Spermatological Characterization of the Cestode Meggittina gerbilli (Cyclophyllidea: Catenotaeniidae), a Parasite of Gerbils, Gerbillus gerbillus and Gerbillus campestris (Rodentia: Muridae) in Tunisia

Simple Summary Ultrastructural characters of spermiogenesis and the spermatozoon of the cestode Meggittina gerbilli, a parasite of gerbils in Tunisia, were studied using transmission electron microscopy. The type III model of spermiogenesis was observed. This model is mainly characterized by a proximodistal fusion of a single flagellum with a cytoplasmic extension. As for the sperm cell, spermatozoon type VI was observed, presenting a single axoneme, a periaxonemal sheath, crest-like bodies, twisted cortical microtubules, and a spiraled nucleus. The results show similarities between Meggittina gerbilli and other studied species within the Catenotaeniidae family. Abstract Ultrastructural characters of spermiogenesis and the mature spermatozoon of the cestode Meggittina gerbilli (Cyclophyllidea: Catenotaeniidae), a parasite of the Lesser Egyptian gerbil (Gerbillus gerbillus) and the North African gerbil (Gerbillus campestris) (Rodentia: Muridae) in the Djebel Dahar (South of Tunisia), were studied using transmission electron microscopy. The spermiogenesis of M. gerbilli is of Bâ and Marchand’s type III, which is mainly characterized by a proximodistal fusion of a single flagellum with a cytoplasmic extension. In this catenotaeniid, the proximal fusion is preceded by a 90° rotation of the flagellum. The spermatozoon is a Levron et al. type VI, which presents a single axoneme with the 9 + ‘1’ trepaxonematan pattern, a periaxonemal sheath, two crest-like bodies, twisted cortical microtubules, and a spiraled nucleus. The obtained results show similarities with the remaining studied catenotaeniids, namely Catenotaenia pusilla and Skrjabinotaenia lobata. The results are compared and discussed according to several characteristics found in the catenotaeniids and other studied cyclophyllideans.

Ultrastructural studies on spermiogenesis and the spermatozoon have been demonstrated as a useful source of characters for phylogenetic inference in diverse parasitic groups of Platyhelminthes [15][16][17][18][19][20][21][22][23][24][25].In the case of cestodes, Świderski [16] described three models of spermiogenesis, namely the pseudophyllidean type, the caryophyllidean type, and the cyclophyllidean type, which were based on the number of flagella/axonemes that originated from the differentiation zone and the presence/absence of the flagella proximodistal fusion.Bâ and Marchand [20] added a fourth model to differentiate spermiogenesis originating a single free flagellum, according to whether the flagellum develops orthogonal or parallel to the cytoplasmic process (spermiogenesis type II or III, respectively).Thus, Bâ and Marchand's type I corresponds to the pseudophyllidean model of Świderski, type II to the caryophyllidean model, and type IV to the cyclophyllidean model.
The new Bâ and Marchand's type III spermiogenesis is also found in cyclophyllidean cestodes.According to the latter authors, the most evident difference between types III and IV, exhibited by cyclophyllideans, is the presence/absence of proximodistal fusion.Thus, in type III spermiogenesis, a flagellum grows externally and parallel to the cytoplasmic process, which is followed by proximodistal fusion.In type IV, an axoneme grows directly into the cytoplasmic process; thus, there is no proximodistal fusion.Justine [21] established several synapomorphies on the basis of ultrastructural characters of spermiogenesis and the spermatozoon for diverse orders of the Eucestoda, e.g., the absence of mitochondrion for the Eucestoda or the spiraled pattern of cortical microtubules for the tetrabothriideans and cyclophyllideans, among others.Finally, considering the available spermatological studies at the time, Levron et al. [23] established seven spermatozoon models in the Eucestoda (I to VII).Types I and II spermatozoa are mainly characterized by having two axonemes, while they are differentiated by the presence/absence of crest-like bodies.The remaining models of sperm cells (types III to VII) have a single axoneme, and they can be distinguished by considering the presence/absence of other characters, such as crest-like bodies, periaxonemal sheath, and intracytoplasmic walls, and the parallel or spiraled pattern of cortical microtubules and nucleus.

