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Tunic phagocytes are involved in allorejection reaction in the colonial tunicate Aplidium yamazii (Polyclinidae, Ascidiacea).

Introduction

Colony specificity (allogeneic recognition between colonies) has been reported in many colonial organisms, such as sponges, hydrozoans, corals, bryozoans, and ascidians. Its occurrence is usually manifested by fusibility, or the lack thereof, between colonies: two colonies either form a single mass (fusion) or do not fuse (rejection) when they come into contact. Colony specificity is thought to be one of the fundamental systems that maintains colonial individuality. In many colonial ascidians, the occurrence of colony specificity has been demonstrated by means of the colony contact assay (Mukai and Watanabe, 1974; Saito et al., 1994). In contrast, colony specificity is not exhibited in some species--for example, Polycitor proliferus (Polycitoridae; Oka and Usui, 1944), Perophora multiclathrata (Perophoridae; Koyama and Watanabe, 1986), and Botryllus horridus (Botryllidae; Saito and Okuyama, 2003). In these species, fusion has never been observed between two growing edges of conspecific or congeneic colonies, whereas fusion is always observed between two artificial cut surfaces of two conspecific colonies.

Colony specificity in colonial ascidians has been studied mainly in species of the family Botryllidae. Oka and Watanabe (1960) and Sabbadin (1962) had suggested that colony specificity might be controlled genetically in Botryllidae. Most recently, De Tomaso et al. (2005) clearly described the existence of genes that control colony fusibility in Botryllus schlosseri. In botryllid ascidians, blood vessels are anastomosed throughout the colonies, and the hemocytes circulating in the vessels play a pivotal role in the allorejection reaction. At the contact site between two allogeneic colonies, morula cells, a type of hemocyte, are released from the tips of the blood vessels and discharge their vacuolar contents into the tissue at the contact site (Hirose, 2003). Phenoloxidase and quinones contained in the vacuole are considered to contribute to the allorejection reaction (Ballarin et al., 1993, 1994, 1995, 1998; Shirae and Saito, 2000; Shirae et al., 2002). On the other hand, little is known about the presence of colony specificity in colonial species that do not have a vascular network.

[FIGURE 1 OMITTED]

Aplidium yamazii (Tokioka, 1949) (Polyclinidae) is a colonial ascidian that does not have a vascular network in its tunic. In the integumentary tissue--that is, the tunic--many tunic cells are freely distributed, and they are classified into seven types based on their morphology (Hirose et al., 1994b). It is thought that tunic cells have various functions for biodefense in the tunic. Among the seven types, tunic phagocytes are the major type of tunic cell, and they are distributed throughout the tunic. These highly motile cells have the ability to phagocytose foreign materials and other tunic cells (Hirose et al., 1994a). The fate of tunic phagocytes has been studied: after engulfing foreign material or other tunic cells, they differentiate into spherical tunic cells (another of the seven types of tunic cells), which are finally expelled from the tunic (Ishii and Hirose, 2003). The results of the present study strongly suggest that tunic phagocytes are the major effector cells in the allorejection reaction in the colonial ascidian A. yamazii.

Materials and Methods

Animals

Colonies of Aplidium yamazii were collected in Nabeta Bay, Shimoda, Shizuoka Prefecture, Japan. They were attached to glass slides with cotton thread and were reared in culture boxes immersed in Nabeta Bay.

Contact assay

Small pieces (each about 20 mm square) of each colony were obtained from larger stock colonies by cutting with a razor blade. Two types of contact assay were carried out: contact between natural growing edges and contact between artificial cut surfaces. In the growing-edge contact assay, two pieces derived from one colony or two pieces from two different colonies were juxtaposed on a glass slide and either incubated in a laboratory tank supplied with continuous running seawater or reared in culture boxes immersed in Nabeta Bay. Within a few days, growth of the two colony pieces of isogenic or allogeneic combinations resulted in contact between their natural growing edges. These slides were observed daily under a binocular stereomicroscope. Some specimens were fixed for histological investigation, as described below. In the cut-surface contact assay, two pieces derived from one colony or two pieces from two different colonies were brought into contact on a glass slide at their artificial cut surfaces. The slides were then treated as above. The water temperature was about 20 [degrees]C in Nabeta Bay and in the running seawater in the laboratory tank during the experimental period.

