Fine structures of the mantle tissue in the pinkish-brown salp <i>Pegea confoederata</i> (Tunicata: Thaliacea)

Plankton Benthos Res 13(3): 129–135, 2018 Plankton & Benthos Research © The Plankton Society of Japan Fine structures of the mantle tissue in the pinkish-brown salp Pegea confoederata (Tunicata: Thaliacea) Euichi hirosE1,* & Jun nishikawa2 1 2 Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903–0213, Japan Department of Marine Biology, School of Marine Science and Technology, Tokai University, Orido, Shimizu, Shizuoka 424– 8610, Japan Received 30 March 2018; Accepted 5 June 2018 Responsible Editor: Dhugal Lindsay doi: 10.3800/pbr.13.129 Abstract: Pegea confoederata is a salp with a pinkish-brown body. The color was retained in the mantle tissue, while the tunic was transparent. We examined the fine structures of the mantle tissue to clarify the cytological basis of the body coloration. Apical cytoplasmic bulges of the epidermal cells were associated with dense tunic fibers, suggesting an involvement in the secretion of the tunic. Light microscopy analysis of the mantle revealed pigment cells that are dendroid-shaped hemocytes filled with brown granules. Five types of hemocytes were recognized in the hemocoel, based on their ultrastructure, and the pigment cells in the present species were hemocytes classified as ʻstorage cellʼ. Additionally, some hemocytes were seen to have migrated into the tunic through the epidermis, and were supposed to be presumptive tunic cells. Key words: Pelagic tunicate, Body color, Electron microscopy, Hemocyte types, Tunic, Pegea Introduction The subphylum Tunicata (Urochordata) is the closest sister group of Vertebrata among the extant metazoans. Tunicates are characterized by a cellulosic tissue that is secreted from the epidermis, called the tunic in Ascidiacea and Thaliacea (Belton et al. 1989, Van Daele et al. 1992, Hirose et al. 1999) and the house in Appendicularia (Kimura et al. 2001). Additionally, the presence of an intracellular, cellulosic matrix was suggested in the tail epidermis in appendicularians (Hirose et al. 2011, Nakashima et al. 2011). Salps (Salpida: Thaliacea) usually have a transparent body̶a trait also seen in many other gelatinous zooplankton̶that probably helps them avoid visual detection by predators. Accordingly, the tunic of salps is expected to have a high transparency and a low reflectance in seawater (e.g., Hirose et al. 2015, Kakiuchida et al. 2017). In contrast to other salps, Pegea confoederata (Forskål, 1775) often has a unique pinkish-brown body coloration. This conspicuous color in the epipelagic layer suggests that a reduction in visibility is not crucial for the survival of * Corresponding author: Euichi Hirose; E-mail, [email protected]. ac.jp this species. In P. confoederata, the pinkish-brown color is retained in the mantle, which is comprised of the epidermis, mesenchymal space (hemocoel), and peribranchial epithelium, while the tunic is transparent and rarely reflective (Sakai et al. 2018). Sessile tunicates (ascidians) often have colorful bodies that are comprised of hemocytes (e.g., Burighel et al. 1983, Hirose et al. 1998) or tunic cells, or both (e.g., Hirose 1992, Turon et al. 2005). Salps are also known to have several types of hemocytes (e.g., Pérès 1943). Cima et al. (2014) described five types of hemocytes in the transparent salp Thalia democratica (Forskål, 1775), based on the ultrastructures, and characterized some functions of each hemocyte type by means of histochemistry and immunohistochemistry. In the present study, we examined the ultrastructure of the mantle of P. confoederata to clarify the cytological basis of its pinkish-brown color. Materials and Methods Floating chains of the salp P. confoederata were collected from the ocean surface in Suruga Bay, Japan, using a scoop net. Collections took place on November 5th, 2016, during research cruise of the R/V Hokuto of Tokai University. Solitary zooids that were separated from the ag- 130 E. HirosE & J. NisHikawa Fig. 1. Tunic and mantle of Pegea