Protandrous Hermaphroditic Reproductive System in the Adult Phases of Mothocya renardi (Bleeker, 1857) (Cymothoidae: Isopoda: Crustacea) - Light and Electron Microscopy Study

© 2020 Academia Sinica, Taiwan Open Access Protandrous Hermaphroditic Reproductive System in the Adult Phases of Mothocya renardi (Bleeker, 1857) (Cymothoidae: Isopoda: Crustacea) – Light and Electron Microscopy Study Panakkool Thamban Aneesh1 and Sudha Kappalli1,2,* 1Post Graduate Department of Zoology and Research Centre, Sree Narayana College, Kannur670 007, India. E-mail: [email protected] (Aneesh) 2Department of Zoology, School of Biological Sciences, Central University of Kerala, Kasaragod671320, Kerala, India. *Correspondence: E-mail: [email protected] (Kappalli) Received 5 May 2020 / Accepted 23 September 2020 / Published 23 November 2020 Communicated by Benny K.K. Chan The reproductive system of Mothocya renardi (Bleeker, 1857), a protandrous hermaphroditic cymothoid that infects the belonid fish Strongylura leiura Bleeker, 1850, is characterized using light and electron microscopy. Three protandrous hermaphroditic adult phases are identified: male, transitional and female. Each phase includes a paired reproductive system, one on either side of the gut. Each consists of three lobed testes, followed by an ovary, then a vas deferens that opens into a penis on the same side. During the male phase, all testis lobes are filled with germ cells at various stages of spermatogenesis and spermeogenesis. Primary and secondary spermatogonial cells are confined to the peripheral side of the testis lobe. The ovary shows peripheral germarium and a large number of yolkless oocytes encircled by follicle cells. The oviduct emerged from the ovary mid laterally and its distal end was found to be sealed. The exceptionally elongated spermatozoon consists of a head and a long filamentous tail. The spermatozoa are found organized into characteristic bundles to form spermatophores, and these are also packed in the vas deferens during the male phase. During the transitional phase, on the other hand, testes appear to be withered, but the vas deferens contains spermatophores. The ovary shows yolky oocytes encircled by follicle cells. During the female phase, the testis lobes appear as thin, empty, and sac like, and the extremity of the vas deferens is closed. Ovaries contain yolky oocytes and more prominent oviducts than male and transitional phases. The present paper also discusses the pattern of correlation between 1) the ovarian and brood cycles and 2) the ovarian and molt cycles. Key words: Protandrous hermaphroditism, Spermatophores, Oocytes, Follicle cells, Ovary cycle. BACKGROUND Isopoda, a crustacean order, comprises over 10,000 species living in diverse habitats, 45% of which are marine organisms that are free living or parasitic. While the majority of the crustacean parasites are dioecious (i.e., have separate male and female sexes), parasitic isopods display both protandrous and protogynous hermaphroditism. Protandrous sex changes have primarily been reported in parasitic isopods like epicarids and cymothoids (Cressey 1983; Brook et al. 1994; Cook and Munguia 2015; Smit et al. 2019; Citation: Aneesh PT, Kappalli S. 2020. Protandrous hermaphroditic reproductive system in the adult phases of Mothocya renardi (Bleeker, 1857) (Cymothoidae: Isopoda: Crustacea) – light and electron microscopy study. Zool Stud 59:61. doi:10.6620/ZS.2020.59-61. Zoological Studies 59:61 (2020) doi:10.6620/ZS.2020.59-61 1 © 2020 Academia Sinica, Taiwan Aneesh et al. 2018). If males of the cymothoid Anilocra frontalis H. Milne Edwards, 1840 happen to infest the host fishes Labrus bergylta Ascanius, 1767 and L. maculates Bloch, 1792 solitarily, it may transition or develop into a functional female. On other hand, if another male settles into the same host as this female, the new male remains the same gender until the female dies or is removed (Legrand 1952). Most studies on the structure of isopod reproductive systems are focused on free living and non-hermaphroditic forms. In Saduria entomon (L.), a marine and free-living isopod, for instance, spermatogenesis does not appear in a synchronized pattern in their testis tubules because the germ cells display different levels of maturation and stay associated with the somatic cells (HryniewieckaSzyfter and Tyczewska 1991). In Oniscus asellus (L.) and Armadillidium vulgare (Latreille 1804), accessory cells