Light and electron microscopy

Specimens were fixed with 10% formalin in seawater for 24 h and were rinsed with tap water. Then they were dehydrated through an ethanol series and embedded in paraffin. All specimens were cut across the contact surface into 8-[micro]m thick sections and stained with hematoxylin and eosin for light microscopy. Some specimens were fixed in 2.5% glutaraldehyde-0.1 mol [l.sup.-1] sodium cacodylate-0.45 mol [l.sup.-1] sucrose (pH 7.4) for 2 h on ice. The specimens were then rinsed in 0.1 mol [l.sup.-1] sodium cacodylate-0.45 mol [l.sup.-1] sucrose (pH 7.4) and postfixed in 1% osmium tetroxide-0.1 mol [l.sup.-1] sodium cacodylate (pH 7.4) for 1 to 1.5 h on ice. The specimens were dehydrated through an ethanol series, cleared with n-butyl glycidyl ether, and embedded in low-viscosity epoxy resins. Thick sections were stained with toluidine blue for light microscopy. For transmission electron microscopy, thin sections were stained with uranyl acetate and lead citrate and were examined in a Hitachi HS-9 transmission electron microscope at 75 kV.

Distribution pattern of tunic cells

To study the distribution pattern of tunic cells in the cut-surface contact assay, some specimens were fixed at about 48 h after the cut surface contact. For the paraffin sections of the cut-surface contact assay, five sections were randomly selected, and 80-[micro]m X 80-[micro]m quadrates were overlaid on these sections every 100 [micro]m far from the contact plane (up to 300 [micro]m). The tunic cells were counted in each quadrate to show the distribution pattern of tunic cells in the area of the contact site.

[FIGURE 2 OMITTED]

Results

The occurrence of colony specificity and allorejection reactions

Contact between two colonies of Aplidium yamazii resulted in fusion or rejection, depending on the specific combination of colonies. In fusible (compatible) combinations, fusion of colonies always occurred at the contact site between growing edges and between cut surfaces, resulting in the formation of a single colony. Isogeneic combinations were always fusible. On the other hand, some allogeneic colony combinations did not result in fusion at their growing edges, and these same colony combinations never resulted in fusion at their cut surfaces. These results clearly show that colonies of A. yamazii exhibit colony specificity. In this study, we used 39 different colonies in 115 paired combinations; 101 combinations resulted in fusion and 14 resulted in rejection.

When two colonies of A. yamazii came into contact at their growing edges, the tunic cuticles of the two colonies made contact and fused, resulting in partial fusion of the two tunics. Then, if the two colonies were compatible, fusion of the colonies proceeded to form a single colony; if the two colonies were incompatible, the allorejection reaction occurred at the contact areas. In the allorejection reaction, many tunic cells were aggregated at the areas of partial fusion of the tunics. Finally, the edges of the two incompatible colonies withdrew from the contact areas, and the aggregates of tunic cells remained as small, tightly packed tunic masses between the two separating colonies (Fig. 1A). Using a binocular stereomicroscope, we observed some tunic masses (arrow and arrowheads in Fig. 1B) between the two incompatible colonies in the allorejection reactions induced by the contact of incompatible colonies at their growing edges.

When two compatible colonies were placed in contact at their cut surfaces, fusion always occurred. When two incompatible colonies were placed in contact at their cut surfaces, partial fusion of their tunics occurred, and then the two colonies separated from each other. In incompatible contacts at cut surfaces, some loosely packed tunic masses, rather than tunic cell aggregates or small, tightly packed tunic masses, sometimes remained in the gap between the two withdrawn colonies (Fig. 1C).