confoederata. A, P. confoederata fixed in glutaraldehyde–seawater. Facing arrows indicate tunic layer. B–D, Histological section of the whole tunic and mantle (B), tunic cuticle (C), and mantle (D). E–I, Hemocytes in the mantle tissue, including the pigment cell (I). co, hemocoel; cu, cuticle; ep, epidermis; gn, gut nucleus; he, hemocyte; ma, mantle; mo, mouth; pbc, peribranchial cavity; pbe, peribranchial epithelium; rp, remnant of placenta; tc, tunic cell; tu, tunic. Scale bars: 5 mm in A; 0.1 mm in B; 20 µm in C and D; 10 µm in E–I. gregate zooids were fixed in 2.5% glutaraldehyde-seawater onboard and stored at 4°C for microscopy analysis (Fig. 1A). Some pieces of mantle tissue covered with tunic (ca. 5×5 mm) were cut from the middle regions of the bodies of the fixed salps using razor blades. For whole-mount observation, the tissue pieces were mounted in water on glass slides, and observed under a light microscope. For histological and electron microscopic observations, the tissue pieces were rinsed with 0.45 M sucrose and 0.1 M cacodylate buffer (pH 7.5) and post-fixed with 1% osmium tetroxide in a 0.1 M cacodylate buffer (pH 7.5) at 4°C for 1.5 h. The tissue pieces were then dehydrated through an ethanol gradient, cleared with n-butyl glycidyl ether, and embedded in an epoxy resin (Epon 812, TAAB Laboratories). Cross sections of the tunic-mantle tissues were prepared with a diamond knife using an ultramicrotome (EM UC6, Leica). Thick sections were stained with toluidine blue for histological observation. Thin sections were stained with uranyl acetate and lead citrate, and were examined using a transmission electron microscope (TEM) (JEM1011, JEOL) at 80 kV. For morphological classification of the hemocytes, we referred to the terminology for the hemocyte types of the salp T. democratica described by Cima et al. (2014). Results Gross Morphology The body of P. confoederata is almost transparent or pinkish-brown in fresh specimens, and transparent, light brown in fixed specimens (Fig 1A), except for the remnant of the placenta and gut nucleus (see ʻrpʼ and ʻgn,ʼ respectively, in Fig. 1A). The body is entirely covered by a transparent, gelatinous tunic (facing arrows in Fig. 1A). The mantle appears light brown in color. In the histological sections, toluidine blue rarely stained Pigmentary cells in a pinkish-brown salp 131 Fig. 2. Electron micrographs of the epithelia and muscle in Pegea confoederata. A, Peribranchial epithelium (pbe). B, Epidermis with cytoplasmic bulge (cb) toward the tunic. C, Enlargement of the apical membrane of the cytoplasmic bulge covered with dense tunic fibers (arrow). D, Cross section of a muscle band in the hemocoel. E, Enlargement of a muscle cell. bl, basal lamina; co, hemocoel; ep, epidermis; mt, mitochondrion; my, myofibril; n, nucleus; pbc, peribranchial cavity; tu, tunic. Scale bars: 2 µm in A, B and D; 0.2 µm in C; 0.5 µm in E. the tunic matrix (Fig. 1B–D), while the outermost layer of the tunic, i.e., the cuticle, was homogeneously stained. The mantle, i.e., the tissue layer beneath the tunic matrix, is comprised of two epithelial layers (the epidermis and peribranchial epithelium) and the hemocoel (or connective tissue) between them (Fig. 1D). Various types of hemocytes were found in the hemocoel, and the tunic cells were found to be sparsely distributed within the tunic matrix. We found several types of hemocytes, including brownpigmented cells, in the whole mount sections of the mantle (Fig. 1E–I). These hemocytes were characterized by amoeboid cell shape (Fig. 1E), some pseudopodia and roundish vacuoles (Fig. 1F), granules that were approximately 2–5 µm in diameter (Fig. 1G) or refractile granules that were about 1 µm in diameter (Fig. 1H). Pigment cells were seen to be dendroid and filled with brown granules that were less than 1 µm in diameter (Fig. 1I). Fine structures Epithelia The peribranchial epithelium is a simple squamous epithelium between the hemocoel and peribranchial cavity (Fig. 2A). The cytoplasm contained mitochondria and endoplasmic reticulum (ER) but no