Testisacs microanatomy and spermatogenesis of Helobdella simplex (Hirudinida: Glossiphoniidae)

ABSTRACT

The aim of this study was to describe the testisac microanatomy and spermatogenesis of Helobdella simplex (Moore, 1911). The leeches were collected in the Sauce Chico River (Tornquist) during April 2012. The collection of material was carried out manually by checking rocks, logs, leaves and artificial substrates in the river bed. Helobdella simplex has six pairs of testisacs located between the gastric caeca. The testisac wall has two structurally and functionally differentiated cell types: the lining mesothelial cells and the germline cells. The lining cells are flat and form a continuous layer. In H. simplex no ciliated cells were observed into the testisacs, nor the formation of spermatogonia due to detachment of the testicular wall. Cell types were microscopically characterized, and five spermatogenesis stages are described, taking into account the maturation of germinal cells, the morphological changes in the cytophore and phagocytic activity. Testicular phagocytes actively participate during spermatogenesis. They are involved both in the removal of defective cells during the early stages of spermatogenesis and in the removal of the cytophore during the reacting reabsorption. Our study adds the knowledge of spermatogenesis in H. simplex and might be useful to differentiate the stages of development during the reproductive cycle, and to interpret aspects of its population dynamics.

KEY WORDS:
Freshwater; gonad; histology; leeches

INTRODUCTION

Helobdella simplex (Moore, 1911) is an endemic leech of the Neotropical region. Its area of distribution comprises Argentina (Entre Ríos, Córdoba, Buenos Aires, Río Negro, Chubut and Santa Cruz); Uruguay; Chile and Peru (Ringuelet 1985, Cristoffersen 2009). It is typically found in lentic environments around hydrophytes (Gullo 1998, 2007, César et al. 2009) and under rocks in areas protected from the river current (Gullo 2015). Also, it can be found in the Limnoperna fortunei (Dunker, 1857) golden mussel byssus (Darrigran et al. 1998) and as an opportunist within the mantle cavity of Pomacea canaliculata (Lamarck, 1822) golden apple snail or channeled apple snail (Damborenea and Gullo 1996).

The spermatogenesis and testicular structure have been studied in different Glossiphoniidae leech species: Glossiphonia complanata (Linnaeus, 1758) (Damas 1965, 1968); Haementeria depressa (É. Blanchard, 1849) (Fernández et al. 1992); Helobdella stagnalis (Linnaeus, 1758) (Martínez-Alós and García-Corrales 1988, Gouda 2013); H. triserialis (É. Blanchard, 1849); H. hyalina Ringuelet, 1942, and H. michaelseni Blanchard, 1900 (Gullo 1995, 2004, 2010); Batracobdella algira (Moquin-Tandon, 1846) (Ahmed et al. 2019).

The spermatogenesis in leeches occurs within testisacs. The germinal cells undergo meiosis, giving rise to isogenic groups, clones (Fernández et al. 1992), germinal poliplasts (Bonet and Molinas 1988) or cysts (Ahmed et al. 2019). These cells remain connected to an anucleated central mass called cytophore by means of cytoplasmic bridges (Martinucci and Felluga 1977) or intercellular bridges (Ahmed et al. 2019). Divisions are synchronous, without cytokinesis. During spermatogenesis many cysts in development, accompanied by free phagocytes, are observed in the lumen of the testisac during testicular activity (Bonet and Molinas 1985). Once spermiogenesis is complete, the spermatozoa travel through vas deferens to the genital atrium. In the case of Glossiphoniidae, the spermatozoa form spermatophores which are transferred through the male gonopore during copulation (Sawyer 1986, Ferraguti 2000).

As knowledge of gametogenesis in leeches is an important tool to analyze the seasonality of the reproductive cycle (Tillman and Barnes 1973, El-Shimy and Davies 1991, Wilken and Appleton 1993, Gullo 2003), the aim of this work was to describe the testisac microanatomy and spermatogenesis of H. simplex, taking into account the maturation of the germinal cells, morphological changes in the cytophore and the phagocytic activity.

