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Insemination of the monogenean Neobenedenia girellae (Capsalidae, Benedeniinae)
Parasitology International 63 (2014) 473–478
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Parasitology International journal homepage: www.elsevier.com/locate/parint
Insemination of the monogenean Neobenedenia girellae (Capsalidae, Benedeniinae) K. Ogawa a,⁎, S. Shirakashi b, H. Ishitani b a b
Meguro Parasitological Museum, 4-1-1, Shimomeguro, Meguro-ku, Tokyo 153-0064, Japan Fisheries Laboratory, Kinki University, Wakayama 649-2211, Japan
a r t i c l e
i n f o
Article history: Received 25 July 2013 Received in revised form 15 October 2013 Accepted 16 October 2013 Available online 24 October 2013 Keywords: Neobenedenia girellae Insemination Spermatophore Monogenea
a b s t r a c t In vitro spermatophore formation and insemination of Neobenedenia girellae (Monogenea: Capsalidae, Benedeniinae) were recorded on video and described for the first time. Upon contact of two individuals, the anterior adhesive discs of the donor firmly attached to the dorsal tegument of the recipient and the donor's fore body strongly contracted such that the genital pore region protruded and the penis was pushed anteriorly to protrude through the genital pore. It is hypothesised that the donor penis mechanically damaged the tegument of the recipient. The sperm and spermatophore matrix were released together through the penis, which was placed under the left anterior attachment disc immediately behind the adhesive pad. The spermatophore matrix containing the spermatozoa became solid and attached to the dorsal surface of recipient's body. When observed under scanning electron microscopy, the spermatophores were irregularly shaped, with a diameter of 52–83 μm. Under light microscopy they consisted of a proximal eosinophilic matrix portion and a distal thin-walled portion containing spermatozoa. Both parts were enclosed with a thin outer casing. Insemination occurred during and after spermatophore formation. Three types of insemination were recorded, unilateral and mutual insemination and self-insemination. The presence of self-insemination indicates that even a single N. girellae on a cultured fish may cause a significant parasite infection in the entire aquaculture system. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction There are several types of reproduction among benedeniine monogeneans (Capsalidae). Kearn & Whittington [1] described mutual crossinsemination with intromission, attachment of spermatophores to other individuals and self-insemination. Of the 14 genera in the subfamily Benedeniinae [2], Neobenedenia is unique in that they do not have a vagina [3]. Reproductive behaviour may be different from other benedeniine monogeneans because of the lack of a vagina, but no information regarding its reproduction is available except for a single case of in vitro observation of possible self-insemination in Neobenedenia melleni [4]. There have been discussions regarding the taxonomy of several Neobenedenia species [4,5]. In Japan, Neobenedenia collected from cultured marine fish has been identified as N. girellae according to Ogawa et al. [5], whereas this species was synonymised with N. melleni by Whittington & Horton [4]. More recently, it was suggested that N. melleni, which is known to infect more than 100 marine fish [4], comprises a species complex. This is based on molecular analysis of the large subunit ribosomal DNA [6]. Currently, it is not possible to identify Neobenedenia at the species level based solely on morphological
⁎ Corresponding author. 1383-5769/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.parint.2013.10.009
characteristics. To avoid further confusion, here, we retain the scientific name N. girellae, instead of ‘Neobenedenia sp.’ because this study deals solely with the reproductive biology of monogeneans. When we observed live Neobenedenia specimens in a Petri dish with seawater under a stereomicroscope, we observed several specimens attached to other specimens for some time and leaving a spermatophore on the dorsal body surface of the latter. The objective of this study is to describe reproductive behaviour of N. girellae for the first time. 2. Materials and methods Parasites were carefully removed with a scalpel from greater amberjack Seriola dumerili cultured at the Fisheries Laboratory, Kinki University, Shirahama, Wakayama Prefecture, Japan. A group of 10 to 20 specimens were transferred to Petri dishes containing clean sand-filtered seawater. They were allowed to move freely in the dish at room temperature and their behaviour was monitored under a stereomicroscope equipped with a digital camera. The insemination behaviour was video recorded and later shown on a computer screen to trace the position of the parasite onto paper. In addition, still pictures were recorded to determine the number of inseminated parasites and to record the location of spermatophores on the recipient body. Within 1 h after insemination, the parasites were fixed for histology or scanning electron microscopy (SEM). The parasites were fixed in 10%
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buffered formalin for histology. Briefly, the parasites were dehydrated, embedded in paraffin, cut into serial sections 4 μm in thickness, and stained with hematoxylin and eosin (H&E). For SEM, the parasites were fixed in 1.5% glutaraldehyde and dehydrated in an ethanol series. 3. Results 3.1. Observation of insemination A total of 13 cases of insemination were recorded on video, in which an entire event was recorded on seven occasions, from the attachment of a donor to a recipient until the separation of the two after the donor planted a spermatophore on the recipient. In nine cases, the fore body of the donor was placed on the recipient (Fig. 1a). In another case in which the recipient was placed on the donor, the left lateral side of the donor's body was partially turned up and its fore body twisted ventrally to attach to the recipient from the dorsal side (Fig. 1b). In this case, the anterior attachment discs of the donor pinched the tegument of the recipient from both sides. Another type of insemination, mutual insemination, was also observed in which two individuals exchanged spermatophores (Fig. 1c). In this case, one worm attached to the other as in Fig. 1a, whereas the other worm maintained a posture similar to the worm in Fig. 1b but without pinching the recipient's tegument. Moreover, there was a case of self-insemination (Fig. 1d) in which the body proper was bent inward towards the middle and the anterior portion twisted and bent inwards again to hold the lateral part of its own body from the dorsal side. In histological sections, both the sperm reservoir and spermatophore matrix reservoir [as accessory gland reservoir [4,5] in the penis sac of the adult parasite is surrounded by well-developed muscle bundles (Fig. 2). When the muscle contracts, it is likely that the spermatozoa and spermatophore matrix are mixed and released together. Throughout the insemination process, the anterior attachment discs of the donor firmly attach to the dorsal body surface of the recipient. Although it was not possible to observe the precise events of each insemination process in the video clips, it is assumed that the genital pore region of the donor protruded and was inserted under the left anterior attachment disc, maintaining the genital pore in a fixed position immediately behind the adhesive pad of the disc. The fore body strongly
Fig. 2. A transverse section of the proximal portion of the penis sac. Well-developed muscle bundles encircle both the sperm reservoir (SR) and spermatophore matrix reservoir (SMR).
