

‡ School of Mathematical and Natural Sciences, Berry College, MountBerry, Georgia 30149-5036.

thyridia were fed by oral pipette to laboratory-reared golden hamsters,Mesocricetus auratus, in which adults developed within 42 days ofexposure. Gravid adult tapeworms were removed from the necropsiedhosts’ small intestines, rinsed briefly in Earle’s balanced salt solution atroom temperature, and immersed whole in 3% glutaraldehyde in sodiumcacodylate buffer (pH 7.4, 375 mOsm/L) at 4 C. Strobilae were slicedinto 1- to 3-mm sections in the fixative, and fixed for 8 hr. They werethen rinsed in buffer and postfixed in 1% osmium tetroxide in caco-dylate buffer for 2 hr. Tissue was then dehydrated in an ascendingaqueous series of ethanol, infiltrated with propylene oxide, embeddedin epoxy on a rotation mixer at room temperature, and polymerized at60 C. Ultrathin sections (50 nm) were cut in a Reichert–Jung UltracutE ultramicrotome, placed on copper grids, and then double-stained withuranyl acetate and lead citrate according to Reynolds (1963).

The Thiery (1967) technique was applied for detection of glycogenat the ultrastructural level. Ultrathin sections of M. lineatus collectedon gold grids were treated with periodic acid, thiocarbohydrazide andsilver proteinate (PA-TCH-SP) during 6-step reactions conducted as fol-lows: (1) 30 min in 10% PA, (2) rinsed in distilled water, (3) 24 hr inTCH, (4) rinsed in acetic solutions and distilled water, (5) 30 min in1% SP in the dark, and (6) rinsed in distilled water.

Grids were examined under a JEOL (Tokyo, Japan) 1010 transmis-sion electron microscope (TEM) at the ‘‘Serveis Cientificotecnics’’ ofthe University of Barcelona, Spain. Voucher specimens of the adultworms were deposited in the Museum of Comparative Zoology, Har-vard University (Cambridge, Massachusetts), and the U.S. National Par-asite Collection (Beltsville, Maryland).

RESULTS

Spermiogenesis in Mesocestoides lineatus is illustrated inFigures 1–13 and 30A–E. The process begins with the forma-tion of a zone of differentiation bordered by cortical microtu-bules (Fig. 1). This zone contains 2 centrioles separated by anintercentriolar body. The intercentriolar body is composed of 3electron-dense layers; the thickest is the central one. These 3layers are separated by 2 electron-lucent layers (Figs. 1, 6).Each centriole is associated with a striated rootlet (Figs. 2, 3).In an initial stage of spermiogenesis, one of the centrioles formsa free flagellum that grows externally and orthogonally to acytoplasmic extension (Fig. 4). The other centriole remains ori-ented in its orthogonal position in a cytoplasmic bud (Figs. 1,2) and aborts after the proximodistal fusion between the freeflagellum and the cytoplasmic extension (Fig. 8). This proxi-modistal fusion occurs after flagellar rotation, which allows thefree flagellum to become parallel to the cytoplasmic extension(Figs. 5–7). Later, the nucleus enlarges, moves across the ringof arched membranes, and initiates its migration along the sper-matid body (Fig. 9). It is interesting to note that the striated

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FIGURES 1–7. Spermiogenesis in Mesocestoides lineatus. (1) Zone of differentiation showing the centrioles (C) and the intercentriolar body(Ib). B, cytoplasmic bud; Cm, cortical microtubules; N, nucleus. Bar � 0.5 �m. (2) Detail of the abortive centriole orthogonally positioned in acytoplasmic bud (B). Sr, striated rootlets. Bar � 0.5 �m. (3) Cross-section of a differentiation zone showing the 2 striated rootlets (Sr). Cm,cortical microtubules. Bar � 0.5 �m. (4) Longitudinal section of a differentiation zone showing the formation of the free flagellum (F). Am,arched membranes; Ce, cytoplasmic extension, N, nucleus. Bar � 1 �m. (5) Longitudinal section of a differentiation zone during flagellar rotation.Am, arched membranes; Ce, cytoplasmic extension; F, flagellum; N, nucleus; Sr, striated rootlets. Bar � 0.3 �m. (6) Longitudinal section of adifferentiation zone after the flagellar rotation of the flagellum (F). Am, arched membranes; Ce, cytoplasmic extension; Ib, intercentriolar body;Sr, striated rootlets. Bar � 1 �m. (7) Cross-sections showing free flagella (F) and cytoplasmic extensions (Ce) before proximodistal fusion. Cm,cortical microtubules. Bar � 0.3 �m.