MATERIAL AND METHODS

The specimens were collected in the Sauce Chico River, 38°03’S; 62°15’W (Tornquist, Buenos Aires, Argentina) during April 2012. The collection of material was carried out manually by checking rocks, logs, leaves and artificial substrates in the river bed (Kutschera 1988). For the microanatomical examination, 33 individuals were fixed in Carnoy 6:3:1 (Gabe 1968), dehydrated in an ascending series of ethanol and embedded in Paraplast^®. Parasagital and cross serial sections (10 µm thick) were cut with a microtome and stained with Haematoxylin and Eosin using standard techniques (Kutschera et al. 2013). Serial sections were examinated under Axiostar Plus Zeiss^® microscope equipped with digital camera. Germinal cells sizes were measured with microscope crossline micrometer 10:100, considering the minor diameter of the nuclei. To prepare figures, Adobe Photoshop 7.0 software was used.

RESULTS

Helobdella simplex has six pairs of testisacs located between the gastric caeca. The testisac wall has two structurally and functionally differentiated cell types: the lining mesothelial cells and the germ line cells. The lining cells are flat and form a continuous layer. They have acidophilic cytoplasm and an oval nucleus. During spermatogenesis, cysts of 16, 32, 64, 128, and 256 germinal cells are observed within the testisac, which remain connected to a central anucleated mass (cytophore) by means of intercellular bridges. The testicular funnel is lined by a ciliated cuboidal epithelium, whose cells are characterized by having a spherical nucleus.

Spermatogenesis was divided into five stages, taking into account the morphological changes of the cytophore, the maturation of the germinal cells and the phagocytic activity (Figs 1-9).

Figures 1-4
Helobdella simplex, cross section of the testisac in stages I, II and III of spermatogenesis: (1) poliplasts in the testisac lumen; (2) Stage II; (3, 4) Stage III of spermatogenesis. (C) Cytophore, (eg) spermatogonia, (em) testicular funnel, (ep) primary spermatocytes, (es) secondary spermatocytes, (et) spermatids, (ez) spermatozoa, (fa) phagocytes, (lu) testisac lumen, (m) mesothelial cell. Scale bars: 1, 3, 4 = 50 µm, 2 = 10 µm.

Figures 5-9
Helobdella simplex, cross section of the testisac in stages IV and V of spermatogenesis: (5-7) Stage IV, mature spermatozoa attached to the cytophore and free in the testisac lumen; initiation of reabsorption processes. (8) Reacting and direct (autolysis) reabsorption. (9) Stage V, nuclei of germinal cells with piknosis and hyperchromatosis. (C) Cytophore, (ca) cytophore in autolysis, (ep) primary spermatocytes, (et) spermatids, (ez) spermatozoa, (fa) phagocytes, (p) piknosis. Scale bars: 50 µm.

Stage I

It is characterized by the proliferation of germinal cells. Spermatogonia divide by mitosis, synchronously, constituting cysts that are released into the testisac lumen. They remain attached to the very small central cytophore. They have an oval nucleus and a prominent nucleolus. The average nuclear size is 4.3 ± 0.4 µm, n = 10.

Stage II

Spermatocytes I initiate the prophase of the first meiotic division. The chromosomes condense and the nucleus shows a marked basophilia. The average size is 5.25 ± 0.6 µm, n = 10. The cytophore increases its size due to the division of the germinal cells.

Stage III

It differs from the previous stage by the presence of spermatids as the most mature cells of the testisac. At the beginning of this stage, the nuclei of the spermatids become elliptical and culminate in the vestigial flagellum. The average size is 1.57 ± 0.24 µm, n = 10. The cytophore size reaches its maximum (50 µm) and subsequently stabilizes. At this stage, cysts reach 256 spermatids and autolytic processes are not observed.

Stage IV

Spermiogenesis is complete. The spermatozoa still remain attached to the cytophore with their flagella facing towards the lumen of the testisac. The elongated nucleus with marked basophilia stands out, with an average size of 1.09 ± 0.17 µm, n = 10. An increase in free phagocytes is observed. Direct reabsorption processes are started inside the cytophore, the presence of acidophilic bodies as a consequence of autolysis is confirmed, and the spermatozoa remain free in the lumen of the testisacs. The cytophore remains are removed by phagocytes during the reacting reabsorption. Phagocytes are cells with an eccentric, spherical, basophilic nucleus, sometimes binucleated; its cytoplasm has eosinophilic granulations and abundant phagocytic vacuoles. The average nuclear size is 23.4 ± 0.56 µm and the average cytoplasmic size is 25 ± 0.52 µm, n = 10.