contracted and the penis sac was pushed anteriorly, such that the distal end of the penis reached the protruding terminal region (Fig. 3a). Then, both the sperm and spermatophore matrix were released from the penis (Fig. 3b–c). The spermatophore matrix containing the spermatozoa became solid and attached to the dorsal surface of recipient's body. Subsequently, the fore body was relaxed and the penis sac returned to the original position (Fig. 3d). After the two parasites separated, the spermatophores, which were shaped irregularly and cylindrical and consisted of a light-coloured proximal part and a larger yellow- to brown-coloured distal part in the video recordings, in no case disintegrated or detached from the recipient. After separation, an oblong mark was left in the donor's left adhesive pad, where the spermatophore was held (Fig. 3e). Both the donors and recipients remained stationary in the Petri dish during insemination. The time required for the entire insemination process ranged from 36 s to 86 s (mean: 56.6 s). A total of 26 spermatophores attached to
Fig. 1. Semi-diagrammatic drawings of insemination in Neobenedenia girellae. Donor parasites (in white) plant a spermatophore on the dorsal side of the recipients (in grey). a, b: unilateral insemination; c: mutual insemination in which parasites (both in white) are donors and recipients; d: self-insemination.
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Fig. 3. Semi-diagrammatic drawings of donor parasites illustrating the process of spermatophore formation. a: initial process, when the genial pore region protrudes and the penis sac moves anteriorly such that its tip is inserted under the left adhesive pad and reaches the tegument of the donor. b: sperm and spermatophore matrix are released from the tip of the penis. Protrusion of the genital pore region becomes less prominent. c: return of the genital pore to the original position as the fore body starts to relax. d: completion of spermatophore formation and separation of the spermatophore from the penis. The penis sac returns to the original position. e: separation of the spermatophore from the donor, leaving a mark (arrow) where the spermatophore is positioned. Note: in a and b, the fore body strongly contracts. All dorsal views.
the body surface of 24 recipients (one to two spermatophores per recipient). These data are plotted in Fig. 4. The spermatophores were all on the dorsal body surface, more frequently on the posterior half of the body proper from the germarium to close to the end of the body proper, with the exception of a spermatophore on the haptor.
3.2. Structure of the spermatophore When observed under SEM, the spermatophores planted on the recipients were irregular in shape, with a size of 52–83 μm (mean 71.6 μm) in diameter (n = 5) (Fig. 5). Based on the histological analysis, the spermatophores consisted of a proximal eosinophilic matrix portion and a distal thin-walled portion containing the spermatozoa, with both parts enclosed within a thin outer casing (Fig. 6a–c). The two parts correspond to the light-coloured proximal and yellow- to brown-coloured distal parts in the video recordings. The spermatozoa were also observed in the proximal portion (Fig. 6b). The spermatophores were connected with the tegument of a recipient through a narrow, short stalk, 22–30 μm (mean 26.0 μm) in width (n = 5) (Fig. 6). The tegument of the recipient connected to the spermatophore was partially disrupted and slightly elevated, suggesting that the tegument was pulled toward the spermatophore. The spermatozoa were observed in the recipient's parenchyma and spermatophore and also in the narrow connecting portion of the latter, indicating that the transfer of spermatozoa occurred after the planting of spermatophores and during spermatophore formation. In histological sections of the spermatophores planted on recipients, spermatozoa and/or eosinophilic granular substance (EGS) were observed in the parenchyma of the recipients (Fig. 6). Apparently, the EGS was from the donor, and based on its stainability and granular nature, it is hypothesised that the EGS is the spermatophore matrix. A total of 13 observed recipients had spermatozoa (n = 5), EGS (n = 5), both spermatozoa and EGS (n=1) or none (n=2) in their parenchyma. 4. Discussion
Fig. 4. Position of the spermatophores planted on the tegument of recipients, based on 26 recorded cases.