granular inclusions. The epidermis is a simple cuboidal epithelium between the tunic and hemocoel (Fig. 2B). The epidermal cells contained 132 E. HirosE & J. NisHikawa Fig. 3. Electron micrographs of hemocytes distributed in the hemocoel of the mantle in Pegea confoederata. A, Hyaline amoebocyte. B, Amoebocyte with large vacuoles. C, Granular cell type-1 (mast cell-like line). D, Granular cell type-2. E, Storage cell and enlargement of the granular inclusions (inset: black arrow, shell; while arrow, central core). F, Amoebocyte with large vacuoles migrating into the tunic through the epidermis. Arrowheads in A indicate small, membrane-bounded vesicles. Arrows in A and B indicate a part of granular cell type-2 shown in D. co, hemocoel; ep, epidermis; mt, mitochondrion; n, nucleus; tu, tunic. Scale bars indicate 1 µm for A–F and 0.5 µm for inset. mitochondria and rough ER but no granular inclusions. In Figure 1D, a cytoplasmic bulge is seen protruding toward the tunic, resulting in a serrated appearance of the epidermis. Bulges are filled with rough ER, and dense tunic fibers are closely associated with the apical membrane of the bulge (Fig. 2C). Muscle The muscle band is located in the hemocoel, and consists of two parts: peripheral myofibrils and the core that contains the nucleus and mitochondria (Fig. 2D). Muscle fibers are multinucleated. It can be seen in Fig. 2E that the core and peripheral parts are not clearly separated, and the nucleus and myofibrils are occasionally contiguous. The nucleus usually has a large nucleolus. Pigmentary cells in a pinkish-brown salp Hemocytes We recognized five hemocyte types based on their ultrastructural features. Hyaline amoebocytes are often fusiform and less differentiated than the other cell types (Fig. 3A). Their cytoplasm contains mitochondria, ER and small membrane-bounded vesicles (arrowheads in Fig. 3A). Amoebocytes with large vacuoles are roundish and their cytoplasm is filled with large vacuoles (up to 1.2 µm in diameter) (Fig. 3B). These vacuoles contain moderately electron-dense, fibrous materials. The hemocytes in Fig. 1E and F may be hyaline amoebocytes and amoebocytes with large vacuoles, respectively. There are two types of granular cells: type-1 and type-2, which correspond to the hemocytes in Fig. 1G and 1H, respectively. The bulk of the cytoplasm of the type-1 cell is occupied by irregularly shaped, membrane-bound granules that consist of a fibrous, electron-dense core surrounded by less electrondense, flocculent materials (Fig. 3C). The granules vary in size and are approximately 2.5 µm in diameter. The type-2 cells are often fusiform and contain round granules (approximately 1 µm in diameter) that are homogeneously and strongly electron-dense (Fig. 3D). Storage cells are irregularly shaped and contain round vesicles of approximately 0.5 µm in diameter (Fig. 3E). These features are consistent with those of the brown pigment cell (Fig. 1I). Within the round vesicles, electron-dense materials form organized structures as a central core and a surrounding shell (Fig. 3E inset). Figure 3F shows an amoebocyte with large vacuoles that is located in the epidermis, suggesting that hemocytes migrate from the hemocoel into the tunic matrix via the epidermis. Discussion Epidermis and tunic synthesis The cuboidal, epidermal cells of P. confoederata have a large amount of rough ER, which suggests active biosynthesis. These cells have a cytoplasmic bulge where the apical membrane is closely associated with dense tunic fibers, and is, therefore, probably the site for tunic synthesis. In ascidians, the apical membrane of the epidermis has a complex of cellulose synthase, i.e., the terminal complex, and cellulose microfibrils are assembled at this site (Kimura & Itoh 1996, 2004). The epidermal morphology of P. confoederata indicates that salps synthesize tunic in the same way as ascidians do. Muscle In the cross sections of the muscle band, the mitochondria-surrounded nuclei form a core, and the myofibrils are located in the periphery (Fig. 1H). This organization has