produce extracellular tubules surrounding the spermatozoa in spermatophores (Reger and FainMaurel 1973; Itaya 1979). Free-living isopods have also been shown to have a unique type of spermatozoon, consisting of a cell body enclosing a long ribbon-like nucleus, above which an acrosomal complex and a long flexible tail are found (Cotelli et al. 1976; Reger et al. 1979). In the females of Saduria entomon (L.) and Asellus aquaticus (L.), the oviduct is modified into a sperm reservoir (Hryniewiecka-Szyfter and Babula 1995; Erkan 1998). Though protandrous hermaphroditism has been reported in epicarids and cymothoids (Brusca 1981; Brook et al. 1994), these studies are based on morphological evidence and little is known about the histological and physiological characteristics associated with hermaphroditism in this group (Brook et al. 1994; Poulin 1995; Kottarathil and Kappalli 2019a b). In the cymothoid Ichthyoxenus fushanensis Tsai and Dai 1999, protandrous hermaphroditism is reportedly associated with a sexual size dimorphism, with females being much larger than males, and the presence of vestigial penes (Tsai and Dai 1999; Tsai et al. 1999). In Ceratothoa oestroides Risso, 1816, Nerocila orbignyi (Guerin-Meneville, 1832), N. bivittata Risso, 1816, Anilocra physodes (L.), A. mediterranea Leach, 1818, and Norileca indica H. Milne Edwards, 1840, the gonad appears with the ovary and testis, along with their ducts (Kottarathil and Kappalli 2019a b). The general anatomy of the protandrous hermaphroditic gonad of N. indica, a host-specific cymothoid that infects the scombrid fish Rastrelliger kanagurta (Cuvier, 1816), has been characterized (Kottarathil and Kappalli 2019a b). Despite these few reports, information on the hermaphroditic reproductive system and its pattern of functioning in parasitic isopods is still incomplete. Cymothoids exhibit both obligatory parasitism and protandrous hermaphroditism, so their reproductive system needs to be structurally characterized based on different hermaphroditic life cycle stages by considering more cymothoid genera and species. At this juncture, this study is relevant because it reports the organization of the hermaphroditic reproductive system in male, transitional, and female phases of a cymothoid, Mothocya renardi (Bleeker, 1857), using light and electron microscopy. Mothocya renardi infects the belonid fish species Strongylura leiura Bleeker, 1850 along the Malabar coast (Kerala, India) in a single-hostspecific manner with high prevalence (92%) (Aneesh et al. 2016). The present paper also demonstrates correlations between the 1) ovarian cycle and brood cycle and 2) ovarian cycle and molt cycle. This basic information will be helpful for understanding the importance of protandric hermaphroditism, if any, to the obligatory parasitic life of cymothoids. MATERIALS AND METHODS Sample Collection Live samples of adult life cycle phases of Mothocya renardi (Bleeker, 1857) parasitizing the fish Strongylura leiura Bleeker, 1850 were collected from the Ayyikkara fish landing center (Lat. 11°51'N, Long. 75°22'E; Malabar Coast, India). Sampling was performed weekly, and soon after collection, the live parasites were brought to the laboratory and maintained in a plastic cistern containing sea or estuarine water. Parasites without a host were found to live for a maximum of six hours. The live samples were observed and processed for further study. The adult male (n = 301; 10.0–19.0 mm), ovigerous female (n = 335; 16.0–34.0 mm), and transitional phases (n = 39; 10.5– 23.0 mm) were identified according to Aneesh et al. (2016). Dissection and photomicrography Mothocya renardi hermaphroditic reproductive system from each adult life cycle phase (male, transitional, and female) was dissected by cutting open the thoracic sternites under a stereomicroscope (LeicaS6D), then quickly rinsing it in sea or estuarine water and placing it on a clean glass slide. Photomicrography was performed using a Leica Stereo Zoom -S6 D with a Canon Power Shot S 50 camera attached and processed with the software LAS EZ (Leica Application Suit-Version 1.7.0). The tissue was then closely observed for morphological characterization under a page 2 of 15Zoological Studies 59:61 (2020) © 2020 Academia Sinica, Taiwan stereomicroscope (Leica S6) and processed for further histological and ultrastructural studies following Kottarathil and Kappalli (2019a b) Identification of ovarian stages The ovary of