Spermatophore formation and insemination were recorded for Neobenedenia girellae for the first time in this study. It is possible that the observed behaviours were a result of the in vitro conditions, in which the parasites were removed from the host and 10–20 were placed together in a Petri dish. This abnormal condition may have stimulated these activities. However, it is reasonable to assume that N. girellae plants spermatophores and inseminates in vivo in the same manner as observed in vitro because these reproductive behaviours were observed among many individuals in Petri dishes on many occasions. Insemination of Neobenedenia spp. has long been a mystery. Several authors hypothesised that the absence of a vagina in this group of monogeneans indicates that the entry route for spermatozoa is via the common genital pore [4] or the uterus [7]. Whittington & Horton [4]
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Fig. 5. SEM photographs of spermatophores planted on a recipient. a: dorsal body of a recipient (arrow: spermatophore; arrowhead: haptor); b: higher magnification of the spermatophore in a; c & d: other spermatophores.
observed a single specimen with its penis directing posteriorly into its own uterus. Such self-insemination remains possible; however, for N. girellae, the four types of insemination shown in this study appear to be more common. Spermatophores were always planted on the dorsal side of the body proper of the recipients, irrespective of differences in the types of insemination. It is postulated that the spermatozoa reach the fertilisation chamber [5] (=intragermarial chamber of Whittington & Horton [4]) within the germarium through vitelline ducts. Reproductive behaviours of benedeniine monogeneans have been categorised into the following three types [1]: 1) intromission as in Benedenia rohdei [8] (as Benedenia sp. 1 [1], 2) external attachment of spermatophores as in Benedenia sp. 2 sensu Kearn and Whittington, 1992 and 3) self-insemination as in Benedeniella macrocopla and Benedeniella posterocolpa. Intromission was also observed in Benedenia seriolae [9]. In the present study, the reproductive behaviours of N. girellae shown in Fig. 1a–c are somewhat similar to the type 2 discussed above. However, fundamental differences exist for the mechanism by which fertilisation occurs. In Benedenia sp. 2, a stalk of the spermatophore was lodged within the distal region of the vagina. It is expected that spermatozoa in the spermatophore later enter through the vaginal duct. By contrast, histological observations of N. girellae insemination demonstrate that the spermatozoa enter into a recipient through the disrupted tegument. Therefore, the insemination in Fig. 1a–c represents a fourth type of insemination for benedeniine monogeneans. In N. girellae, there is another type of insemination, the self-insemination shown in Fig. 1d, and this mode of self-insemination is different from the known types. In N. girellae, the adhesive pads held its own tegument by bending and twisting the body during insemination, whereas in B. macrocopla and B. posterocolpa, the penis was inserted into its own uterus. It is apparent that insemination by N. girellae is a new type of self-insemination, and there are therefore five types of insemination among the benedeniine monogeneans. Further studies are necessary to determine whether other Neobenedenia species have the same or similar insemination behaviours. The insemination process of N. girellae occurred for 36–86 s, which is much shorter than Entobdella soleae, another capsalid (Entobdellinae),
which took as long as 30 min [10]. N. girellae shows constant breathing movements (Ogawa, unpublished observation) as observed in E. soleae [11]. Insemination during the constant undulating movements of a recipient will require firm and continuous attachment to the recipient's body by the anterior adhesive pads of the donor. The present observations suggest that the fixed position of the spermatophores just behind the left adhesive pad will secure successful insemination by the donor. With the characteristic insemination behaviour, spermatozoa passed into a recipient through the tegument, whereas the spermatozoa remained in the spermatophore. During the insemination process, the donor must have damaged the recipient's tegument to allow passage of the spermatozoa because it is highly unlikely that the spermatozoa can penetrate the intact tegument of the recipient. Histological sections of spermatophores (Fig. 6) indicate that the tegument of the recipient sustained mechanical damage. Presumably the tip of the donor parasite penis reached the tegument of the recipient at the start of insemination when the fore body of the donor strongly contracted to push up the penis sac and the genital pore region protruded. It is possible that physical contact of the penis may have mechanically damaged the recipient’'s tegument. The EGS observed in the parenchyma of the recipient was similar to the spermatophore matrix in stainability and granular nature. It is not clear whether it plays a role in spermatozoan movement to the fertilisation chamber in the germarium. In the penis sac, welldeveloped muscle bundles encircle both the sperm reservoir and spermatophore matrix reservoir. When the muscle contracts, it is likely that the spermatozoa and spermatophore matrix is mixed and released together. Because no other secretion is thought to be involved in spermatophore formation, it is possible that the EGS together with the spermatozoa enter into the recipient through the damaged tegument. Many monogenean species form spermatophores, which are categorised into two types. In the more common type, the spermatophores are capsules with or without a stalk, such as Benedenia sp. 2 sensu Kearn and Whittington, 1992, Neoentobdella spp. (N. diadema [=Entobdella diadema after Llewellyn & Euzet [12], N. apicolpos, N. garinei, N.