Specimens
Specimens of the cestode Meggittina gerbilli (Cyclophyllidea, Catenotaeniidae) were recovered from the intestinal tract of 11 naturally infected gerbils captured in the Djebel Dahar (South of Tunisia) in March, April, and June 2023.Of the 11 infected gerbils, seven were North African gerbils G. campestris, which had been trapped in Ksar El Hallouf, Zammour, Zmerten, and Zmerten Kef Ennsoura, while the remaining four were lesser Egyptian gerbils G. gerbillus, which had been trapped in Sakrana, Zammour, Zmerten, and Zmerten Kef Ennsoura (Figure 1).The studied gerbils were captured using Manufrancetype (Saint-Étienne, France) or Firobind-type (Besançon, France) wire-mesh traps and sacrificed by cervical dislocation before being examined for intestinal helminths using a stereomicroscope.
periaxonemal sheath, and intracytoplasmic walls, and the parallel or spiraled pa ern of cortical microtubules and nucleus.

Specimens
Specimens of the cestode Meggi ina gerbilli (Cyclophyllidea, Catenotaeniidae) were recovered from the intestinal tract of 11 naturally infected gerbils captured in the Djebel Dahar (South of Tunisia) in March, April, and June 2023.Of the 11 infected gerbils, seven were North African gerbils G. campestris, which had been trapped in Ksar El Hallouf, Zammour, Zmerten, and Zmerten Kef Ennsoura, while the remaining four were lesser Egyptian gerbils G. gerbillus, which had been trapped in Sakrana, Zammour, Zmerten, and Zmerten Kef Ennsoura (Figure 1).The studied gerbils were captured using Manufrancetype (Saint-Étienne, France) or Firobind-type (Besançon, France) wire-mesh traps and sacrificed by cervical dislocation before being examined for intestinal helminths using a stereomicroscope.

Species Identification
Several cestodes were fixed in the field in ethanol at 70% and later, in the laboratory, they were stained with Semichon's acetic carmine, dehydrated in an ethanol series and with 2-propanol, cleared in clove oil, and, finally, mounted on slides with Canada balsam (Figure 2).Specimens were identified as M. gerbilli, in agreement with specialized litera-ture [3,6,8,9], using a Leica DMLB light microscope (Leica Microsystems, Wetzlar, Germany) at magnifications of 100× and 400×.

Species Identification
Several cestodes were fixed in the field in ethanol at 70% and later, in the laboratory, they were stained with Semichon's acetic carmine, dehydrated in an ethanol series and with 2-propanol, cleared in clove oil, and, finally, mounted on slides with Canada balsam (Figure 2).Specimens were identified as M. gerbilli, in agreement with specialized literature [3,6,8,9], using a Leica DMLB light microscope (Leica Microsystems, Wedlar, Germany) at magnifications of 100× and 400×.

Transmission Electron Microscopy Study
Some living adult cestodes were rinsed with a 0.9% NaCl solution immediately upon removal from the intestinal tract of both gerbil species.Then, they were fixed in 2.5% glutaraldehyde at 4 °C in a 0.1 M sodium cacodylate buffer (pH 7.4) for a minimum of 2 h.After rinsing in a 0.1 M sodium cacodylate buffer (pH 7.4), the specimens were postfixed in 1% osmium tetroxide at 4 °C with 0.9% potassium ferricyanide in the same buffer for 1 h.After rinsing in Milli-Q water (Millipore Gradient A10, Millipore Co., Merck KGaA, Darmstadt, Germany), the dehydration process was started by using an ethanol series and propylene oxide.The specimens were finally embedded in Spurr's epoxy resin and polymerized at 60 °C for 72 h.Semithin sections (1 µm thick) were obtained using a Leica Reichert-Jung Ultracut E ultramicrotome (Leica Microsystems, Wedlar, Germany), placed on slides, and stained with a mixture of 1% methylene blue-1% borax.Semithin sections were used to locate the study area (testes and vas deferens) (Figure 3).Ultrathin sections (60 nm thick) were obtained using a Leica Reichert-Jung Ultracut E ultramicrotome (Leica Microsystems), placed on gold grids, and double-stained with uranyl acetate and lead citrate, as in Reynolds [35].Stained ultrathin sections were observed under a JEOL 1010 transmission electron microscope (JEOL Ltd., Tokyo, Japan) operated at an accelerating voltage of 80 kV in the "Centres Científics i Tecnològics de la Universitat de Barcelona (CCiTUB)".