female-phase M. renardi was classified into seven stages based on the oocyte size: a Pre-vitellogenic stage (Pvs), five Vitellogenic stages (Vg1–Vg5), and a Spent stage (Ss). Identification of molt and brood stages The maxillule was used to identify the molt stages following the procedure described by Aneesh and Kappalli (2020). Brood stages such as Zygotic stage, Embryonic stages (ES I/ES II/ES III), and larval stages (Manca-1 and Manca-11) were classified according to Aneesh et al. (2016). Histology The dissected reproductive systems (consisting of testis, ovary, and vas deferens, along with their respective ducts) from male, transitional, and female phases of M. renardi were immediately fixed in Carnoy fixative (mixture of ethanol and acetic acid in the ratio of 3:1); microtome (paraffin) sections 3–5 µm thick were stained in Harris haematoxylin-eosin (after Pearse 1968). DPX (Merck) mounted slides were observed under the Leica Research Microscope (DM 750), and photomicrography was performed using a Leica ICC50 camera attached; the microscopic images were processed with the software LAS EZ (Leica Application Suit-Version 1.7.0). Electron microscopy The dissected tissues (ovary, testes, and vas deferens of the male phase and ovary of the female phase of M. renardi) were fixed in Karnovsky’s fixative (4% paraformaldhyde and 3% glutaraldehyde at pH 7.2) for 24 h, then processed following the procedure described by Williams and Carter (1997). The tissue was washed twice in phosphate buffer (pH 7.4) for 15 minutes and subsequently post-fixed in 1% osmium tetroxide. After washing (with phosphate buffer, pH 7.4) and dehydrating, the tissue was transferred to an enblock stain (2% uranyl acetate in 95% ethyl alcohol), followed by dehydration and clearing. Then the tissue was embedded in araldite. Semithin (1.0 µm) and ultrathin (0.5 µm) sections were cut using a Leica Em Uc6 ultra microtome fixed with a glass knife and placed in glass slides and copper grids, respectively. The semithin sections stained with methylene blue were used for the light microscopic observation. The ultrathin sections mounted in the copper grid were stained with Uranyl acetate followed by lead citrate. The stained specimens were subjected to observation under a Transmission Electron Microscope (Tecnai G2) in the NIMHANS facility (Bangalore). Correlation between stages of ovary cycle and brood cycle The stages of the ovary cycle and stages of the brood cycle of female-phase M. renardi were closely observed individually (n = 34) to assess the correlation, if any, between these cycles. Correlation between ovary cycle and biphasic molt cycle The ovary and molt cycle stages of female-phase M. renardi were closely observed individually (n = 42) to assess correlation, if any, between these cycles. Statistical analysis The mean value was calculated using the standard statistical software InStat (Graphpad InStat, Version 2.00, 2007). RESULTS Reproductive system of the male-phase M. renardi Gonads and gametes: light microscopic observations The paired and symmetrical reproductive system of M. renardi is located dorsally on either side of the gut, and each consisted of three testis lobes, one ovary, and their respective ducts (vas deferens and oviduct) (Fig. 1A). The testis lobes and ovary appeared to be closely associated structures, and the ovary formed a simple tubule 1850–2300 µm long and 350–500 µm wide. Three testis lobes were positioned at the anterolateral border of the ovary (Fig. 1A). The spermatozoa were organized in bundles to form spermatophores (Fig. 1B–D), each of which contained 84.57 ± 14.99 (n = 6) spermatozoa organized in such a way that spermatozoan tails aligned parallel to the long axis of the bundle (Fig. 1D). Mature spermatozoon appeared as an elongated structure consisting of a head containing a ribbon shaped nucleus and a long filamentous nonpage 3 of 15Zoological Studies 59:61 (2020) © 2020 Academia Sinica, Taiwan motile tail 0.06 ± 0.001 mm and 1.35 ± 0.25 mm long, respectively. The sperm head contained the nucleus; the bulb-like acrosome connected the head and tail (Fig. 1F). Gonads and gametes - Histology and ultrastructure Testis and germ cells Each testis lobe of M. renardi was composed of germ cells undergoing spermatogenesis and somatic accessory cells (Figs. 2, 3). The testis wall was composed of a basal lamina supported by connective tissue and a muscle layer (Fig. 2A–C). All germ cells occupying