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Fig. 6. Transverse sections of parasites with spermatophores planted on the tegument. The spermatophores are enclosed with a thin outer casing, consisting of the proximal eosinophilic matrix portion and the distal portion containing spermatozoa. a: a spermatophore at a lower magnification. Note that the tegument is disrupted and slightly elevated at the connection site with the spermatophore (arrow). b: higher magnification of a. Note that the spermatozoa are observed in the eosinophilic matrix of the spermatophore and at the connection site with the recipient (arrows); c: Another spermatophore at high magnification. d: spermatozoa (arrow) and eosinophilic material, apparently the same as the eosinophilic matrix of spermatophore, in the parenchyma of the recipient. egs: eosinophilic material in the parenchyma of a recipient; em: eosinophilic matrix portion of the spermatophore; oc: a thin outer casing of spermatophore; sp: spermatozoa in the parenchyma of a recipient; ss: spermatozoa in the distal portion of a spermatophore.
taiwanensis), Diplectanum aequans, Dendromonocotyle spp. (D. octodiscus, D. californica, D. centrourae, D. cortesi, D. ukuthena and possibly D. akajeii), Dermopristis cairae and possibly Dioncopseudobenedenia kala [1,7,12–19]. In the other type, the spermatophores are a mixture of jelly-like material and spermatozoa, such as Entobdella soleae, Neoentobdella natans, Acanthocotyle greeni and A. lobianchi [10,20,21]. The spermatophores formed by N. girellae have characteristics in common with the former type in that they are encased within a thin wall and with the latter in that the spermatozoa and spermatophore matrix are mixed and released together to form a spermatophore. However, it is not clear whether the thin-walled outer casing of N. girellae is equivalent to the capsules in the former, and the spermatophores of N. girellae are not jelly-like as in the latter. N. girellae is rather unique in that upon insemination, it damages the tegument of the recipient before the sperm and spermatophore matrix are released. Among spermatophore-forming monogeneans, spermatophores are planted close to the vaginal opening for E. soleae, D. kala and possibly D. cairae [10,17,18], placed partially inside the vagina for Benedenia sp. 2 sensu Kearn and Whittington, 1992, N. diadema, N. apicolpos, N. garinei, N. taiwanensis and D. aequans [7,13,16], or placed entirely inside vagina for D. octodiscus, D. californica, D. centrourae, D. cortesi, D. akajeii and D. ukuthena [14,15]. None of these monogeneans damages the tegument to plant spermatophores. Among the spermatophore-forming monogeneans, Acanthocotyle spp. and N. girellae in this study are exceptional for their absence of a vagina. Spermatophores of acanthocotylids produce jelly-like spermatophores [20], but the mechanism by which sperm transfer occurs in acanthocotylids remains largely unknown. It is hypothesised that in acanthocotylids, the spermatophores are picked up by the uterine arm and passed into the uterus [20]. If the spermatophores are planted on the tegument randomly such as N. girellae, it is unlikely that the spermatozoa can penetrate the tegument because acanthocotylids have no sclerotised or muscular penis.
The mode of insemination is more similar to that of the monogenean Gyrodactylus wageneri in that this monopisthocotylean, which has no vagina, directly inseminates through the tegument of the recipient [22]. Unlike the mode of fertilisation of N. girellae, in which the spermatozoa enter the vitelline ducts before reaching the oviduct, the spermatozoa of G. wageneri travel through parenchyma to the oocyte [22]. The polyopisthocotylean monogeneans Gastrocotyle trachuri, Diclidophora merlangi and Heterobothrium okamotoi, also lacking vagina, inseminate just the same way as G. wageneri [23–26]. There are some discussions about how the spermatozoa eventually reach the female tract of the recipient. In G. trachuri and H. okamotoi, the inserted spermatozoa invade the vitelline ducts and eventually enter into the oviduct for fertilisation [24–26]. MacDonald and Caley [23] claimed that in D. merlangi the spermatozoa travel through parenchyma and eventually perforate the wall of seminal receptacle, though Llewellyn [23] cast doubt on their interpretation. Rather, it is plausible that the spermatozoa of these three polyopisthocotyleans reach the oviduct through the vitelline ducts, as in N. girellae in this study. The self-insemination observed in this study indicates that N. girellae can reproduce without a mate to receive spermatozoa. This method of reproduction is particularly favourable for cases of infection with a single parasite. This is very different from Benenenia seriolae, an important capsalid parasite of cultured amberjacks, Seriola spp., because B. seriolae reproduces only by intromission (Ogawa, unpublished observation). This suggests that N. girellae infection can be established more easily and rapidly than B. seriolae in the culture system.
Acknowledgment The authors thank Ms. Chihaya Hirano for help with the collection of live N. girellae specimens from amberjack.