Transmission Electron Microscopy Study
Some living adult cestodes were rinsed with a 0.9% NaCl solution immediately upon removal from the intestinal tract of both gerbil species.Then, they were fixed in 2.5% glutaraldehyde at 4 • C in a 0.1 M sodium cacodylate buffer (pH 7.4) for a minimum of 2 h.After rinsing in a 0.1 M sodium cacodylate buffer (pH 7.4), the specimens were postfixed in 1% osmium tetroxide at 4 • C with 0.9% potassium ferricyanide in the same buffer for 1 h.After rinsing in Milli-Q water (Millipore Gradient A10, Millipore Co., Merck KGaA, Darmstadt, Germany), the dehydration process was started by using an ethanol series and propylene oxide.The specimens were finally embedded in Spurr's epoxy resin and polymerized at 60 • C for 72 h.Semithin sections (1 µm thick) were obtained using a Leica Reichert-Jung Ultracut E ultramicrotome (Leica Microsystems, Wetzlar, Germany), placed on slides, and stained with a mixture of 1% methylene blue-1% borax.Semithin sections were used to locate the study area (testes and vas deferens) (Figure 3).Ultrathin sections (60 nm thick) were obtained using a Leica Reichert-Jung Ultracut E ultramicrotome (Leica Microsystems), placed on gold grids, and double-stained with uranyl acetate and lead citrate, as in Reynolds [35].Stained ultrathin sections were observed under a JEOL 1010 transmission electron microscope (JEOL Ltd., Tokyo, Japan) operated at an accelerating voltage of 80 kV in the "Centres Científics i Tecnològics de la Universitat de Barcelona (CCiTUB)".

Spermiogenesis
In M. gerbilli, spermiogenesis begins with the formation of a differentiation zone in the spermatid.This differentiation zone is a conical area surrounded by a submembranous layer of cortical microtubules, and it is delimited by a ring of arching membranes in its basal part (Figures 4a and 5a).The nucleus and two orthogonally oriented centrioles are present in the differentiation zone (Figures 4a and 5a,b).A cytoplasmic extension elongates from the differentiation zone.One of the centrioles gives rise to a free flagellum that grows more or less orthogonally to the cytoplasmic extension (Figures 4b and 5c,d), whereas the other centriole aborts later.At this stage of spermiogenesis, the elongation of the nucleus in the spermatid is already observed (Figures 4b and 5d).Thereafter, the free flagellum rotates and becomes parallel to the cytoplasmic extension (Figures 4c and 5e,f), while fusion occurs by the so-called proximodistal fusion (Figures 4d and 5f,h).Both in the cytoplasmic extension before the proximodistal fusion and in the spermatid after proximodistal fusion, cortical microtubules are parallel to the hypothetical long axis of the spermatid (Figure 5f).However, progressively, the submembranous layer of the cortical microtubules becomes twisted (Figure 5g).During the final stages of spermiogenesis, a densification in the cytoplasm is observed (Figure 6a-c), and crest-like bodies are formed (Figures 4e and 6b-e).Spermiogenesis finishes with the constriction of the ring of arching membranes (Figures 4e and 6e) in order to liberate the spermatozoon.