a particular region of the testis lobe were at the same stage of development (Fig. 2A). The differentiated primary and secondary spermatogonia cells were found confined to the periphery of the testis. Relatively large primary spermatogonia were 12.02 ± 0.08 µm in width and their spherical large nuclei were characterized by peripherally distributed condensed chromatin and one or two centrally located nucleoli (Fig. 2F). Secondary spermatogonia measured 9.05 ± 0.04 µm in width, had less cytoplasm than the primary one, and had a nucleus with scattered chromatin and a prominent nucleolus (Fig. 2B, F). Spermatocytes (Figs. 2A–D, 3A, 4A1) measured 7.95 ± 0.08 µm in width and appeared under different divisional stages, mostly meiotic prophase and metaphase (Figs. 2B, 3A). The spermatids (Fig. 3B–D) were relatively small (4.92 ± 0.1 µm in width) and highly distinct from other developing germ cells. Those under more advanced stages of spermiogenesis lay close to the somatic accessory cells lying marginal to the testis wall (Fig. 3B–E). Under the electron microscope, the chromatinrich nucleus of the spermatids appeared to be highly electron-dense (Fig. 4A2). In the longitudinal section, the sperm tail appeared as an elongated structure showing transverse striations and a central lumen filled with electron-dense material (Fig. 4D1–3). The outer margin of the tail was covered with a double-layered membrane (Fig. 4D3). The ultrastructural view showed that the spermatophore also contained an abundance of massive extracellular tubules (Figs. 4B3, 4C3, 4D1). The wedge-shaped acrosome appeared to be highly electron-dense (Figs. 4B1–2, 4C1–3). The cytoplasmic area between the acrosome and nucleus contained mitochondria (Fig. 4B2). Fig. 1. Mothocya renardi (Bleeker, 1857): Reproductive system during the male phase. A, Hermaphroditic reproductive system showing testis lobes, ovary, vas deferens, and oviduct (4X); B, spermatophores: unstained (10X); C, spermatozoa: unstained (100X). O: ovary, TL: testis lobe, VE: vas deferens, OD: oviduct, AVD: anterior vas deferens, PVD: posterior vas deferens, S: spermatophore, T: tail, A: acrosome, H: head. page 4 of 15Zoological Studies 59:61 (2020) © 2020 Academia Sinica, Taiwan Ovary and germ cells The histological sections of the ovary of malephase M. renardi showed a narrow channel running along its lateral margin and lumen with an abundance of small oocytes with a prominent nucleus and one or two nucleoli (Fig. 5A–B). Each oocyte was encircled by a layer of follicle cells (Fig. 5A–B). Vas deferens Both anterior and posterior vas deferens of male-phase M. renardi had many spermatophores resembling those found in the testis lobe. In the electron microscopic view, the lumen of the vas deferens had an abundance of extracellular tubules, and their density was higher in the vas deferens than the testis lobe (Fig. 6A–C). Reproductive system in the transitional-phase M. renardi During the transitional phase, the sac like testis lobes appeared withered and occasionally contained several spermatophores (Fig. 7A). Histologically, the posterior part of the vas deferens was filled with spermatophores (Fig. 7B), but its posterior extremity was sealed throughout this phase. Histologically, the ovary showed the presence of relatively large oocytes with a nucleus and one or two nucleoli; each oocyte was encircled by a follicular layer (Fig. 7C). Though the oviduct was relatively wider than that found during the male stage, its extremity remained sealed throughout the transitional phase. Ovary in the female-phase M. renardi The kidney-shaped ovary consisted of 8–10 cone-shaped pouches, each containing 42–62 oocytes (Fig. 8C). During the Pvs (previtellogenic stage), the relatively small ovary contained a large number of proliferating oocytes ranging from 1–150 µm long (Fig. 9A), each with a large nucleus and nucleolus. The wall of the previtellogenic ovary contained highly branched foldings throughout its length (Fig. 10A and B). During the vitellogenic stages (Vg1–Vg5), the ovary contained yolky oocytes—as was evident by the presence of highly basophilic yolk globules—which showed a Fig. 2. Mothocya renardi (Bleeker, 1857): Testis lobe during the male phase. A–D, Semithin sections (CS; 1 µm: toluidine blue stained). A, Acini and androgenic gland (100X); B, wall showing muscle band (100X); C, somatic accessory cells and germ cells during different stages of maturation (40X); D, enlarged