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References [1] Kearn GC, Whittington ID. Diversity of reproductive behaviour in platyhelminth parasites: insemination in some benedeniine (capsalid) monogeneans. Parasitology 1992;104:489–96. [2] Whittington ID. The Capsalidae (Monogenea: Monopisthocotylea): a review of diversity, classification and phylogeny with a note about species complexes. Folia Parasitol 2004;51:109–22. [3] Systema Helminthum Yamaguti S. Monogenea and Aspidocotylea, vol. 4. New York: Interscience Publ.; 1963. [4] Whittington ID, Horton MA. A revision of Neobenedenia Yamaguti, 1963 (Monogenea: Capsalidae) including a redescription of N. melleni (MacCallum, 1927) Yamaguti, 1963. J Nat Hist 1996;30:1113–56. [5] Ogawa K, Bondad-Reantaso MG, Fukudome M, Wakabayashi H. Neobenedenia girellae (Hargis, 1955) Yamaguti, 1963 (Monogenea: Capsalidae) from cultured marine fishes of Japan. J Parasitol 1995;81:223–7. [6] Whittington ID, Deveney MR, Morgan JAT, Chisholm LA, Adlard RD. A preliminary phylogenetic analysis of the Capsalidae (Platyhelminthes: Monogenea: Monopisthocotylea) inferred from large subunit rDNA sequences. Parasitology 2004;128:511–9. [7] Kearn GC, Whittington ID, Euzet L. The handling and fate of spermatophores in Neoentobdella diadema and N. apiocolpos (Monogenea: Capsalidae: Entobdellinae). Folia Parasitol 2006;53:57–62. [8] Whittington ID, Kearn GC, Beverley-Burton M. Benedenia rohdei n. sp. (Monogenea: Capsalidae) from the gills of Lutjanus carponotatus (Perciformes: Lutjanidae) from the Great Barrier Reef, Queensland, Australia, with a description of the oncomiracidium. Syst Parasitol 1994;28:5–13. [9] Kearn GC. Mating in the capsalid monogenean Benedenia seriolae, a skin parasite of the yellowtail, Seriola quinqueradiata. Publ Seto Mar Biol Lab 1992;35:273–80. [10] Kearn GC. The production, transfer and assimilation of spermatophores by Entobdella soleae, a monogenean skin parasite of the common sole. Parasitology 1970;60:301–11. [11] Kearn GC. Breathing movements in Entobdella soleae (Trematoda, Monogenea) from the skin of the common sole. J Mar Biol Assoc U K 1962;42:93–104. [12] Llewellyn J, Euzet L. Spermatophores in the monogenean Entobdella diadema Monticelli from the skin of sting-rays, with a note on the taxonomy of the parasite. Parasitology 1964;54:337–44. [13] Paling JE. The functional morphology of the genitalia of the spermatophoreproducing monogenean parasite Diplectanum aequans (Wagener) Diesing, with a note on the copulation of the parasite. Parasitology 1966;36:367–83.
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Parasitology International journal homepage: www.elsevier.com/locate/parint
Insemination of the monogenean Neobenedenia girellae (Capsalidae, Benedeniinae) K. Ogawa a,⁎, S. Shirakashi b, H. Ishitani b a b
Meguro Parasitological Museum, 4-1-1, Shimomeguro, Meguro-ku, Tokyo 153-0064, Japan Fisheries Laboratory, Kinki University, Wakayama 649-2211, Japan
a r t i c l e
i n f o
Article history: Received 25 July 2013 Received in revised form 15 October 2013 Accepted 16 October 2013 Available online 24 October 2013 Keywords: Neobenedenia girellae Insemination Spermatophore Monogenea
a b s t r a c t In vitro spermatophore formation and insemination of Neobenedenia girellae (Monogenea: Capsalidae, Benedeniinae) were recorded on video and described for the first time. Upon contact of two individuals, the anterior adhesive discs of the donor firmly attached to the dorsal tegument of the recipient and the donor's fore body strongly contracted such that the genital pore region protruded and the penis was pushed anteriorly to protrude through the genital pore. It is hypothesised that the donor penis mechanically damaged the tegument of the recipient. The sperm and spermatophore matrix were released together through the penis, which was placed under the left anterior attachment disc immediately behind the adhesive pad. The spermatophore matrix containing the spermatozoa became solid and attached to the dorsal surface of recipient's body. When observed under scanning electron microscopy, the spermatophores were irregularly shaped, with a diameter of 52–83 μm. Under light microscopy they consisted of a proximal eosinophilic matrix portion and a distal thin-walled portion containing spermatozoa. Both parts were enclosed with a thin outer casing. Insemination occurred during and after spermatophore formation. Three types of insemination were recorded, unilateral and mutual insemination and self-insemination. The presence of self-insemination indicates that even a single N. girellae on a cultured fish may cause a significant parasite infection in the entire aquaculture system. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction There are several types of reproduction among benedeniine monogeneans (Capsalidae). Kearn & Whittington [1] described mutual crossinsemination with intromission, attachment of spermatophores to other individuals and self-insemination. Of the 14 genera in the subfamily Benedeniinae [2], Neobenedenia is unique in that they do not have a vagina [3]. Reproductive behaviour may be different from other benedeniine monogeneans because of the lack of a vagina, but no information regarding its reproduction is available except for a single case of in vitro observation of possible self-insemination in Neobenedenia melleni [4]. There have been discussions regarding the taxonomy of several Neobenedenia species [4,5]. In Japan, Neobenedenia collected from cultured marine fish has been identified as N. girellae according to Ogawa et al. [5], whereas this species was synonymised with N. melleni by Whittington & Horton [4]. More recently, it was suggested that N. melleni, which is known to infect more than 100 marine fish [4], comprises a species complex. This is based on molecular analysis of the large subunit ribosomal DNA [6]. Currently, it is not possible to identify Neobenedenia at the species level based solely on morphological
⁎ Corresponding author. 1383-5769/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.parint.2013.10.009