Spermiogenesis
In M. gerbilli, spermiogenesis begins with the formation of a differentiation zone in the spermatid.This differentiation zone is a conical area surrounded by a submembranous layer of cortical microtubules, and it is delimited by a ring of arching membranes in its basal part (Figures 4a and 5a).The nucleus and two orthogonally oriented centrioles are present in the differentiation zone (Figures 4a and 5a,b).A cytoplasmic extension elongates from the differentiation zone.One of the centrioles gives rise to a free flagellum that grows more or less orthogonally to the cytoplasmic extension (Figures 4b and 5c,d), whereas the other centriole aborts later.At this stage of spermiogenesis, the elongation of the nucleus in the spermatid is already observed (Figures 4b and 5d).Thereafter, the free flagellum rotates and becomes parallel to the cytoplasmic extension (Figures 4c and 5e,f), while fusion occurs by the so-called proximodistal fusion (Figures 4d and 5f,h).Both in the cytoplasmic extension before the proximodistal fusion and in the spermatid after proximodistal fusion, cortical microtubules are parallel to the hypothetical long axis of the spermatid (Figure 5f).However, progressively, the submembranous layer of the cortical microtubules becomes twisted (Figure 5g).During the final stages of spermiogenesis, a densification in the cytoplasm is observed (Figure 6a-c), and crest-like bodies are formed (Figures 4e and 6b-e).Spermiogenesis finishes with the constriction of the ring of arching membranes (Figures 4e and 6e) in order to liberate the spermatozoon.

Spermatozoon
In the spermatozoon of M. gerbilli, five regions (I to V) can be considered from the anterior to the posterior extremity.These five regions are distinguished by their ultrastructural characteristics.
Region I (Figures 7 and 8a-h) is the anterior extremity of the spermatozoon.It has a maximum width of around 360 nm.Region I is capped with an electron-dense apical cone, which is more than 1150 nm long (Figures 7 and 8a).This anterior region is also characterized by the presence of two helical crest-like bodies of unequal length (Figures 7 and 8a-h).The two crest-like bodies appear at the level of the apical cone, with one appearing before the other (Figures 7 and 8a-c).Thus, in the anterior part of region I, there are two crest-like bodies (Figure 8c-e), whereas in the posterior part, there is only one (Figure 8f-h).The maximum thickness of the crest-like bodies is around 70 nm, which was observed in the part of region I where only one crest-like body is present (Figure 8f,h).Other ultrastructural characteristics of this region include the presence of a periaxonemal sheath (Figure 8e-g) and a continuous and submembranous layer of cortical microtubules, which are twisted at a 30 • angle in relation to the hypothetical long axis of the spermatozoon (Figure 8a,d-h).Region II (Figures 7 and 9a-c) is characterized by the disappearance of the crest-like bodies.Its maximum width is around 410 nm.Region II shows the axoneme surrounded by the periaxonemal sheath (Figures 7 and 9b,c) and the spiraled cortical microtubules arranged as a discontinuous submembranous layer (Figures 7 and 9a,c).
Region III (Figures 7 and 9d-f) is the nuclear region of the spermatozoon.Its maximum width is around 495 nm.The nucleus is placed helicoidally around the axoneme (Figures 7 and 9d,e).In the cross-sections, the nucleus appears with a round to a horse-shoe shape (Figure 9d,f).The periaxonemal sheath that surrounds the axoneme seems to be interrupted when the nucleus is present (Figure 9d-f).Finally, as in region II, in the nuclear region, the twisted cortical microtubules are arranged in a discontinuous submembranous layer (Figure 9d,f).
Region IV (Figures 7 and 9g,h) has a maximum width of 400 nm and is characterized by the absence of both the nucleus and the periaxonemal sheath (Figures 7 and 9g,h).Moreover, cortical microtubules progressively disappear and become parallel to the hypothetical sperm axis (Figures 7 and 9g,h).
Region V (Figures 7 and 9i,j) constitutes the posterior extremity of the spermatozoon.Its maximum width is around 240 nm.It is characterized by the sole presence of the axoneme (Figures 7 and 9i), which progressively disorganizes into doublets and singlets (Figures 7 and 9j).