view showing somatic accessory cells (100X). E, Histological section (CS; 4 µm: haematoxylin-eosin) showing spermatogonial cells (100X). F, Primary and secondary spermatogonia (100X). Sc: spermatocytes, Sac: somatic accessory cell, W: wall, Ag: androgenic gland, M: muscle layer, SSg: secondary spermatogonia, Sg: spermatogonia, N: nucleus, Psg: primary spermatogonia. page 5 of 15 Zoological Studies 59:61 (2020) © 2020 Academia Sinica, Taiwan concomitant increase in number from Vg1 through Vg5; the oocytes contained a visible nucleus and nucleolus, and each oocyte was encircled by a layer of follicle cells throughout all the stages (Fig. 9B–E). The oviduct (Fig. 8A and C) arising from the posterolateral margin of each ovary was opened at the 6th thoracic segment. After oviposition, the spent staged (Ss) ovary appeared as an opaque and flaccid structure containing few residual eggs (Fig. 8F). Histologically, the Ss ovary contained residual follicle cells of the preceding clutch of oocytes Fig. 3. Mothocya renardi (Bleeker, 1857): Histology of testis lobes during the male phase. A, Semithin section (LS; 1 µm: toluidine blue) showing spermatocytes during meiotic division. B–E, Cross sections (4 µm: haematoxylin-eosin) showing the spermatids during different maturation stages. Sc: spermatocytes, Sd: spermatid, ImS: immature spermatozoa, T: tail, N: nucleus, H: head, Sac: somatic accessory cell. page 6 of 15Zoological Studies 59:61 (2020) © 2020 Academia Sinica, Taiwan (Fig. 9F). Correlation between ovarian cycle and brood development in M. renardi during the female phase While the female M. renardi was brood carrying, a second clutch of oocytes started developing in the ovary. Interestingly, the stages in the succeeding ovarian cycle showed a perfect synchrony with the brood development (Fig. 11A). Immediately after oviposition, the spent stage ovary appeared as an opaque, thin, and flaccid structure; the eggs remained in the brood pouch during the zygotic stage (uncleaved stage). While the ovary was in the Pvs, eggs in the marsupium completed cleavage and reached any of the three embryonic stages (ES I/ES II/ES III). When the ovary entered the Vg1, the ES III-stage eggs in the marsupium hatched into the first larvae, Manca-1. When the ovary reached Vg2, the brood pouch of the mother animal contained the next stage of larvae, Manca-II, or was empty (after releasing the Manca-II). When the female molted to remove the old brood pouch, oocytes were at Vg3. No brood pouch was found in females, while their ovary was at stage Vg5. When the ovary reached late Vg5 (oocytes 1230 µm in width), the female possessed a partially or completely formed brood pouch to receive the next clutch of eggs. Fig. 4. Mothocya renardi (Bleeker, 1857): Electron micrograph of the testis lobes during the male phase: A1, Spermatocyte (4800X); A2, spermatids (9300X); A3, somatic accessory cells and portion of spermatophore (2900X); B1, cross section through spermatozoan head (4800X); B2, longitudinal section through spermatophore (4800X); B3, cross section through spermatozoan tail (6800X); C1, spermatozoan head region (6800X); C2, spermatozoan tail, acrosome and extracellular tubules (6800X); C3, spermatozoan acrosome and tail (18500X); D1, spermatozoan tail surrounded by extracellular tubules (11000X); D2, spermatozoan tail (30000X) showing transverse striations and central lumen (insight: wall of spermatozoan tail with double membrane); D3, a portion of spermatozoan tail showing central lumen filled with electron-dense granules (68000X). Sc: spermatocyte, N: nucleus, M: mitochondria, Pm: plasma membrane, Sd: spermatid, T: tail, H: head, Sac: somatic accessory cell, A: acrosome, Ect: extracellular tubule, Sz: spermatozoa, S: spermatophore, Edg: electron-dense granules, Ts: transverse striations, Dm: double membrane. page 7 of 15Zoological Studies 59:61 (2020) © 2020 Academia Sinica, Taiwan Correlation between ovary cycle and biphasic molt cycle in M. renardi during the female stage In M. renardi each ovary cycle was accompanied by two molt cycles (one to remove the old brood pouch and the other to form the new brood pouch) (Fig. 11B). Furthermore, we found a correlation between the molt stages and ovary stages of M. renardi. When the ovary was at the Ss/Pvs/Vg1 stages, the female remained in the intermolt stage. As the ovary stage advanced to Vg2, the female reached the early premolt stages (D0– D2) at its posterior