Stage V

It occurs after egg-laying. Testicular activity continues, although new spermatozoa will not form since the cysts are reabsorbed by phagocytes. Germinal cells undergo apoptosis, and it is common to observe nuclei with hyperchromatosis and piknosis, which are also affected by reacting reabsorption processes.

DISCUSSION

Spermatogenesis of H. simplex follows the general pattern of sperm production that is found in annelids (Ferraguti 2000) and the early stages of spermatogenesis are basically the same for Clitellata (Sawyer 1986). The spermatogenesis occurs in the lumen of fluid-filled coelomic testisacs. Inside it, the germinal cells are connected to the cytophore by intercellular bridges and develop functional units called cysts. In H. simplex we found that the testisac wall has two structurally and functionally differentiated cell types: the lining mesothelial cells and the germline cells. The lining cells are flat and form a continuous layer called coelothelium (Sawyer 1986). The H. simplex testisac wall and the germline cells are comparable to those of other Glossiphiniidae species (Gullo 1995, 2004, 2010). Unlike what was observed in H. stagnalis by Martínez-Alós and García-Corrales (1988) and Batracobdella algira (Ahmed et al. 2019), in H. simplex no ciliated cells were observed in the testisacs, nor the formation of spermatogonia due to detachment of the testicular wall.

Microanatomical examination about serial sections in testisacs of H. simplex revealed sequence of spermatogenic stages within the testisacs. Spermatogonia divide by mitosis, synchronously, constituting cysts that are released into the testisac lumen. They remain attached to the very small central cytophore. During spermatogenesis, cysts are observed within the testisac, which remain connected to a central cytophore by means of intercellular bridges. The changes in the appearance and volume of the cytophore registered in H. simplex have been mentioned for other Rhyncobdellida species, namely: Glossiphonia complanata (Damas 1968), H. stagnalis (Martínez-Alós and García-Corrales 1988), H. triserialis, H. hyalina and H. michaelseni (Gullo 1995, 2004, 2010), Helobdella stagnalis (Gouda 2013) and Batracobdella algira (Ahmed et al. 2019).

When the spermiogenesis is complete, the spermatozoa still remain attached to the cytophore with their flagella facing towards the lumen of the testisac. Direct reabsorption processes are started inside the cytophore, and the spermatozoa remain free into the lumen of the testisacs. The cytophore remains are removed by phagocytes during the reacting reabsorption. The direct reabsorption (autolysis) and reacting processes recorded in this work were described in H. triserialis, H. hyalina, and H. michaelseni (Gullo 1995, 2004, 2010). Likewise, the phagocytic activity during spermatogenesis was confirmed in several Helobdella species (Bonet and Molinas 1985, Martínez-Alós and García-Corrales 1988, Fernández et al. 1992).

In H. simplex, the processes of hyperchromatosis and pyknosis observed during stage V (after egg-laying) indicate the reabsorption of cysts into the testisacs lumen and therefore gonadal restoration is not possible. This particularity has been observed in other South American Helobdella species (Gullo 1995, 2004, 2010) and is consistent with an annual life cycle and semelpary as a reproductive strategy. Despite the importance of direct and reactional resorption processes, these have only been recorded in South American Helobdella species.

Testicular phagocytes actively participate during spermatogenesis, developing macrophage activity (Bonet and Molinas 1985, Martínez-Alós and García-Corrales 1988, Fernández et al. 1992). Helobdella simplex phagocytes are larger compared to those of other Helobdella species (Gullo 1995, 2004, 2010). They are involved both in the removal of defective cells during the early stages of spermatogenesis and in the removal of the cytophore during the reacting reabsorption. In H. simplex, the participation of phagocytes in the removal of normal spermatozoa is not excluded.

The criteria proposed to differentiate the stages of spermatogenesis might be useful to interpret the life cycle and the population dynamics of Helobdella species.

ACKNOWLEDGEMENTS

We wish to thank the reviewers for their helpful suggestions. This work was subsidized by Project 11/802-2016-2019, accredited by the National University of La Plata (UNLP, Argentina).

