Reproductive System in the Male Phase of a Parasitic Isopod (Crustacea) - Morphological, Histological and Ultrastructural Evidence for Sequential Protandrous Hermaphroditic Changes

© 2019 Academia Sinica, Taiwan Open Access Reproductive System in the Male Phase of a Parasitic Isopod (Crustacea) – Morphological, Histological and Ultrastructural Evidence for Sequential Protandrous Hermaphroditic Changes Helna Ameri Kottarathil1 and Sudha Kappalli2,* 1Post Graduate Department of Zoology and Research Centre, Sree Narayana College, Kannur 670 007, India. E-mail: [email protected] 2Department of Animal Science, School of Biological Sciences, Central University of Kerala, Kasaragod, Kerala, India. *Correspondence: E-mail: [email protected] Received 5 October 2018 / Accepted 28 February 2019 / Published 27 March 2019 Communicated by Benny K.K. Chan This paper reports the protandric hermaphroditic changes in the reproductive system of the malephased Norileca indica, a cymothoid that parasitizes the scombrid fish Rastrelliger kanagurta. Each part of N. indica’s paired reproductive system lies on either side of the gut. This study considers the three successive size classes of the male phase – designated as M1, M2 and M3 – using light microscopy and ultrastructural methods. The testis comprises of three bulged sac-like lobes labelled t1, t2 and t3, all of which open into the ovary of their respective side. The vas deferens, which emerges as a posterior extension of the ovary, opens into the penis and the distal end of each oviduct leads to a sealed gonopore on their respective sides. Each testis lobe (t1/t2/t3) displays clusters of germ cells undergoing stage-specific differentiation. Spermatids undergoing sequential changes associated with spermiogenesis keep close proximity to somatic accessory cells. The characteristic histological changes associated with protandric hermaphroditism are visible in the ovaries of sequential size classes (M1, M2 and M3). In early M1, besides spermatophores, the ovary has abundant polymorphic nuclei; in the mid/late M1, the posterior ovary has abundant spermatophores, anterior displayed oogonia, previtellogenic oocytes and two distinct forms of follicle cells. In M2, the anterior ovary shows compactly arranged oocytes while the posterior region accommodates spermatophores – fewer, however, than during M1. The entire ovary during M3 is crowded with previtellogenic oocytes, which marginalize the spermatophore passage. The vas deferens of the smallest M1 lack spermatophores. As the size class progresses through late M1 into M2 and M3, the posterior vas deferens is filled with spermatophores, which closely associate with the glandular epithelial lining. Key words: Norelica indica, Protandric hermaphroditism, Male reproductive system, Testis, Ovary, Spermatogenesis, Spermiogenesis. Citation: Kottarathil HA, Kappalli S. 2019. Reproductive system in the male phase of a parasitic isopod (Crustacea) – morphological, histological and ultrastructural evidence for sequential protandrous hermaphroditic changes Zool Stud 58:4. doi:10.6620/ZS.2019.58-04. BACKGROUND Cymothoids, the parasitic isopod crustaceans, are known to be obligatory parasites of fishes from diverse ecosystems and cause deleterious impacts on them (Trilles et al. 2011 2012; Elshahawy and Zoological Studies 58: 4 (2019) doi:10.6620/ZS.2019.58-04 1 © 2019 Academia Sinica, Taiwan Desouky 2012; Hadfield et al. 2013; Aneesh et al. 2013 2014 2015a b 2018). They have also been well recognized as protandrous hermaphrodites, spending the first part of their adult life as males and the later part as females with profuse breeding (Cressey 1983; Brook et al. 1994; Subramoniam 2013, 2016; Ghiselin 1969; Cook 2012). Morphological evidence of protandrous hermaphroditism has been reported in a few cymothoids infesting teleost fishes (Legrand 1952; Juchault 1965; Tsai et al. 1999). The female members of Ichthyoxenus fushanensis, a flesh burrowing cymothoid, have a vestigial penis signifying that the species is protandrous hermaphrodite (Tsai et al. 1999). In Cymothoa oestroides, Nerocila maculata, N. bivittata, Anilocra