part. When the ovary stage reached Vg3, the parasite entered late premolt stages (D3/D4) at its posterior part or completed the ecdysis (of posterior body part). During the period between Vg4 and Vg5, the parasite completed the ecdysis of the anterior body part. By the end of this biphasic molt cycle, the old brood pouch had disappeared and the individual entered into the second molt cycle (to form the new brood pouch). When this molting process was over, the females were found to have an ovary at Vg5. Oviposition ensued when the females reached the intermolt stage. DISCUSSION Isopods use different modes of reproduction and mating strategies, and these have been explored widely in free living forms (Shuster and Wade 1991 2003). Parasitic isopods like epicarids and cymothoids display protandrous hermaphroditism, and the underlying transitions from male to female phases are critical for these parasites. In all three identified hermaphroditic sexual forms of M. renardi (male, transitional, and female), the paired and symmetrical gonad is intersexed and each consists of three testis lobes, an ovary, and ducts in the vas deferens and oviduct that lead to the respective gonopore. Throughout the male phase, the ovary contains yolkless oocytes with nuclei and more than one nucleolus, indicating that they are not in a Fig. 5. Mothocya renardi (Bleeker, 1857): Ovary during the male phase. A, Semi thin section (LS; 1 µm; toluidine blue) showing germarium and previtellogenic oocytes encircled by follicle cells (100 X). B, Electron micrograph of ovary (LS; 0.5 µm) showing previtellogenic oocyte encircled by follicle cells (1400X). N: nucleus, n: nucleolus, Pm: plasma membrane, Oo: oocyte, Gz: germinal zone, Ow: ovarian wall, C: cytoplasm, Fc: Follicle cell, V: vacuole. Fig. 6. Mothocya renardi (Bleeker, 1857): Electron micrographs of the cross sections of the vas deferens during the male phase showing spermatophore. A, Head regions (3200X); B, tail regions (6800X); C, tail regions showing central lumen with electron-dense granules (30000X). N: nucleus, T: tail, H: head, Ect: extracellular tubule, S: spermatophore, Edg: electron-dense granules, L: lumen. page 8 of 15Zoological Studies 59:61 (2020) © 2020 Academia Sinica, Taiwan dormant state (Fig. 11). When the male enters the transitional phase, oocytes increase in both number and size, and yolk deposition occurs. The transformation of the ovary from the previtellogenic stage into the vitellogenic stage through the transitional phase indicates that M. renardi undergoes a gradient of feminization, which agrees with the observations of the freshwater crayfish Parastacus brasiliensis von Martens 1869 (Almeida and Buckup 1997), the caridean shrimp Samastacus spinifrons Philippi, 1882 (Rudolph 1999), and the cymothoid N. indica (Kottarathil and Kappalli 2019a b). In M. renardi, when the members transform from the male to transitional phase through molting, gonopores (opening of oviduct and vas deferens) remain sealed and the penes become rudimentary, indicating the termination of the male phase. The subsequent female phase contains the brood, and the ovary also contains yolky oocytes, while the testis lobe and vas deferens contain leftover spermatophores. The present morphological observation together with histological studies on the gonad of M. renardi confirm the existence of sequential protandrous hermaphroditism similar to that reported in N. indica (Kottarathil and Kappalli 2019a b) and P. brasiliensis, in which the protandrous hermaphroditic sexual stages are termed intersexed males, transitionals, and intersexed females. In another crayfish species, Parastacus nicoleti Philippi, 1882, the male gonad contains the previtellogenic ovary, but it is not distinct from the testis and appears as an ootestis (Rudolph 1999). Contrary to this pattern, in Fig. 7. Mothocya renardi (Bleeker, 1857): Reproductive system during the transitional phase. A, Reproductive system showing oocyte-filled ovary, spermatophore-filled vas deferens, and oviduct (4X); B, histological section (LS; 4 µm; hematoxylin-eosin) through vas deferens showing spermatophores (20X); C, histological section (LS; 4 µm; hematoxylin-eosin) through ovary showing yolky oocytes (20X). O: ovary, TL: testis lobe, OD: oviduct, AVD: anterior vas deferens, PVD: posterior vas deferens, Oo: oocyte, N: nucleus, n: nucleolus, Fc: follicle cell, S: spermatophore. page 9 of 15Zoological Studies 59:61 (2020)