also been reported in other salps, such as Iasis zonaria (Pallas, 1774) and T. democratica (Bone & Ryan 1973, Bone 1998). In the present species̶P. confoederata̶the core and periphery are not clearly separated and the nu- 133 cleus, mitochondria, and myofibrils are occasionally seen overlapping one another. Hemocyte types and their possible functions Cima et al. (2014) described five types of hemocytes from the transparent-bodied salp T. democratica: 1. undifferentiated cells (lymphocyte-like cells), 2. hyaline amoebocytes, 3. amoebocytes with large vacuoles, 4. granular cells, and 5. storage cells (nephrocytes). Based on TEM observations, we also recognized five types of hemocytes in P. confoederata, many of which are morphologically similar to those in T. democratica. We, therefore, followed the terminology of Cima et al. (2014) to classify the five different hemocyte types in P. confoederata. Although the undifferentiated cells (lymphocyte-like cells) in Cima et al. (2014) were characterized by small hemocytes with an oval nucleus and scanty cytoplasm, we did not find these hemocytes in the present specimens. This cell-type morphologically is consistent with the hemoblasts or lymphocytelike cells described in various ascidians (e.g., Burighel & Cloney 1997) and are supposed to serve as hematopoietic stem cells, producing other cell types in the hemocoel. It is possible that undifferentiated cells are localized in a particular site within the hemocoel in P. confoederata, and that we did not encounter the site during our examinations. In T. democratica, hyaline amoebocytes are characterized by the massive presence of small membrane-bounded vesicles in the cytoplasm (Cima et al. 2014), whereas the vesicles are not abundant in P. confoederata. Cima et al. (2014) histochemically demonstrated that both hyaline amoebocytes and amoebocytes with large vacuoles in T. democratica contain acid phosphatase, non-specific esterase, and peroxidase that are generally involved in phagocytosis, and are labeled with Narcissus pseudonarcissus agglutinin that serves as a specific marker of mammalian macrophages. These hemocytes may also serve as phagocytes in P. confoederata, although we did not conduct such histochemical analyses in this study. The granular cell type-1 in the present species is consistent with the granular cells in T. democratica, in that the granules in both hemocytes have heterogeneous electron densities. Cima et al. (2014) demonstrated by means of immunoelectron microscopy that the granules in T. democratica contain heparin and histamine, which classifies the granular cell as a mast cell-like hemocyte. The granular cell type-2 was not consistent with any of the five hemocytes described in T. democratica (Cima et al. 2014), and the function(s) of this hemocyte is uncertain. In contrast to the transparent body of T. democratica, P. confoederata is pinkish-brown, and therefore, the refractile granules in granular cell type-2 may possibly contribute to its body color. The brown pigment cells in P. confoederata are usually dendroid in shape, and have numerous brown, round granules throughout the cytoplasm (Fig. 1I). Because these characteristics are consistent with the ultrastructural fea- 134 E. HirosE & J. NisHikawa tures of storage cells in Cima et al. (2014), we conclude that the brown pigment cells seen via light microscopy are storage cells. The dendroid cell shape of pigment cells suggests that the cells adhere and extend pseudopodia on to the inner wall of the mantle epithelia. In ascidian hemocytes, nephrocytes and pigment cells have a large vacuole that contains granules that are often crystalline, geometric, or tubular in form (e.g., Burighel & Cloney 1997). Storage cells in T. democratica have vacuoles of various sizes that contain tubular, crystalline granules, and Cima et al. (2014) classified this cell type as a nephrocyte. Storage cells in P. confoederata have small vesicles that contain electrondense granules that form an organized structure. The difference in structure of granular inclusions