MIQUEL ET AL.—SPERM ULTRASTRUCTURE OF M. LINEATUS 547

FIGURES 8–13. Spermiogenesis in Mesocestoides lineatus. (8) Longitudinal section of a differentiation zone after the proximodistal fusion.Am, arched membranes; Ax, axoneme; N, nucleus; Sr, striated rootlets. Bar � 1 �m. (9) Longitudinal section of a differentiation zone during thenuclear migration. Am, arched membranes; N, nucleus; Sr, striated rootlets. Bar � 1 �m. (10) Cross-sections of spermatids during the nuclearmigration. N, nucleus. Bar � 0.5 �m. (11) Longitudinal section of a differentiation zone in final stages of nuclear migration. Note the disorga-nization of the striated rootlets (arrowhead). Am, arched membranes; N, nucleus. Bar � 1 �m. (12) Cross-section of a spermatid in final stage ofspermiogenesis. Am, arched membranes; Cb, crested body. Bar � 0.3 �m. (13) Longitudinal section of a differentiation zone in final stage ofspermiogenesis after the formation of the crested body (Cb). Note the disorganization of striated rootlets (arrowheads). Am, arched membranes.Bar � 1 �m.

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→

FIGURES 14–28. Spermatozoon of Mesocestoides lineatus. (14) Longitudinal section of region I showing the presence of a single, crested body(Cb). Bar � 1 �m. (15) Detail of region I near the anterior spermatozoon extremity. Ac, apical cone; C, centriole. Bar � 0,5 �m. (16) Cross-section of region I. Cb, crested body. Bar � 0.2 �m. (17) Cross-sections of region I showing the row of 6 parallel cortical microtubules (Cm).Cb, crested body. Bar � 0.2 �m. (18) Thiery’s cytochemical test showing glycogen granules in a cross-section through region II. Bar � 0.2 �m.(19) Longitudinal section through region II showing the spiraled arrangement of glycogen (arrowheads). Bar � 1 �m. (20) Cross-sections ofregion II showing glycogen (G) located between the row of parallel cortical microtubules (Cm) and the axoneme. Bar � 0.2 �m. (21) Cross-section of region III showing the roughly annular shaped nucleus (N). Cm, cortical microtubules; G, glycogen. Bar � 0.2 �m. (22) Longitudinalsection showing the transition between regions II and III. G, glycogen; N, nucleus. Bar � 1 �m. (23) Longitudinal section of region III. G,glycogen; N, nucleus. Bar � 1 �m. (24) Cross-section of region III showing the circular-shaped nucleus (N). Cm, cortical microtubules; G,glycogen. Bar � 0.2 �m. (25) Longitudinal section showing the transition between regions III and IV. Ax, axoneme; N, nucleus. Bar � 0.5 �m.(26) Cross-section of the region IV. Notice the absence of cortical microtubules around the axoneme. Bar � 0.2 �m. (27) Cross-section of regionIV showing the disorganization of the axoneme. D, doublets. Bar � 0.2 �m. (28) Cross-section of the region IV near the posterior spermatozoonextremity showing the presence of singlets (S) and glycogen (G). Bar � 0.2 �m. (29) Longitudinal section of region IV showing the posteriorspermatozoon extremity (Pse). D, doublets; G, glycogen; S, singlets. Bar � 0.5 �m.

rootlet is still present at the base of the incorporated axoneme(Fig. 9). During nuclear migration, the nucleus encircles theaxoneme, and the spermatid increases in section diameter (Fig.10). Moreover, at this point of spermiogenesis, the striated root-lets degenerate at the base of the spermatid (Fig. 11). Thesedegenerated structures remain through the final stages of sper-miogenesis, when it is already possible to observe the crestedbody (Figs. 12, 13).

The ultrastructural organization of the mature spermatozoonof M. lineatus is illustrated in Figures 14–29 and 31I–IV. Themale gamete is a filiform cell that lacks a mitochondrion. It ismainly characterized by the presence of (1) a single, spiraled,crested body; (2) a single axoneme of the 9�‘1’ pattern ofTrepaxonemata; (3) a parallel and reduced row of submem-branous cortical microtubules; and (4) a spiraled band of gly-cogen granules. In accordance with the observation of numer-ous longitudinal and cross-sections, it is possible to establish 4regions with distinctive ultrastructural characters.