physodes, A. mediterranea and Mothocya renardi, the gonad appears with the ovary and testis, along with their ducts (Bullar 1876; Aneesh 2014). Despite these sporadic reports, information on the hermaphroditic reproductive system in parasitic isopods and its pattern of functioning are still lacking. Norileca indica, a cymothoid that profusely infects the Indian mackerel Rastrelliger kanagurta, displays characteristic morphological features associated with sequential protandrous hermaphroditism from the juvenile to the adult phase; these comprise three successive phases such as male, transitional and female (Helna 2016). While this cymothoid (N. Indica) is in its male phase, no female morphological characters are expressed. An interesting question about N. indica is if the reproductive system in the male phase is devoted exclusively to the production of male gametes, or if the gonad simultaneously begins to prepare for the female gametes. This paper demonstrates for the first time the sequential protandrous hermaphroditic changes in N. indica’s reproductive system during its male phase. The reproductive system of three sequential and morphologically distinct size classes during the male phase—designated M1, M2 and M3—were considered for the present light microscopic and ultrastructural studies. An attempt was also made to gather data on spermatogenesis, spermiogenesis, sperm discharge and the structure of spermatozoa in the candidate species. MATERIALS AND METHODS Collecting Norileca indica Norileca indica was collected from the branchial cavity of its host fish, Rastrelliger kanagurta, obtained from the Ayikkara Fish Landing Centre (11°51'33"N, 75°22'30"E; Malabar Coast, India) from July 2011-July 2015. Additional collections were made from March 2017-May 2018. Three sequential and morphologically distinct size classes (M1, M2, M3) of the N. indica adult male phase were categorized using body length and width as criteria. M1 (early size male formed right from the juvenile through molting) has 9.0-12.9 mm long and 3.5-4.5 mm wide. The size of M2 ranges from 13.0-17.9 mm in length and maximum 7.0 mm in width and that of M3 is 18.0-20.0 mm in length and maximum 12.0 mm in width. Morphological study of reproductive systems and gametes The hermaphroditic reproductive system from all size classes (M1, M2 and M3) of male N. indica was dissected out by cutting open the terga in 0.9% saline solution under a stereo dissection microscope (LeicaS6D) and their stage-specific morphological features were observed. Gonad size was measured and visible cells counted using micrometry. The gonad was incised using a fine needle and the spermatozoa that oozed out were observed under a Leica microscope (DM 750). Histology and histochemistry of the reproductive system Ovary, testis and their respective ducts were separated from the formol-alcohol fixed hermaphroditic reproductive system derived from M1, M2 and M3. The tissues were paraffin wax embedded, sectioned (3-4 µm thickness) and double stained with Harri’s haematoxylin and 1% alocoholic eosin (Humason 1967). For the purpose of light microscopic observation, semi-thin sections (1.0 µm) of Karnovisky-fixed (4% paraformaldehyde and 3% glutaraldehyde at pH 7.2) gonads (ovary and testis) were also stained with methylene blue. The histochemistry was done using Mercuric Bromophenol Blue (proteins), PAS (carbohydrates), Sudan Black (lipids) and Basic Fuchsin (DNA) (Pearse 1968). Electron microscopy The hermaphroditic reproductive system from M1, M2 and M3 was fixed in Karnovsky’s fixative for 24 hours and processed according to the procedure described by Williams and Carter (1996). Briefly, the fixed tissue was washed with phosphate buffer (pH 7.4) for approximately 15 minutes and post-fixed in 1% osmium tetroxide (OsO4). After further washing with phosphate buffer for half an hour, the tissue was dehydrated in ethanol grades followed by en bloc staining (2% uranyl acetate in 95% ethyl alcohol) and dehydration. Clearing and embedding were carried out in propylene oxide and araldite, respectively. Ultrathin page 2 of 16Zoological Studies 