LITERATURE CITED

• Ahmed RB, Malota K, Jarosz N, Świątek P (2019) Microscopic analysis of spermatogenesis and mature spermatozoa in the amphibian leech Batracobdella algira (Annelida, Clitellata, Hirudinida). Protoplasma 256(6): 1609-1627. https://doi.org/10.1007/s00709-019-01407-w
  » https://doi.org/10.1007/s00709-019-01407-w
• Bonet S, Molinas M (1985) Estudio ultraestructural del fagocito testicular de Dina lineata (Hirudinea, Erpobdellidae). Scientia Gerundensis 11: 29-37.
• Bonet S, Molinas M (1988) Anatomía e histología del aparato reproductor de Dina lineata (O.F. Müller, 1974) (Hirudinea, Erpobdellidae). Boletín de la Real Sociedad Española de Historia Natural (Biología) 84(1-2): 73-89.
• César II, Martín SM, Gullo BS, Liberto R (2009) Biodiversity and ecology of Hirudinea (Annelida) from the Natural Reserve of Isla Martín García Río de la Plata, Argentina. Brazilian Journal of Biology 69(4): 1107-1113. https://doi.org/10.1590/S1519-69842009000500014
  » https://doi.org/10.1590/S1519-69842009000500014
• Christoffersen ML (2009) A catalogue of Helobdella (Annelida, Clitellata, Hirudinea, Glossiphoniidae), with a summary of leech diversity from South America. Neotropical Biology and Conservation 4(2): 89-98. https://doi.org/10.1073/pnas.0907931107
  » https://doi.org/10.1073/pnas.0907931107
• Damas D (1965) Mode de nutrition des cellules males et femelles de Glossiphonia complanata L. (Hirudinée) durant la spermatogenèse et l’ovogenèse. Bulletin de la Societé Zoologique de France 90: 337-338.
• Damas D (1968) Les cellules germinales males de Glossiphonia complanata (Hirudinée, Rhyncobdelle): Origine, evolution et structure. Bulletin de la Societé Zoologique de France 93: 375-385.
• Damborenea MC, Gullo BS (1996) Hirudíneos asociados a la cavidad paleal de Pomacea canaliculata (Lamarck, 1822) (Gasteropoda: Ampullariidae) del Balneario Bagliardi, Río de la Plata, Argentina. Neotrópica 42(107-108): 97-101.
• Darrigran G, Martín SM, Gullo BS, Armendáriz LC (1998) Macroinvertebrates associated with Limnoperna fortunei (Dunker, 1857) (Bivalvia, Mytilidae) in Río de la Plata, Argentina. Hydrobiologia 367: 223-230. https://doi.org/10.1023/A:1003244603854
  » https://doi.org/10.1023/A:1003244603854
• El-Shimy NA, Davies RW (1991) The life-cycle, ecology and host specificity of the freshwater leech Alboglossiphonia polypompholyx (Glossiphoniidae) in Egypt. Hydrobiologia 222: 173-178. https://doi.org/10.1007/BF00016156
  » https://doi.org/10.1007/BF00016156
• Fernández J, Téllez V, Olea N (1992) Hirudinea. In: Harrison FW, Gardiner SL (Eds) Microscopic anatomy of invertebrates. Annelida. Wiley-Liss, New York, 323-394.
• Ferraguti M (2000) Euclitellata. In: Adiyodi KG, Adiyodi RG (Eds) Reproductive biology of invertebrates: Progress in Male Gamete, Ultrastructure and Phylogeny. John Wiley & Sons, London, 125-182.
• Gabe M (1968) Tecniques histologiques. Masson et Cie, Paris, 1113 pp.
• Gouda HA (2013) Spermatogenesis, spermiogenesis and sperm transfer in two freshwater leeches from Assiut, Egypt. Research in Zoology 3(2): 45-55. https://doi.org/10.5923/j.zoology.20130302.01
  » https://doi.org/10.5923/j.zoology.20130302.01
• Gullo BS (1995) Microanatomía de la gónada y vías de evacuación masculinas de Helobdella triserialis (Hirudinea, Glossiphoniidae). Neotrópica 41(105-106): 67-75.
• Gullo BS (1998) Hirudíneos Glossiphoniidae asociados a Lemnaceas, en Los Talas (Pdo. de Berisso), Buenos Aires. Neotrópica 44(111-112): 65-68.