Region I (Figs. 14–17) constitutes the anterior extremity ofthe spermatozoon, which is capped by an electron-dense apicalcone (Fig. 15). This region is characterized by the presence ofa single, spiraled, crested body (Figs. 14–17), with a maximumthickness of 150 nm. The submembranous layer of cortical mi-crotubules occurs in a row of 6 parallel, densely packed micro-tubules (Figs. 16, 17).

Region II (Figs. 18–20, 22) is characterized by the presenceof glycogen granules between the parallel row of submembran-ous cortical microtubules and the axoneme of the 9�‘1’ patternof trepaxonematan Platyhelminthes (Fig. 20). This layer of gly-cogen granules forms a longitudinal band, which is coiledaround the axoneme (Figs. 18–20, 22).

Region III (Figs. 21–25) is the nuclear region of the sper-matozoon where the nucleus is spiraled around the axoneme(Figs. 21–23) and cross-sections show that the nucleus variesfrom circular to annular shape (Figs. 21, 24). Granules of gly-cogen are also present (Figs. 21–24).

Region IV (Figs. 25–29) constitutes the postnuclear area andposterior extremity of the spermatozoon. In this region, it isalso possible to see the axoneme (Figs. 25, 26), which pro-gressively disorganizes toward the end of the cell. Indeed, thecentral core disappears (Fig. 27), and the doublets become dis-located and transform into singlets (Fig. 28). The posterior sper-matozoon tip contains granules of glycogen (Figs. 28, 29).

DISCUSSION

Some of the characters described in an earlier precursive re-port on Mesocestoides lineatus by Conn (2001) differ fromthose observed in the present study, particularly with respect tothe presence of striated rootlets in the differentiation zone, fla-gellar rotation, and proximodistal fusion. In the present study,it was possible to perform a more extensive examination of thesame material analyzed by Conn (2001), thus resolving theseaspects. Spermiogenesis in M. lineatus is, in fact, similar to thatdescribed in M. litteratus by Miquel et al. (1999) and corre-sponds to the ‘‘caryophyllidean type’’ proposed by Swiderski(1986) for Glaridacris catostomi or to Type II in the classifi-cation established by Ba and Marchand (1995). This pattern ischaracterized by the orthogonal growth of a single free flagel-lum, followed by flagellar rotation, and proximodistal fusionwith the cytoplasmic extension. Type II spermiogenesis hasbeen described in species of cestodes that belong to the Cary-ophyllidea, Diphyllidea, Tetraphyllidea (Phyllobothriidae), Te-trabothriidea, and in the congeneric M. litteratus (Cyclophyllid-ea) (Mokhtar–Maamouri, 1979; Azzouz–Draoui and Mokhtar–Maamouri, 1986/88; Swiderski, 1986; Stoitsova et al., 1995;Miquel et al., 1999; Swiderski and Mackiewicz, 2002; Brun-anska and Poddubnaya, 2006).

The intercentriolar body in the differentiation zone of M. li-neatus has a much reduced number of layers, similar to thecase of M. litteratus (Miquel et al., 1999). In spite of this re-duction common for both Mesocestoides spp., the most impor-tant difference between these 2 species is that the intercentriolarbody of M. lineatus is composed of 3 electron-dense plates,whereas in M. litteratus, it was described as formed by a singleplate (Miquel et al., 1999). The morphology of the intercentrio-lar body in the representatives of this family probably corre-sponds to an intermediate state between the primitive and themore evolved Platyhelminthes (see Miquel et al., 2005, 2006).Indeed, this character is also reduced to a single layer in theproteocephalideans (Sene et al., 1997; Brunanska et al., 2003,2004, 2005) and to 3 layers in the pseudophyllideans (Brun-anska et al., 2001; Levron et al., 2005, 2006).

In M. lineatus, the migration of the nucleus along the sper-matid body is observed after the proximodistal fusion of thefree flagellum with the cytoplasmic expansion. This contrastswith the previous report of nuclear migration into the cytoplas-mic expansion before proximodistal fusion with the free fla-

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FIGURES 30A–E. Spermiogenesis in Mesocestoides lineatus. (A) Initial stage of spermiogenesis. Am, arched membranes; C, centriole; Cm,cortical microtubules; Ib, intercentriolar body; N, nucleus; Sr, striated rootlets. (B) More advanced stage in which 1 centriole develops a freeflagellum (F) and the other centriole remains oriented in a cytoplasmic bud (B). Ce, cytoplasmic expansion. (C) Later stage when flagellar rotationoccurs. Ce, cytoplasmic expansion; N, nucleus. (D) More advanced stage after proximodistal fusion occurs. Note the degeneration of striatedrootlets. Am, arched membranes. (E) Final stage of spermiogenesis showing the appearance of crested bodies (Cb) and the constriction of ringof arched membranes (arrows). The degenerated striated rootlets remain in the residual cytoplasm (Rc).

gellum in the congeneric M. litteratus described by Miquel etal. (1999). However, the prior nuclear migration in M. litteratusmust be considered with caution because that nuclear migrationwas shown on a single cross-section only, which could, in fact,correspond to a basal section near the arched membranes.