58: 4 (2019) © 2019 Academia Sinica, Taiwan sections (grey to silver) stained with uranyl acetate and lead citrate were examined under a Transmission Electron Microscope (Tecnai G2) in the facility extended from the National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore. Documentation Photomicrography was done using a Leica ICC50 camera (attached to a Leica research microscope -DM750) and Canon Power Shot S 50 camera (attached to a Leica Stereo Zoom -S6 D) and microscopic image capturing and processing software (LAS EZ, Leica Application Suit-Version 1.7.0). RESULTS Morphology of hermaphroditic reproductive system in male-phased N. indica In male-phased N. indica (Fig. 1A), the paired hermaphroditic reproductive system lies one on either side of the gut below the nerve tract and extends between the 4th and 6th pereonites. Both the ovary and testis on each side form a continuous structure in which the ovary appears simple and elongated while the testis comprises three bulged sac-like lobes labelled t1, t2 and t3, located on the antero-lateral side of the ovary (Fig. 1B). The vas deferens emerges as a posterior extension of the ovary and opens into the penis located Fig. 1. Norileca indica and its protandrous hermaphroditic reproductive system. A. Norileca indica – male; B. paired structure of gonad (20 X); C-E. Gonad in different male stages (C: M1, D: M2, E: M3) (40 X); F. testes lobes showing different staged cells and germarium. t1, t2, t3testis lobes 1-3, oovary, odoviduct, vdvas deferens, avdanterior vas deferens, pvdposterior vas deferens, Oooocytes, spspermatophore, agandrogenic gland. A B C EF D page 3 of 16Zoological Studies 58: 4 (2019) © 2019 Academia Sinica, Taiwan at the sternite 7. The slender and inconspicuous tubulelike oviduct (Figs. 1 B-E) extends laterally from the mid ovary and ends in the sealed gonopore located at the 6th pereonite (Fig. 1D); the average length of the reproductive system found in M1, M2 and M3 is 1.38 mm, 1.39 mm, and 1.40 mm, respectively (Table 1). Testis Testis was significantly different between M1, M2 and M3 (Table 1). Irrespective of size class, all the three testis lobes (t1, t2 and t3) were bulged and t1 was relatively large in size compared to t2 and t3 (Table 1). All three lobes tapered posteriorly and while t1 and t2 were close to each other, sharing a common duct leading to the antero-lateral side of the ovary; t3 was slightly isolated from the duo, and light microscopic observations showed it had a separate duct (Figs. 1C; 2A). The androgenic gland was seen attached to the antero-lateral side of t1 (Fig. 1D, E). Germ cells undergoing different stages of differentiation were clearly visible while viewing the gonad under the light microscope (Fig. 1F). The testis in M1 had abundant primary and secondary spermatogonia (Fig. 2B) and, in the successive stage (M2), spermatocytes—as well as spermatogonia—were also visible at different meiotic stages (Figs. 2C, D). In M3, the testis had abundant spermatogonia, spermatocytes, spermatids, spermatozoa and spermatophores (Fig. 2E). Both primary and secondary spermatogonia usually occupy the periphery of the testis, irrespective of the lobe (t1, t2 and t3). In our close observation, it is also evident that each testis lobe displays clusters of germ cells undergoing specific stage of differentiation; for instance, in t1, while one half portion is abundant with spermatozoa/spermatophores, the other half houses the germ cells undergoing spermatogenesis, of which meiotically dividing spermatocytes are dominant (Fig. 3A). Most of the spermatophores were found to be closely associated with somatic accessory cells (Fig. 3B). The histological section of t2 (of the same testis) shows the presence of spermatogonia as well as spermatocytes, whereas t3 not only has abundant spermatogonia and spermatocytes but also spermatids (Figs. 3C, D). The spermatids (in t3) were also seen at different stages of spermiogenesis (Fig. 3D). Histochemically, the testis lobes of M1, M2 and M3 tested positively for carbohydrate, protein and lipid (Fig. 4). Germ cells undergoing differentiation: Light