• Gullo BS (2003) Ciclo reproductor de Helobdella triserialis (Hirudinea, Glossiphoniidae), Provincia de Buenos Aires, Argentina. Revista del Museo de La Plata Zoología 15(167): 1-7.
• Gullo BS (2004) Histología del sistema reproductor masculino de Helobdella hyalina (Hirudinea, Glossiphoniidae) en Argentina. Iheringia, Série Zoologia 94(1): 13-18. https://doi.org/10.1590/S0073-47212004000100002
  » https://doi.org/10.1590/S0073-47212004000100002
• Gullo BS (2007) Hirudíneos asociados a hidrófitas en la laguna Los Patos, Buenos Aires, Argentina. Revista del Museo de La Plata Zoología 18(172): 11-18. http://hdl.handle.net/10915/67290
  » http://hdl.handle.net/10915/67290
• Gullo BS (2010) Microanatomía de los testisacos y espermatogénesis de Helobdella michaelseni (Hirudinea, Glossiphoniidae). Neotropical Biology and Conservation 5(3): 167-171. https://doi.org10.4013/nbc.2010.5355
  » https://doi.org10.4013/nbc.2010.5355
• Gullo BS (2015) Nuevos registros de especies de la familia Glossiphoniidae (Annelida: Clitellata: Hirudinida) en la Comarca de Sierra de la Ventana, provincia de Buenos Aires, Argentina. Natura Neotropicalis 46(1-2): 25-40. https://doi.org/10.14409/natura.vli46.4910
  » https://doi.org/10.14409/natura.vli46.4910
• Kutschera U (1988) A new leech species from North America, Helobdella californica nov. sp. (Hirudinea: Glossiphoniidae). Zoologischer Anzeiger 220: 173-178.
• Kutschera U, Langguth H, Kuo D-H, Weisblat DA, Shankland M (2013) Description of a new leech species from North America, Helobdella austinensis n. sp. (Hirudinea: Glossiphoniidae), with observations on its feeding behavior. Zoosystematics and Evolution 89: 239-246. https://doi.org/10.1002/zoos.201300010
  » https://doi.org/10.1002/zoos.201300010
• Martínez-Alós S, García-Corrales P (1988) Anatomía e histología del aparato reproductor de Helobdella stagnalis L. (Annelida, Hirudinea, Rhyncobdellida, Glossiphoniidae). Boletín de la Real Sociedad Española de Historia Natural (Biología) 84(1-2): 15-31.
• Martinucci GB, Felluga B (1977) Early development of cytophorus in premeiotic male gonial cells of Eisenia foetida (Sav.). Bollettino di Zoologia 42: 271-273. https://doi.org/10.1080/11250007509431442
  » https://doi.org/10.1080/11250007509431442
• Ringuelet RA (1985) Annulata. Hirudinea. En: Castellanos ZA (Ed.) Fauna de Agua Dulce de la República Argentina 27(1): 1-321.
• Sawyer RT (1986) Leech biology and behavior. Feeding biology, ecology and systematics. Clarendon Press, Oxford, vol. 2, 375 pp.
• Tillman DL, Barnes JR (1973) The reproductive biology of the leech Helobdella stagnalis in Utah Lake, Utah. Freshwater Biology 3: 137-145.
• Wilken GB, Appleton CC (1993) Gametogenesis in the leech Asiaticobdella buntonensis (Meyer, 1951) (Hirudinidae). South African Journal of Zoology 28(1): 1-5. https://doi.org/10.1080/02541858.1993.11448281
  » https://doi.org/10.1080/02541858.1993.11448281

ADDITIONAL NOTES

• Zoobank register

  http://zoobank.org/C6DCFC86-3239-4C58-812A-4B0E84FE03BF

• How to cite this article

  Gullo BS, Lopretto EC (2021) Testisacs microanatomy and spermatogenesis of Helobdella simplex (Hirudinida: Glossiphoniidae). Zoologia (Curitiba) 38: e21019. https://doi.org/10.1590/S1984-4689.v38.e21019

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Editorial responsibility

Carolina Arruda Freire

Publication Dates

• Publication in this collection
  26 Nov 2021
• Date of issue
  2021

History

• Received
  18 Aug 2021
• Accepted
  06 Nov 2021