Spermatozoon
In the spermatozoon of M. gerbilli, five regions (I to V) can be considered from the anterior to the posterior extremity.These five regions are distinguished by their ultrastructural characteristics.
Region I (Figures 7 and 8a-h) is the anterior extremity of the spermatozoon.It has a maximum width of around 360 nm.Region I is capped with an electron-dense apical cone, which is more than 1150 nm long (Figures 7 and 8a).This anterior region is also characterized by the presence of two helical crest-like bodies of unequal length (Figures 7 and  8a-h).The two crest-like bodies appear at the level of the apical cone, with one appearing before the other (Figures 7 and 8a-c).Thus, in the anterior part of region I, there are two crest-like bodies (Figure 8c-e), whereas in the posterior part, there is only one (Figure 8fh).The maximum thickness of the crest-like bodies is around 70 nm, which was observed in the part of region I where only one crest-like body is present (Figure 8f,h).Other ultrastructural characteristics of this region include the presence of a periaxonemal sheath (Figure 8e-g) and a continuous and submembranous layer of cortical microtubules, which are twisted at a 30° angle in relation to the hypothetical long axis of the spermatozoon (Figure 8a,d-h).Region II (Figures 7 and 9a-c) is characterized by the disappearance of the crest-like bodies.Its maximum width is around 410 nm.Region II shows the axoneme surrounded by the periaxonemal sheath (Figures 7 and 9b,c) and the spiraled cortical microtubules arranged as a discontinuous submembranous layer (Figures 7 and 9a,c).Region III (Figures 7 and 9d-f) is the nuclear region of the spermatozoon.Its maximum width is around 495 nm.The nucleus is placed helicoidally around the axoneme (Figures 7 and 9d,e).In the cross-sections, the nucleus appears with a round to a horseshoe shape (Figure 9d,f).The periaxonemal sheath that surrounds the axoneme seems to be interrupted when the nucleus is present (Figure 9d-f).Finally, as in region II, in the nuclear region, the twisted cortical microtubules are arranged in a discontinuous submembranous layer (Figure 9d,f).
Region IV (Figures 7 and 9g,h) has a maximum width of 400 nm and is characterized by the absence of both the nucleus and the periaxonemal sheath (Figures 7 and 9g,h).Moreover, cortical microtubules progressively disappear and become parallel to the hypothetical sperm axis (Figures 7 and 9g,h).
Region V (Figures 7 and 9i,j) constitutes the posterior extremity of the spermatozoon.Its maximum width is around 240 nm.It is characterized by the sole presence of the axoneme (Figures 7 and 9i), which progressively disorganizes into doublets and singlets (Figures 7 and 9j).