and Electron microscopic view The primary spermatogonia (7.0-11.0 µm) possessed a relatively large nucleus with nucleolusand peripherally-distributed chromatin; in the nucleus of secondary spermatogonia (5.0-9.0 µm), the chromatin was found to be more or less scattered (Fig. 2B). Under the light microscope, both primary and secondary spermatocytes (size 6.90 ± 0.42 µm) were characterized by the presence of highly condensed chromatin, and many of them were found with meiotic stages (Figs. 2C, D). Ultrastructurally, spermatocytes were rich in mitochondria and membrane-bound vesicles (Fig. 5A). Relatively small (3.79 ± 0.39 µm) spermatids with centrally placed spherical nuclei along with its nucleoli were distinct (Figs. 2E; 3D). In both light and electron microscopic view, many of the spermatids kept close in proximity to the somatic accessory cells and were likely under the process of spermiogenesis. By the onset of spermiogenesis, the nucleus of the newly-formed spermatids conical bulges, apparently for the formation of acrosomal vesicle (Figs. 2E, F), and the heterochromatin condenses and remains close to the nuclear envelope, resulting in the formation of incomplete rings of variable thickness. With the progress of spermiogenesis, the bulged nuclear pole elongates while the opposite pole remains vesicular. The Table 1. Micrometric measurements of reproductive structures in N. indica’s male phase Male stages Reproductive structures with size Testis Ovary Vas deferens Oviduct M1 t1: 344.704 ± 96.41 µm in length t2: 244.55 ± 53.86 µm in length t3: 228.67 ± 42.95 µm in length 791.405 ± 12.92 µm in length 1650 ± 0.95 µm in length, 32.4 ± 2.07 µm in width 272 ± 2.64 µm in length, 19.07 ± 0.69 µm in width M2 t1: 352.48 ± 82.96 µm in length t2: 283.35 ± 106.57 µm in length t3: 256.012 ± 102.76 µm in length 1800.58 ± 0.62 µm in length 2941.007 ± 59.93 µm in length, 98 ± 0.5 µm in width 277.66 ± 19.39 µm in length, 19.23 ± 0.05 µm in width M3 t1: 387 ± 142.14 µm in length t2: 277.81 ± 219.74 µm in length t3: 259.85 ± 123.28 µm in length 3407 ± 26.14 µm in length 2680.14 ± 65.32 µm in length, 289.25 ± 120.9 µm in width 275.2 ± 14.23 µm in length, 23.25 ± 0.35 µm in width page 4 of 16Zoological Studies 58: 4 (2019) © 2019 Academia Sinica, Taiwan gradual elongation of the nuclear pole eventually results in the formation of a ribbon-shaped nucleus with the simultaneous reduction of cytoplasm and subsequent degeneration of the vesicular part (Fig. 2F). Overall transformation culminates in the formation of uniqueshaped spermatozoon with ribbon-like nuclear head and apexed acrosome thickly wrapped with extracellular tubules (Figs. 2H; 5C). The cell boundaries gradually disappear and the nuclei are seen in the common cytoplasmic mass; many of these nuclei found clustered Fig. 2. Norileca indica – hermaphroditic gonad in male stages. A. anterior gonad showing the connection between the testis lobes and the ovary (200 X); B. testis (M1) showing spermatogonia (1000 X); C. testis (M2) showing spermatocytes at equatorial plate forming stage of meiosis and somatic accessory cells (1000 X); D. spermatocytes at different meiotic stages (1000 X) (semithin LS: 1 µm; Methylene blue). Testes showing E. regionalization of germ cells undergoing spermatogenesis and spermiogenesis (M3) (200 X); F. spermiogenesis (arrow showing elongation of spermatid nucleus) (1000 X); G. somatic accessory cell types (1000 X); H. somatic accessory cell, spermatophores and spermatozoa (400 X). t1, t2, t3 - testis lobes 1-3, Oooocyte, wwall, psgprimary spermatogonia, ssgsecondary spermatogonia, scspermatocyte, sacsomatic accessory cell, oovary, fcfollicle cell, sacsomatic accessory cell, sdspermatid, scspermatocyte, nnucleus, spspermatophore, sspermatozoa, sac 1, sac 2, sac 3 - somatic accessory cell types 1-3, wwall, Mmuscle. A C F E B D G H page 5 of 16Zoological Studies 58: 4 (2019) © 2019 Academia Sinica, Taiwan and the cytoplasmic extremities become more elongated to form tails and their further association culminates in the formation of the characteristic sperm bundle designated as spermatophore (Figs. 4A-D). Spermatophore