Allorejection in the growing edge contact assay: ultrastructural study

The allospecific rejection reaction occurred at the contact site between the growing edges of incompatible colonies. As seen in a thick resin section, many tunic cells were aggregated at the areas of partial fusion of the tunics (Fig. 2A). After two incompatible colonies withdrew from each other, isolated small, tightly packed tunic masses, which contained many aggregates of tunic cells, were seen at the contact areas (Fig. 2B). In the electron micrograph, there were many electron-dense filaments along the border between the two colonies (Fig. 2C, arrows). Partial fusion of the tunics occurred between two incompatible colonies by fusion between their tunic cuticles (Fig. 2D, arrowheads). Many aggregates of tunic cells (Fig. 2D, asterisks) were seen in an area that becames a small, tightly packed tunic mass (left side of Fig. 2D) in the following stage of rejection, surrounded by the electron-dense layer, which might be thickened cuticles (Fig. 2D, double arrows). Figure 2E shows the small, tightly packed tunic mass that has been separated from the colony. Many aggregating tunic cells were identified as tunic phagocytes because of their irregular shapes and the presence of phagosomes (Fig. 2E).

Distribution pattern of the tunic cells in the cut-surface contact assay

In the cut-surface contact assay, the number of tunic cells per 1 [mm.sup.3] in the contact areas was determined after contact occurred (Fig. 2F, G). In isogeneic combinations, the number of tunic cells was almost constant among the quadrates, regardless of the distance from the contact site (Fig. 2F). In contrast, in incompatible colony combinations, the number of tunic cells per unit area was smallest at the contact site (Fig. 2G).

Discussion

The occurrence of colony specificity has been reported in various colonial ascidians that have vascular networks (Mukai and Watanabe, 1974; Saito et al., 1994). In these species, the allorejection reaction was promoted by hemocytes that infiltrated from the blood vessels (Taneda and Watanabe, 1982; Hirose et al., 1990, 1997a). In this study, we demonstrated the occurrence of colony specificity in the colonial ascidian Aplidium yamazii. To our knowledge, this is the first report of the occurrence of allorejection in a colonial ascidian that lacks a vascular network. The histological investigation showed that tunic phagocytes may play an important role in the allorejection reaction in this species.

In botryllid ascidians, two types of rejection reaction are known to be mediated by hemocytes. One type of rejection reaction is promoted mainly by the morula type of hemocytes (M-type rejection), and the other type of rejection reaction is promoted mainly by the phagocyte type of hemocytes (P-type rejection) (Shirae et al., 1999). Among botryllid ascidians, P-type rejection is found exclusively in Botryllus scalaris; the other botryllids so far studied show M-type rejection. Moreover, Symplegma reptans (Polyzoinae), which is thought to be the species having the closest phylogenetic relationship to botryllid ascidians (Saito et al., 2001), shows M-type rejection. In Perophora sagamiensis (Perophoridae), Koyama and Watanabe (1982) reported that amoebocytes and lymphocyte-like cells were involved in allorejection reactions. These amoebocytes were spherical, flattened, or irregular in shape. Thus, we regard Perophora sagamiensis as having P-type rejection. In A. yamazii, although morula-like cells also exist in the tunic (Hirose et al., 1994b), we did not find morula-like tunic cells in allorejection lesions. In the allorejection reaction of A. yamazii, tunic phagocytes may play a key role, as do hemocytes in other colonial ascidians that have a vascular network in the tunic. The allorejection reaction in A. yamazii appears to be comparable to the P-type rejection.

Tunic phagocytes are the most abundant cells in the tunic. They move throughout the tunic and are thought to have various functions, besides phagocytosis, in the tunic (Hirose et al., 1994a, b). When a part of the tunic of A. yamazii was damaged by the injection of distilled water or 5% NaOH, a cuticular boundary appeared in the tunic, separating the damaged and undamaged tunic (Hirose et al., 1997b). The formation of the cuticular layer, derived from electron-dense fibers, was accompanied by aggregation of tunic phagocytes along the boundary on the side of undamaged tunic. After the boundary formation was completed, the tunic of the damaged area peeled off (Hirose et al., 1997b). The tunic phagocytes might be involved in the aggregation of the electron-dense filaments to form the cuticular layer. Therefore, we propose that tunic phagocytes may control tunic separation in the allorejection reaction. In the cut-surface contact assay between two incompatible colonies, the number of tunic cells per unit volume was smallest in the contact boundary. It seemed that tunic phagocytes moved from the tunic area that became partially fused with the paired allogeneic colony, but it is not yet known whether the decrease in tunic cell density at the contact boundary might promote the formation of the cuticular layer or, instead, be triggered by it. As shown in the area marked by an asterisk in st. 3 of Figure 1C, the area of decreased tunic cell density might be degenerated into some loosely packed tunic masses. Then, new tunic cuticles were made in the gap between the two withdrawn colonies. These reactions of the tunic cells were seen not only in the artificial cut-surface repair reaction but also in the allorecognition reaction.