Apart from the previous data on M. lineatus presented byConn (2001), some aspects of the spermatozoon, such as thenuclear area and non-nuclear area, were also presented in an-other ultrastructural investigation on fertilization in this meso-cestoidid (Swiderski and Conn, 1999).

The present study is, however, in general agreement with theprevious analysis of the mature spermatozoon of M. litteratus(Miquel et al., 1999). Both mesocestoidids exhibit several sim-ilar ultrastructural characters: (1) an anterior extremity of thespermatozoon with a single and spiraled crested body with amaximum thickness of 150 nm; (2) a reduced row of parallel,peripheral microtubules; (3) a spiraled cordon of glycogen gran-ules; and (4) a spiraled nucleus that in certain areas encirclesthe axoneme. Moreover, the posterior extremity of the sper-matozoon of M. lineatus contains several axonemal singlets.Similar observations were made of M. litteratus by Miquel et

al. (1999), despite the larger posterior tip of the spermatozoonand the presence of glycogen granules in M. lineatus.

The systematic position of mesocestoidids within the Cyclo-phyllidea constitutes a controversial subject. Indeed, membersof the Mesocestoididae exhibit biological (3-host life cycle) andmorpho-anatomical characters (genital atrium and vitellinegland), which differ from those observed in the representativesof the remaining cyclophyllidean families (see Rausch, 1994).From a molecular point of view, the analysis of sequence datafrom 18s rDNA reveals that mesocestoidids are basal to theTetrabothriidea and suggests the exclusion of both taxa fromthe Cyclophyllidea to consider this order as monophyletic (Mar-iaux, 1998). Hoberg et al. (1999), in a phylogenetic analysis ofcyclophyllideans at the family level and based on comparativemorphology, including sperm characters (flagellar rotation,number of axonemes, and twisting of cortical microtubules),placed the Mesocestoididae and Nematotaeniidae as sister taxa,basal to the Cyclophyllidea. Therefore, according to Hoberg etal. (1999), the postulated monophyly of the Cyclophyllidea,based on comparative morphology and molecular analysis ofthe orders of the Eucestoda (Hoberg et al., 1997; Mariaux,

MIQUEL ET AL.—SPERM ULTRASTRUCTURE OF M. LINEATUS 551

FIGURES 31I–IV. Diagram showing the ultrastructural organizationof the mature spermatozoon of Mesocestoides lineatus. Aae, axonemalanterior extremity; Ac, apical cone; Ape, axonemal posterior extremity;Ase, anterior spermatozoon extremity; Ax, axoneme; Cb, crested bod-ies; Cm, cortical microtubules; D, doublets; G, granules of glycogen;N, nucleus; Pm, plasma membrane; Pse, posterior spermatozoon ex-tremity; S, singlets.

1998) and the relationships among the basal taxa of the Cyclo-phyllidea, require further examination. Finally, based on sper-matozoal characters exclusively, Mesocestoides has been placedbasal to the Tetrabothriidea � Cyclophyllidea by Justine (2001).

The present study confirms the presence, in a second speciesbelonging to the Mesocestoididae, of the plesiomorphic condi-tion of several features in the mature spermatozoon and duringspermiogenesis. These characters are (1) the spermiogenesis

pattern; (2) the presence of typical striated rootlets associatedto centrioles; (3) the presence of an intercentriolar body; and(4) the presence of parallel cortical microtubules in the sper-matozoon. Furthermore, the presence of a single, thick, crestedbody and the absence of periaxonemal sheath should also beconsidered additional plesiomorphic characters.

ACKNOWLEDGMENTS

Authors thank ‘‘Serveis Cientificotecnics’’ of the University of Bar-celona for their support in the ultramicrotomy and in the TEM obser-vation of materials. This study was partially supported by the Project2005-SGR-00576 from the ‘‘DURSI (Generalitat de Catalunya).’’

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