and spermatozoon: Light and electron microscopic view In eosin-hematoxylin stained smear preparation, the spermatophore’s nuclei were basophilic, while the tails were strongly acidophilic (Fig. 4F). Under the electron microscope, the longitudinal and cross sections of spermatophore tails appeared as a cluster of discs and each disc (representing the cross/oblique section of a sperm tail) showed the presence of a central lumen filled with electron light and dense materials (Figs. 5D, E). Also, the cross sections of spermatophore’s nuclei were seen as electron dense discs. The spermatophores, closely associated with the somatic accessory cells were found to be enriched with both RER and mitochondria (Figs. 5F, G). Spermatozoon was characterized by its long, non-motile tail (length 0.90 ± 0.06 mm) and ribbon-like nucleus (0.06 ± 0.01 mm), joined together with an acrosome to form the head. The head projected laterally from the tail at an acute angle, giving the spermatozoon a flag-like appearance, tapering at its distal end (Figs. 4D, E; 5H). The tail expressed a high degree of histochemical affinity to MBB and PAS tests but poor affinity towards Sudan Black. On the other hand, the nucleus was strongly/moderately positive for all the tested histochemical stains (Basic Fuchsin, MBB, Sudan Black and PAS) (Figs. 4G-I). The nuclei of the spermatophore were embedded in a large number of microtubules (Fig. 5C) and the mitochondria were found between the nucleus and the acrosome (Fig. 5G). Somatic accessory cell: Light and Electron microscopic view In the testes of N. indica, a somatic accessory cell (sac) appears to be large, with an irregular and polymorphic nucleus and its three forms were identified and labelled sac 1, sac 2 and sac 3 (Figs. 2G, H). Sac 1 occupied the periphery of the testes and possessed an oval/spherical basophilic nucleus 16.5 ± 2.10 µm large; under the electron microscope, the nucleoplasm Fig. 3. Histological section of testicular lobes (t1, t2, t3) of male N. indica (LS: 3 µm, Haematoxylin-Eosin) depicting asynchrony in spermatogenesis (1000 X) A. t1 showing spermatocytes and spermatophores; B. t1 showing the association between spermatophores and somatic accessory cells; C. t2 showing spermatocytes at pachytene stage and spermatogonia; D. t3 showing spermatocytes at equatorial plate forming stage of meiosis (arrow), spermatid and somatic accessory cells. sp - spermatophore, scspermatocytes, sacsomatic accessory cells, sgspermatogonia, sdspermatid. A C B D page 6 of 16Zoological Studies 58: 4 (2019) © 2019 Academia Sinica, Taiwan was enriched with electron-dense heterochromatin that adhered to the membrane (Figs. 5B, F). The nucleus of sac 2 possessed a distinct nucleolus 9.14 ± 1.48 µm in size (Fig. 2G). sac 3 appeared to have a more elongated spindle-shaped nucleus 14.21 ± 0.98 µm large and its pattern of chromatin condensation was different from the former ones (Fig. 2H). Fig. 4. N. indica male A. spermatophores (unstained) (100 X); B. methylene blue stained single spermatophore (200 X); C. spermatophore heads showing positivity to methylene blue (1000 X); D. spermatophore (unstained) (1000 X); E. spermatozoon showing distinct nucleus, acrosome and tail (1000 X); F. spermatophores (Haematoxylin-Eosin) (400 X); G. spermatophore heads showing positivity to basic fuchsin (1000 X); H. spermatophore showing positivity to bromophenol blue (1000 X); I. spermatophore heads showing positivity to Sudan Black (1000 X). spspermatophore, ttail, aacrosome, nnucleus, hhead. A DE G H I F B C page 7 of 16Zoological Studies 58: 4 (2019) © 2019 Academia Sinica, Taiwan Fig. 5. Electron micrographs of testis in male stage of N. indica showing A. spermatocyte (arrow) at meiotic stage (equatorial plate formation) (2900 X), B. somatic accessory cells (2900 X), C extracellular tubules and nuclei of spermatozoa (6800 X), D. tails of spermatozoa showing striations and central lumen (13000 X), E. clustered spermatophore tails (13000 X), F. spermatophore associated with somatic accessory cells (4800 X), G. somatic accessory cell cytoplasm showing cell organelles (30000 