Spermiogenesis
In M. gerbilli, flagellar rotation and proximodistal fusion are the major events that characterize spermiogenesis.The process of spermiogenesis in M. gerbilli is quite similar to that observed in another studied catenotaeniid, Catenotaenia pusilla [33].Thus, both species show a growing free flagellum that fuses proximodistally with the cytoplasmic extension.Unfortunately, no data are available concerning spermiogenesis in Skrjabinotaenia lobata [34], the only catenotaeniid with information on the ultrastructure of the sperm cell that belongs to the same subfamily (Skrjabinotaeniinae) as M. gerbilli (Table 1).In C. pusilla, the flagellum develops at an angle of about 45 • to the cytoplasmic extension [33].However, an angle of about 90 • was observed in the present study.Świderski [16] and, posteriorly, Bâ and Marchand [20] characterized spermiogenesis in cestodes, establishing three and four models, respectively.In cyclophyllidean cestodes, two types of spermiogenesis were described: III and IV.These two models differ in the presence or absence of a proximodistal fusion [20].The two catenotaeniids studied to date follow a "modified" type III of Bâ and Marchand.Type III was defined based on a free flagellum growing parallel to the cytoplasmic extension and, consequently, lacking flagellar rotation [20].However, the Catenotaeniidae representatives, C. pusilla [33] and M. gerbilli, both present flagellar rotation (Table 1).Likewise, other cyclophyllideans did not fit the traditional type III spermiogenesis proposed by Bâ and Marchand [20].These are the paruterinids Anonchotaenia globata, Notopentorchis sp., and Triaenorhina rectangula [28,36,37], and the taeniids Taenia parva and Taenia taeniaeformis [38,39].Finally, a doubtful slight flagellar rotation, unsupported by the published TEM micrographs, has been mentioned in the metadilepidid Skrjabinoporus merops [40].Other characteristics of spermiogenesis in M. gerbilli include the absence of striated rootlets associated with centrioles and the absence of an intercentriolar body.These structures are typically associated with cestode spermiogenesis types I and II, which are observed in species of other cestode orders [16,20].However, striated rootlets or similar structures are observed in some cyclophyllideans presenting spermiogenesis types III or IV.This is the case for the dipylidiids Dipylidium caninum, Joyeuxiella echinorhynchoides, and Joyeuxiella pasqualei, the paruterinids A. globata, Notopentorchis sp., and T. rectangula, and the taeniid T. taeniaeformis, which present striated rootlets or vestigial striated rootlets associated with the centrioles in a type III spermiogenesis [28,36,37,39,41,42].Moreover, these structures are also present in the anoplocephalids Anoplocephaloides dentata, Gallegoides arfaai, and Mosgovoyia ctenoides, which follow a type IV spermiogenesis [43][44][45].
The cytoplasm densification observed in advanced stages of M. gerbilli spermiogenesis could be the origin of the periaxonemal sheath or the crest-like bodies.Unfortunately, no TEM micrographs illustrating this process were obtained.Ndiaye et al. [42] described the formation of the periaxonemal sheath in J. echinorhynchoides (Dipydidiidae), which was initiated by cytoplasm densification in the periphery of the spermatid and showed a progressive displacement towards surrounding the axoneme.
In M. gerbilli, as in all the Eucestoda, the spermatozoon lacks mitochondrion, which was postulated as a synapomorphy for this subclass [17,21,22].Contrarily, the representatives of the subclass Cestodaria, which includes the orders Amphilinidea and Gyrocotylidea, are the only cestodes with mitochondria in their male gametes [76,77].

Conclusions
Spermiogenesis of Meggittina gerbilli follows Bâ and Marchand's type III, which is characterized by a proximodistal fusion of a single flagellum with a cytoplasmic extension.However, the observed flagellar rotation in M. gerbilli constitutes a characteristic that was also mentioned in Catenotaenia pusilla, the other catenotaeniid in which spermiogenesis has been studied to date.Spermiogenesis type III in these two catenotaeniids can be distin-guished from spermiogenesis type II by the absence of striated rootlets and intercentriolar body.The spermatozoon of M. gerbilli is of Levron et al.'s type VI, characterized by having one axoneme, a periaxonemal sheath, crest-like bodies, twisted cortical microtubules, and a spiraled nucleus.Moreover, the mature spermatozoon in the three studied catenotaeniids have two crest-like bodies of unequal length and similar maximum thicknesses (between 60 and 80 nm).Thus, despite the scarce ultrastructural data on this family, the currently available ultrastructural results emphasize the similarities in the spermatological characteristics of the Catenotaeniidae.

Figure 1 .
Figure 1.Sampling locations for North African gerbils and lesser Egyptian gerbils containing the parasite Meggi ina gerbilli in the Djebel Dahar (Tunisia).Images captured from Google Earth Pro and modified using Adobe Illustrator 2024 software (Adobe, San José, CA, USA).

Figure 1 .
Figure 1.Sampling locations for North African gerbils and lesser Egyptian gerbils containing the parasite Meggittina gerbilli in the Djebel Dahar (Tunisia).Images captured from Google Earth Pro and modified using Adobe Illustrator 2024 software (Adobe, San José, CA, USA).

Table 1 .
Main ultrastructural characteristics of spermiogenesis and the spermatozoon in Catenotaeniidae species.