characteristics. To avoid further confusion, here, we retain the scientific name N. girellae, instead of ‘Neobenedenia sp.’ because this study deals solely with the reproductive biology of monogeneans. When we observed live Neobenedenia specimens in a Petri dish with seawater under a stereomicroscope, we observed several specimens attached to other specimens for some time and leaving a spermatophore on the dorsal body surface of the latter. The objective of this study is to describe reproductive behaviour of N. girellae for the first time. 2. Materials and methods Parasites were carefully removed with a scalpel from greater amberjack Seriola dumerili cultured at the Fisheries Laboratory, Kinki University, Shirahama, Wakayama Prefecture, Japan. A group of 10 to 20 specimens were transferred to Petri dishes containing clean sand-filtered seawater. They were allowed to move freely in the dish at room temperature and their behaviour was monitored under a stereomicroscope equipped with a digital camera. The insemination behaviour was video recorded and later shown on a computer screen to trace the position of the parasite onto paper. In addition, still pictures were recorded to determine the number of inseminated parasites and to record the location of spermatophores on the recipient body. Within 1 h after insemination, the parasites were fixed for histology or scanning electron microscopy (SEM). The parasites were fixed in 10%
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buffered formalin for histology. Briefly, the parasites were dehydrated, embedded in paraffin, cut into serial sections 4 μm in thickness, and stained with hematoxylin and eosin (H&E). For SEM, the parasites were fixed in 1.5% glutaraldehyde and dehydrated in an ethanol series. 3. Results 3.1. Observation of insemination A total of 13 cases of insemination were recorded on video, in which an entire event was recorded on seven occasions, from the attachment of a donor to a recipient until the separation of the two after the donor planted a spermatophore on the recipient. In nine cases, the fore body of the donor was placed on the recipient (Fig. 1a). In another case in which the recipient was placed on the donor, the left lateral side of the donor's body was partially turned up and its fore body twisted ventrally to attach to the recipient from the dorsal side (Fig. 1b). In this case, the anterior attachment discs of the donor pinched the tegument of the recipient from both sides. Another type of insemination, mutual insemination, was also observed in which two individuals exchanged spermatophores (Fig. 1c). In this case, one worm attached to the other as in Fig. 1a, whereas the other worm maintained a posture similar to the worm in Fig. 1b but without pinching the recipient's tegument. Moreover, there was a case of self-insemination (Fig. 1d) in which the body proper was bent inward towards the middle and the anterior portion twisted and bent inwards again to hold the lateral part of its own body from the dorsal side. In histological sections, both the sperm reservoir and spermatophore matrix reservoir [as accessory gland reservoir [4,5] in the penis sac of the adult parasite is surrounded by well-developed muscle bundles (Fig. 2). When the muscle contracts, it is likely that the spermatozoa and spermatophore matrix are mixed and released together. Throughout the insemination process, the anterior attachment discs of the donor firmly attach to the dorsal body surface of the recipient. Although it was not possible to observe the precise events of each insemination process in the video clips, it is assumed that the genital pore region of the donor protruded and was inserted under the left anterior attachment disc, maintaining the genital pore in a fixed position immediately behind the adhesive pad of the disc. The fore body strongly
Fig. 2. A transverse section of the proximal portion of the penis sac. Well-developed muscle bundles encircle both the sperm reservoir (SR) and spermatophore matrix reservoir (SMR).
contracted and the penis sac was pushed anteriorly, such that the distal end of the penis reached the protruding terminal region (Fig. 3a). Then, both the sperm and spermatophore matrix were released from the penis (Fig. 3b–c). The spermatophore matrix containing the spermatozoa became solid and attached to the dorsal surface of recipient's body. Subsequently, the fore body was relaxed and the penis sac returned to the original position (Fig. 3d). After the two parasites separated, the spermatophores, which were shaped irregularly and cylindrical and consisted of a light-coloured proximal part and a larger yellow- to brown-coloured distal part in the video recordings, in no case disintegrated or detached from the recipient. After separation, an oblong mark was left in the donor's left adhesive pad, where the spermatophore was held (Fig. 3e). Both the donors and recipients remained stationary in the Petri dish during insemination. The time required for the entire insemination process ranged from 36 s to 86 s (mean: 56.6 s). A total of 26 spermatophores attached to
Fig. 1. Semi-diagrammatic drawings of insemination in Neobenedenia girellae. Donor parasites (in white) plant a spermatophore on the dorsal side of the recipients (in grey). a, b: unilateral insemination; c: mutual insemination in which parasites (both in white) are donors and recipients; d: self-insemination.
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Fig. 3. Semi-diagrammatic drawings of donor parasites illustrating the process of spermatophore formation. a: initial process, when the genial pore region protrudes and the penis sac moves anteriorly such that its tip is inserted under the left adhesive pad and reaches the tegument of the donor. b: sperm and spermatophore matrix are released from the tip of the penis. Protrusion of the genital pore region becomes less prominent. c: return of the genital pore to the original position as the fore body starts to relax. d: completion of spermatophore formation and separation of the spermatophore from the penis. The penis sac returns to the original position. e: separation of the spermatophore from the donor, leaving a mark (arrow) where the spermatophore is positioned. Note: in a and b, the fore body strongly contracts. All dorsal views.
the body surface of 24 recipients (one to two spermatophores per recipient). These data are plotted in Fig. 4. The spermatophores were all on the dorsal body surface, more frequently on the posterior half of the body proper from the germarium to close to the end of the body proper, with the exception of a spermatophore on the haptor.