X), H. head of spermatozoon (13000 X). sacsomatic accessory cell, scspermatocyte, csomatic accessory cell cytoplasm, nsomatic accessory cell nucleus, nsomatic accessory cell nucleus, tspermatophore tail, tstransverse striations, mmitochondria, RERRough endoplasmic reticulum, llumen, nnucleus, llumen, hhead, crchromatin, ectextracellular tubules, aacrosome. A C E G B D F H page 8 of 16Zoological Studies 58: 4 (2019) © 2019 Academia Sinica, Taiwan Ovary M1: Ovary (average length - 568.0 µm) of the prestaged M1 (size - 8.8 mm) showed the presence of cells with large vacuoles and deeply stained peripheral nuclei (Fig. 6A). In M1 (size 9.0 mm), the ovary (size 1.05 ± 0.21 mm) had abundant oogonial cells and spermatophores (Fig. 7B). The histological sections displayed a large number of randomly distributed polymorphic nuclei (size 4.7 - 7.5 µm) with condensed chromatin, which are apparently the precursors of the prospective oocytes or follicle cells (Figs. 6B; 7A). Our histological observations concluded that the epithelial wall of the ovarian lobe is secretory (Fig. 7G). During the mid and late stages of M1, spermatophores were found aggregated at the posterior ovary, orienting their heads invariably toward the mouth of the vas deferens (Fig. 7E). Meanwhile, the anterior ovary had abundant previtellogenic oocytes (size 276.02 ± 44.53 µm) with large nuclei (size 135.82 ± 18.71 µm) and nucleoli. Antero-laterally, the ovary was occupied by high prolific germarium and differentiating/differentiated oogonial cells (Fig. 1E). Some of these cells (size 121.56 ± 10.66 µm) possessed a large nuclei (size 82.48 ± 9.30 µm) and the cytoplasm—filled with vesicles containing dense basophilic granules—were displaced towards the posterior ovary (Fig. 7C). Two forms of follicle cells (labelled fc1 and fc2) with size ratio 2:1 were found to be associated with the oocytes; fc1 (size 6.94 ± 1.07 µm) appeared with a highly basophilic round/oval nucleus (size 5.9 ± 0.26 µm) and a rim of cytoplasm; the nucleus of most fc1 cells also had relatively large condensed chromatin granules and nucleolus (Fig. 7F). The relatively elongated fc2 cells displayed a distinct nucleus (8.9 µm) and highly condensed chromatin, but no distinct nucleolus (Figs. 7B, F). Ultrastructurally, the cytoplasm of both fc1 and fc2 were enriched with RER, golgi, free ribosomes, mitochondria and secretory vesicles and some of the vesicles were found fused with the plasma membrane (Figs. 8C-E). Two subpopulations of fc1 were also recognized; one with a rim of cytoplasm and a large, oval nucleus showing diffused electron lucent euchromatin and peripheral electron-dense heterochromatin; the other with spherical large nucleus had thread-like heterochromatin and a high proportion of euchromatin (Fig. 8A). M2: The ovary in M2 attained a length of 1.86 ± 0.62 mm and accommodated a large number of yolkless (previtellogenic) oocytes (size 121.56 ± 10.66 µm) with a distinct nucleus (size 82.48 ± 9.30 µm) and a Fig. 7. Histological and histochemical observation of ovarian germ cells and oocytes (N. indica) during male phase of N. indica. (LS: 4 µm; Haematoxylin-Eosin; 1000 X) A. germ cells (in M1); B. spermatophore and germ cells (in M1); C. follicle cells enveloping the oocytes (in M2); D. oocytes (in late M3); semithin LS: 1 µm; methylene blue showing E. spermatophores (M1) (400 X); F. follicle cells, previtellogenic oocytes and spermatophores (M2 1000 X); G. bulged ovarian wall (arrow) (400 X); H. ovarian wall showing involution (M2) (arrow) (400 X). ggerm cells, spspermatophore, Oooocytes, fcfollicle cells. spspermatophore, ttail, Oooocytes, fc1follicle cell type 1, fc2follicle cell type 2, nnucleus, ssperm, wwall. Fig. 6. Histology of ovary in male stages (M1, M2, M3). A. prestaged M1 ovary B. M1 ovary. A B A E B F C G D H page 9 of 16Zoological Studies 58: 4 (2019) © 2019 Academia Sinica, Taiwan Gabala E. 2008. Ultrastructure of spermiogenesis in the marine isopod Saduriaentomon (Crustacea, Isopoda). Invertebr Reprod Dev 51(1):33–47. doi:10.1016/j.asd.2007.11.004. Ghiselin MT. 1969. 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