In the allorecognition system of colony specificity, little is known about the site of allorecognition. In botryllid ascidians that have a vascular network in their tunic, allorecognition sites are thought to be in the tunic or in the vascular network containing hemocytes (or in both locations) (Hirose, 2003). In ovoviviparous botryllid ascidians, when two incompatible colonies are brought into contact at their cut surfaces, an intense rejection reaction is induced. In contrast, in viviparous botryllid ascidians, when two colonies are brought into contact at their cut surfaces, fusion always occurs, even between incompatible combinations of colonies in which allorejection is induced by growing-edge contact. This cut-surface fusion between incompatible colonies results in fusion of their vascular networks and formation of a chimeric colony ("surgical fusion": Hirose et al., 1988, 1994c; Okuyama et al., 2002). The occurrence of this surgical fusion might suggest that the distribution of allorecognition sites is different between ovoviviparous and viviparous botryllid ascidians. In ovoviviparous botryllid ascidians, the vascular network in the tunic might be able to exhibit allorecognition, whereas this might not be the case in viviparous botryllids. In viviparous botryllids, the vascular network might be involved in the allorejection reaction but perhaps not in the allorecognition stage. As pointed out by Hirose (2003), in viviparous botryllids, it is possible that only tunic cells bear allorecognition sites but that morula cells play a pivotal role in the allorejection reaction. On the other hand, in A. yamazii, tunic phagocytes promote the allorejection reaction. We propose that tunic phagocytes are not only effector cells in allorejection reactions but also bear the sites of allorecognition. We speculate that these highly motile tunic phagocytes "seek out" and recognize allogeneic elements and work in the allorejection site. In A. yamazii, colony specificity exists, and the allorejection reaction is mediated primarily by tunic phagocytes. This study demonstrated that self/nonself recognition occurs even in a colonial ascidian with no vascular network in its tunic and that tunic cells promote the rejection reaction in this species.

In Diplosoma listrianum, a colonial ascidian that does not possess a vascular network, allogeneic fusion often occurred and resulted in stable chimera colonies (Bishop and Sommerfeldt, 1999; Sommerfeldt and Bishop, 1999; Sommerfeldt et al., 2003). Colonial fusion may provide economic benefits for each zooid, because the respiration of each zooid is smaller in larger colonies (Nakaya et al., 2003): a larger colony can conserve more nutrient/energy resources than a smaller colony. On the other hand, nonselective fusion may lose the individuality of the colonies, and as speculated by Buss (1982), zooids of a genotype can be parasitic on other zooids through unequal sharing of the energetic cost to maintain the chimera colony. Although allogeneic rejection occurred in very low frequency (14 out of 115 combinations) in the present study, the capacity for colonial allorecognition would be essential for survival of A. yamazii.

Acknowledgments

We gratefully acknowledge Dr. Yasunori Saito and the staff of the Shimoda Marine Research Center (SMRC), University of Tsukuba, for their assistance and hospitality. Most of this study was performed at SMRC. We also acknowledge Mrs. Yuki Kawata for her assistance with histological observations. This study was supported in part by the 21st Century COE program of the University of the Ryukyus. This is contribution no. 736 from SMRC.

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TERUHISA ISHII (1,*), EUICHI HIROSE (2), AND YASUHO TANEDA (3)

(1) Division of Biology, Department of Natural and Environmental Sciences, Faculty of Education and Human Studies, Akita University, Akita 010-8502, Japan; (2) Department of Chemistry, Biology and Marine Science, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan; and (3) Division of Science Education, Faculty of Education and Human Sciences, Yokohama National University, Yokohama 240-8501, Japan

Received 15 May 2007; accepted 8 January 2008.

* To whom correspondence should be addressed. E-mail: tishii@ipc.akita-u.ac.jp
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Author:Ishii, Teruhisa; Hirose, Euichi; Taneda, Yasuho
Publication:The Biological Bulletin
Article Type:Report
Geographic Code:9JAPA
Date:Apr 1, 2008
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