3.2. Structure of the spermatophore When observed under SEM, the spermatophores planted on the recipients were irregular in shape, with a size of 52–83 μm (mean 71.6 μm) in diameter (n = 5) (Fig. 5). Based on the histological analysis, the spermatophores consisted of a proximal eosinophilic matrix portion and a distal thin-walled portion containing the spermatozoa, with both parts enclosed within a thin outer casing (Fig. 6a–c). The two parts correspond to the light-coloured proximal and yellow- to brown-coloured distal parts in the video recordings. The spermatozoa were also observed in the proximal portion (Fig. 6b). The spermatophores were connected with the tegument of a recipient through a narrow, short stalk, 22–30 μm (mean 26.0 μm) in width (n = 5) (Fig. 6). The tegument of the recipient connected to the spermatophore was partially disrupted and slightly elevated, suggesting that the tegument was pulled toward the spermatophore. The spermatozoa were observed in the recipient's parenchyma and spermatophore and also in the narrow connecting portion of the latter, indicating that the transfer of spermatozoa occurred after the planting of spermatophores and during spermatophore formation. In histological sections of the spermatophores planted on recipients, spermatozoa and/or eosinophilic granular substance (EGS) were observed in the parenchyma of the recipients (Fig. 6). Apparently, the EGS was from the donor, and based on its stainability and granular nature, it is hypothesised that the EGS is the spermatophore matrix. A total of 13 observed recipients had spermatozoa (n = 5), EGS (n = 5), both spermatozoa and EGS (n=1) or none (n=2) in their parenchyma. 4. Discussion
Fig. 4. Position of the spermatophores planted on the tegument of recipients, based on 26 recorded cases.
Spermatophore formation and insemination were recorded for Neobenedenia girellae for the first time in this study. It is possible that the observed behaviours were a result of the in vitro conditions, in which the parasites were removed from the host and 10–20 were placed together in a Petri dish. This abnormal condition may have stimulated these activities. However, it is reasonable to assume that N. girellae plants spermatophores and inseminates in vivo in the same manner as observed in vitro because these reproductive behaviours were observed among many individuals in Petri dishes on many occasions. Insemination of Neobenedenia spp. has long been a mystery. Several authors hypothesised that the absence of a vagina in this group of monogeneans indicates that the entry route for spermatozoa is via the common genital pore [4] or the uterus [7]. Whittington & Horton [4]
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Fig. 5. SEM photographs of spermatophores planted on a recipient. a: dorsal body of a recipient (arrow: spermatophore; arrowhead: haptor); b: higher magnification of the spermatophore in a; c & d: other spermatophores.
observed a single specimen with its penis directing posteriorly into its own uterus. Such self-insemination remains possible; however, for N. girellae, the four types of insemination shown in this study appear to be more common. Spermatophores were always planted on the dorsal side of the body proper of the recipients, irrespective of differences in the types of insemination. It is postulated that the spermatozoa reach the fertilisation chamber [5] (=intragermarial chamber of Whittington & Horton [4]) within the germarium through vitelline ducts. Reproductive behaviours of benedeniine monogeneans have been categorised into the following three types [1]: 1) intromission as in Benedenia rohdei [8] (as Benedenia sp. 1 [1], 2) external attachment of spermatophores as in Benedenia sp. 2 sensu Kearn and Whittington, 1992 and 3) self-insemination as in Benedeniella macrocopla and Benedeniella posterocolpa. Intromission was also observed in Benedenia seriolae [9]. In the present study, the reproductive behaviours of N. girellae shown in Fig. 1a–c are somewhat similar to the type 2 discussed above. However, fundamental differences exist for the mechanism by which fertilisation occurs. In Benedenia sp. 2, a stalk of the spermatophore was lodged within the distal region of the vagina. It is expected that spermatozoa in the spermatophore later enter through the vaginal duct. By contrast, histological observations of N. girellae insemination demonstrate that the spermatozoa enter into a recipient through the disrupted tegument. Therefore, the insemination in Fig. 1a–c represents a fourth type of insemination for benedeniine monogeneans. In N. girellae, there is another type of insemination, the self-insemination shown in Fig. 1d, and this mode of self-insemination is different from the known types. In N. girellae, the adhesive pads held its own tegument by bending and twisting the body during insemination, whereas in B. macrocopla and B. posterocolpa, the penis was inserted into its own uterus. It is apparent that insemination by N. girellae is a new type of self-insemination, and there are therefore five types of insemination among the benedeniine monogeneans. Further studies are necessary to determine whether other Neobenedenia species have the same or similar insemination behaviours. The insemination process of N. girellae occurred for 36–86 s, which is much shorter than Entobdella soleae, another capsalid (Entobdellinae),
which took as long as 30 min [10]. N. girellae shows constant breathing movements (Ogawa, unpublished observation) as observed in E. soleae [11]. Insemination during the constant undulating movements of a recipient will require firm and continuous attachment to the recipient's body by the anterior adhesive pads of the donor. The present observations suggest that the fixed position of the spermatophores just behind the left adhesive pad will secure successful insemination by the donor. With the characteristic insemination behaviour, spermatozoa passed into a recipient through the tegument, whereas the spermatozoa remained in the spermatophore. During the insemination process, the donor must have damaged the recipient's tegument to allow passage of the spermatozoa because it is highly unlikely that the spermatozoa can penetrate the intact tegument of the recipient. Histological sections of spermatophores (Fig. 6) indicate that the tegument of the recipient sustained mechanical damage. Presumably the tip of the donor parasite penis reached the tegument of the recipient at the start of insemination when the fore body of the donor strongly contracted to push up the penis sac and the genital pore region protruded. It is possible that physical contact of the penis may have mechanically damaged the recipient’'s tegument. The EGS observed in the parenchyma of the recipient was similar to the spermatophore matrix in stainability and granular nature. It is not clear whether it plays a role in spermatozoan movement to the fertilisation chamber in the germarium. In the penis sac, welldeveloped muscle bundles encircle both the sperm reservoir and spermatophore matrix reservoir. When the muscle contracts, it is likely that the spermatozoa and spermatophore matrix is mixed and released together. Because no other secretion is thought to be involved in spermatophore formation, it is possible that the EGS together with the spermatozoa enter into the recipient through the damaged tegument. Many monogenean species form spermatophores, which are categorised into two types. In the more common type, the spermatophores are capsules with or without a stalk, such as Benedenia sp. 2 sensu Kearn and Whittington, 1992, Neoentobdella spp. (N. diadema [=Entobdella diadema after Llewellyn & Euzet [12], N. apicolpos, N. garinei, N.