in the vacuoles is probably attributable to the difference of the components in the granules. In botryllid ascidians, the colonies of the differently-colored strains have distinct structures of the pigment cell granules (Burighel et al. 1983), and polychromatic colonies have several types of pigment cells characterized by different forms of granules in the vacuole (Hirose et al. 1998, Cima et al. 2015). Accordingly, the difference in the granular contents of the storage cells is probably the reason for the difference in coloration between P. confoederata (pinkish-brown) and T. democratica (transparent or blueish). Notably, the pigments are included in multiple vacuoles in salps, whereas they are contained in a single, large vacuole in ascidians. Tunic cells Tunic cells are free mesenchymal cells that are distributed in the tunic matrix and perform various roles that depend on the cell types (Hirose 2009). In salps, tunic cells are usually amoeboid and the cell densities in the tunic are much lower when compared with those in the ascidians and pyrosomas (Hirose et al. 1999). This is consistent with the findings of the present study. On the contrary, phagocytic cells that were alive were found even in the ʻbarrelʼ made from a salp tunic that was processed by the amphipod Phronima sedentaria (Forskål, 1775) (Hirose et al. 2005). The tunic cells in salps are probably involved in innate immunity. We found an amoebocyte with large vacuoles in the epidermis (Fig. 3F). This hemocyte probably migrated from the hemocoel into the tunic by crossing the epidermis. This migration suggests that tunic cells originated from hemocytes. Migration of hyaline amoebocytes into the tunic through the epidermis has also been reported in T. democratica (Seeliger 1893, Pérès 1943, Cima et al. 2014), suggesting that trans-epidermal migration of amoebocytes is common in salps. Conclusion The present study showed that P. confoederata and T. democratica share many morphological features, such as muscle organization and certain types of hemocytes. According to the molecular phylogeny of thaliaceans based on 18S rDNA sequences, these genera are relatively specialized and similar to the ancestral condition inside salpids (Govindarajan et al. 2011). As a remarkable difference, P. confoederata displays an unique body color. It originates from the pigment cells located in the hemocoel of the mantle. This hemocyte is classified as a storage cell based on the ultrastructural features. The pigment is located in small vacuoles as crystalline contents that are brown in P. confoederata but are transparent in T. democratica. Considering visual detection by predators, a colored body should be unfavorable for gelatinous planktons that occur in the epipelagic layer. Because the present species is often distributed in the euphotic zone during the daytime, the protection from harmful solar radiation may be a possible function of the color. However, other salp species distributed in similar depths in the daytime usually have transparent body, and ultraviolet radiation permeates well through their transparent tunic (e.g., Hirose et al. 2015, Kakiuchida et al. 2017). Therefore, the biological significance of this conspicuous coloration of P. confoederata remains unknown. Acknowledgements This study was supported in part by the Okinawa Research Core for Highly Innovative Discipline Science (ORCHIDS) project to EH. The specimens were obtained during the research cruise of the SURUME (SUruga bay Research for Understanding Marine Ecosystems) project. The authors thank the captain and crew of R/V Hokuto, and the scientists and students onboard who helped with sampling. References Belton PS, Tanner SF, Cartier N, Chanzy H (1989) High-resolution solid-state carbon-13 nuclear magnetic resonance spectroscopy of tunicin, an animal cellulose. Macromolecules 22: 1615–1617. Bone Q, Ryan KP (1973) The structures and innervation of the locomotor muscle of salps (Tunicata: Thaliacea). J Mar Biol Assoc UK 53: 873–883. Bone Q (1998) Locomotion, locomotor muscles, and buoyancy. In: The Biology of the Pelagic Tunicates (ed Bone Q). Oxford University Press, Oxford, pp. 35–53. Burighel P, Milanesi C, Sabbadin A (1983) Blood cell ultrastructure of the ascidian Botryllus schlosseri L. 2. pigment cells. Acta Zool 64: 15–23. Burighel P, Cloney RA (1997) Urochordata: Ascidiacea. In: Microscopic anatomy of invertebrates, Vol. 15. Hemichordata, Chaetognatha, and the invertebrate chordates (eds Harrison FW, Ruppert E). Wiley-Liss, Inc., New York, pp. 221–347. Cima F, Caicci F, Sordino P (2014) The haemocytes of the salp Thalia democratica (Tunicata, Thaliacea): An ultrastructural and histochemical study in the oozoid. Acta Zool 95: 375–391. Cima F, Ballarin L, Caicci F, Franchi N, Gasparini F, Rigon F, Pigmentary cells in a pinkish-brown salp Schiavon F, Manni L (2015). Life history and ecological genetics of the colonial ascidian Botryllus schlosseri. Zool Anz 257: 54–70. Govindarajan AF, Bucklin A, Madin LP (2011) A molecular phylogeny of the Thaliacea. J Plankton Res 33: 843–853. Hirose E (1992) Tunic cells in Leptoclinides echinatus (Didemnidae, Ascidiacea): An application of scanning electron microscopy for paraffin embedding specimens. Hiyoshi Review Natur Sci 11: 5–8. Hirose E (2009) Ascidian tunic cells: morphology and functional diversity of free cells outside the epidermis. Invertebr Biol 128: 83–96. Hirose E, Yoshida T, Akiyama T, Ito S, Iwanami Y (1998) Pigment cells representing polychromatic colony color in Botrylloides simodensis (Ascidiacea, Urochordata): Cell morphology and pigment substances. Zool Sci 15: 489–497. Hirose E, Kimura S, Itoh T, Nishikawa J (1999) Tunic morphology and cellulosic components of pyrosomas, doliolids, and salps (Thaliacea, Urochordata). Biol Bull 196: 113–120. Hirose E, Aoki MN, Nishikawa J (2005) Still alive? Fine structure of the barrels made by Phronima (Crustacea: Amphipoda). J Mar Biol Assoc UK 85: 1435–1439. Hirose E, Nakashima K, Nishino A (2011) Is there intracellular cellulose in the appendicularian tail epidermis? A tale of the adult tail of an invertebrate chordate. Commun Integr Biol 4: 768–771. Hirose E, Sakai D, Shibata T, Nishii J, Mayama H, Miyauchi A, Nishikawa J (2015) Does the tunic nipple array serve to camouflage diurnal salps? J Mar Biol Assoc UK 95: 1025–1031. Kakiuchida H, Sakai D, Nishikawa J, Hirose E (2017) Measurement of refractive indices of tunicatesʼ tunic: light reflection of 135 the transparent integuments in an ascidian Rhopalaea sp. and a salp Thetys vagina. Zool Lett 3: 7. http://rdcu.be/s6bz Kimura S, Itoh T (1996) New cellulose synthesizing complexes (terminal complexes) involved in animal cellulose biosynthesis in the tunicate Metandrocarpa uedai. Protoplasma 194: 151– 163. Kimura S, Ohshima C, Hirose E, Nishikawa J, Itoh T (2001) Cellulose in the house of the appendicularian Oikopleura rufescens. Protoplasma 216: 71–74. Kimura S, Itoh T (2004) Cellulose synthesizing terminal complexes in the ascidians. Cellulose 11: 377–383. Nakashima K, Nishino A, Hirose E (2011) Forming a tough shell via an intracellular matrix and cellular junctions in the tail epidermis of Oikopleura dioica (Chordata: Tunicata: Appendicularia). Naturwissenschaften 98: 661–669. Pérès JM (1943) Recherches sur le sang et les organes neuraux des Tuniciers. Ann Institut Oceanogr Monaco 21: 229–359. Sakai D, Kakiuchida H, Nishikawa J, Hirose E (2018) Physical properties of the tunic in the pinkish-brown salp Pegea confoederata (Tunicata: Thaliacea). Zool Lett, 4: 7. https://rdcu. be/LqUJ Seeliger O (1893) Einige Beobachtungen über die Bildung des äußeren Mantels der Tunicaten. Z Wiss Zool 56: 488–505. Turon X, López-Legentil S, Banaigs B (2005) Cell types, microsymbionts, and pyridoacridine distribution in the tunic of three color morphs of the genus Cystodytes (Ascidiacea, Polycitoridae). Invertebr Biol 124: 355–369. Van Daele Y, Revol J, Gaill F, Goffinet G (1992) Characterization and supramolecular architecture of the cellulose-protein fibrils in the tunic of the sea peach (Halocynthia papillosa, Ascidiacea, Urochordata). Biol Cell 76: 86–97.