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Fig. 6. Transverse sections of parasites with spermatophores planted on the tegument. The spermatophores are enclosed with a thin outer casing, consisting of the proximal eosinophilic matrix portion and the distal portion containing spermatozoa. a: a spermatophore at a lower magnification. Note that the tegument is disrupted and slightly elevated at the connection site with the spermatophore (arrow). b: higher magnification of a. Note that the spermatozoa are observed in the eosinophilic matrix of the spermatophore and at the connection site with the recipient (arrows); c: Another spermatophore at high magnification. d: spermatozoa (arrow) and eosinophilic material, apparently the same as the eosinophilic matrix of spermatophore, in the parenchyma of the recipient. egs: eosinophilic material in the parenchyma of a recipient; em: eosinophilic matrix portion of the spermatophore; oc: a thin outer casing of spermatophore; sp: spermatozoa in the parenchyma of a recipient; ss: spermatozoa in the distal portion of a spermatophore.
taiwanensis), Diplectanum aequans, Dendromonocotyle spp. (D. octodiscus, D. californica, D. centrourae, D. cortesi, D. ukuthena and possibly D. akajeii), Dermopristis cairae and possibly Dioncopseudobenedenia kala [1,7,12–19]. In the other type, the spermatophores are a mixture of jelly-like material and spermatozoa, such as Entobdella soleae, Neoentobdella natans, Acanthocotyle greeni and A. lobianchi [10,20,21]. The spermatophores formed by N. girellae have characteristics in common with the former type in that they are encased within a thin wall and with the latter in that the spermatozoa and spermatophore matrix are mixed and released together to form a spermatophore. However, it is not clear whether the thin-walled outer casing of N. girellae is equivalent to the capsules in the former, and the spermatophores of N. girellae are not jelly-like as in the latter. N. girellae is rather unique in that upon insemination, it damages the tegument of the recipient before the sperm and spermatophore matrix are released. Among spermatophore-forming monogeneans, spermatophores are planted close to the vaginal opening for E. soleae, D. kala and possibly D. cairae [10,17,18], placed partially inside the vagina for Benedenia sp. 2 sensu Kearn and Whittington, 1992, N. diadema, N. apicolpos, N. garinei, N. taiwanensis and D. aequans [7,13,16], or placed entirely inside vagina for D. octodiscus, D. californica, D. centrourae, D. cortesi, D. akajeii and D. ukuthena [14,15]. None of these monogeneans damages the tegument to plant spermatophores. Among the spermatophore-forming monogeneans, Acanthocotyle spp. and N. girellae in this study are exceptional for their absence of a vagina. Spermatophores of acanthocotylids produce jelly-like spermatophores [20], but the mechanism by which sperm transfer occurs in acanthocotylids remains largely unknown. It is hypothesised that in acanthocotylids, the spermatophores are picked up by the uterine arm and passed into the uterus [20]. If the spermatophores are planted on the tegument randomly such as N. girellae, it is unlikely that the spermatozoa can penetrate the tegument because acanthocotylids have no sclerotised or muscular penis.
The mode of insemination is more similar to that of the monogenean Gyrodactylus wageneri in that this monopisthocotylean, which has no vagina, directly inseminates through the tegument of the recipient [22]. Unlike the mode of fertilisation of N. girellae, in which the spermatozoa enter the vitelline ducts before reaching the oviduct, the spermatozoa of G. wageneri travel through parenchyma to the oocyte [22]. The polyopisthocotylean monogeneans Gastrocotyle trachuri, Diclidophora merlangi and Heterobothrium okamotoi, also lacking vagina, inseminate just the same way as G. wageneri [23–26]. There are some discussions about how the spermatozoa eventually reach the female tract of the recipient. In G. trachuri and H. okamotoi, the inserted spermatozoa invade the vitelline ducts and eventually enter into the oviduct for fertilisation [24–26]. MacDonald and Caley [23] claimed that in D. merlangi the spermatozoa travel through parenchyma and eventually perforate the wall of seminal receptacle, though Llewellyn [23] cast doubt on their interpretation. Rather, it is plausible that the spermatozoa of these three polyopisthocotyleans reach the oviduct through the vitelline ducts, as in N. girellae in this study. The self-insemination observed in this study indicates that N. girellae can reproduce without a mate to receive spermatozoa. This method of reproduction is particularly favourable for cases of infection with a single parasite. This is very different from Benenenia seriolae, an important capsalid parasite of cultured amberjacks, Seriola spp., because B. seriolae reproduces only by intromission (Ogawa, unpublished observation). This suggests that N. girellae infection can be established more easily and rapidly than B. seriolae in the culture system.
Acknowledgment The authors thank Ms. Chihaya Hirano for help with the collection of live N. girellae specimens from amberjack.
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