A new solitary entoproct, Loxosomella plakorticola sp. nov., was found on a sponge, Plakortis sp., on a coral reef slope on the western coast of Okinawa Island, Ryukyu Archipelago, Japan. This species has a medium-sized body (up to about 1.2 mm), slender proportion (the stalk is 0.83–1.76 times longer than the calyx), a slug-like foot with a foot gland and foot groove, and 14 to 18 tentacles. Small black pigment granules are visible only in the living stage in the calyx, stalk, and buds. This is the first report of a commensal loxosomatid from the Ryukyu Archipelago and the second species inhabiting sponges reported from Japan. Detailed morphological observations indicate that this species attaches to sponges by narrowing the foot groove; the sponge surface is pinched in the deepest part of the groove, which is free of the cuticle layer but covered by microvilli of epidermal cells. The accessory cells lining the foot groove have long been believed to be gland cells, but they are not gland cells in ultrastructure, at least in this species.
Entoprocts are small benthic animals that live mostly in marine waters. Scientists currently recognize about 180 species, of which 50 are colonial and the remainder solitary. All solitary species belong to the family Loxosomatidae, and most loxosomatids associate with larger animals such as polychaetes, sponges, bryozoans, and sipunculans (Nielsen, 1964; Soule and Soule, 1965). Commensal entoprocts attach to the body surface of animals or the inner surface of animals’ tubes. Commensal association benefits entoprocts by providing a safe habitat (Iseto, 2005) and water currents generated by the host animals, which provide loxosomatids with food particles and fresh water for respiration (Nielsen, 1964). However, recent reports have indicated that non-commensal species are also common in this taxon (Iseto, 2005) and are found on stones, shells, algae, and man-made materials such as plastic panels and glass slides. On the shallow coral reef shores of Okinawa Island, the abundance of non-commensal loxosomatids exceeded 1800 individuals per glass slide in August (Iseto et al., 2007).
Loxosomatids have a “foot” attached to the substratum. Because this foot is the only organ that has direct contact with the substratum, it likely plays a pivotal role in substrate selection and attachment. For commensal species, the foot may establish and maintain the specific association with the host. Moreover, the foot is key in the generic classification of loxosomatids: Each genus is characterized by a specific foot structure and budding mode (see Iseto, 2002). Therefore, investigating the detailed structure of the foot is important for better understanding the ecology and taxonomy of entoprocts.
All 12 species reported to date from Okinawa Island and its vicinity are non-commensal and have been found on stones, dead coral fragments, shell remains, and glass slides immersed along the coast (Iseto, 2001, 2002, 2003, 2006). Here we provide the first report of a commensal loxosomatid from the Ryukyu Archipelago: a new Loxosomella species found densely on a sponge on a reef slope at a depth of about 10–15 m. Foot structure was studied histologically and ultrastructurally to determine how the loxosomatid attaches to the sponge surface.
MATERIALS AND METHODS
Loxosomatids were collected from a demosponge, Plakortis sp., at a depth of about 11 m on a reef slope at Manza, Onna Village, on the western coast of Okinawa Island (26°30’13.70”N, 127°50’32.39”E) by SCUBA diving. Underwater photographs of colonies of loxosomatids on the host sponge were taken in the natural habitat. Loxosomatids with the host sponge were anesthetized by adding 0.37 M MgCl2 to the seawater and then fixed with 2% formalin in seawater (Iseto, 2001). All drawings were made from fixed specimens under a light microscope equipped with a camera lucida. Type specimens were stored in 2% formalin in seawater and deposited in the National Science Museum, Tokyo (NSMT). Dr. Yuji Ise (Misaki Marine Biological Station, University of Tokyo) kindly identified the host sponge to genus.
Specimens were fixed with 2.5% glutaraldehyde with 0.45 M sucrose in 0.1 M cacodylate buffer (pH 7.4) for 1 h at room temperature, briefly rinsed with 0.45 M sucrose–0.1 M cacodylate, and postfixed with 1% OsO4 in the same buffer on ice. The specimens were dehydrated through a graded series of ethanol treatments. For scanning electron microscopy, the specimens were immersed in t-butanol and freeze dried. The dried specimens were sputter-coated with gold and examined under a scanning electron microscope (JEOL JEM-6060LV). For histology and transmission electron microscopy, specimens were embedded in low-viscosity epoxy resin, sectioned to 0.5–1 μm thick, and stained with 1% toluidine blue. Ultra-thin sections were stained with uranyl acetate and lead citrate and examined under a transmission electron microscope (JEOL 1011).
DESCRIPTION OF THE NEW SPECIES
Genus Loxosomella Mortensen, 1911
Loxosomella plakorticola sp. nov. lseto & Sugiyama
Holotype: NSMT-Ka 87. Found on Plakortis sp. 10 March 2002, on a reef slope at Manza (Onna Village), Okinawa Island, Japan, at a depth of 11 m.
Paratypes: NSMT-Ka 88; 23 adults collected with the holotype. NSMT-Ka 89; one adult collected with the holotype.
Type host: NSMT-Po 1342. An individual of Plakortis sp. on which the holotype and paratypes were attached.
Lives at high densities on sponges on reef slopes (Fig. 1A–C). In the living animals, black pigment granules (ca. 2 μm in diameter) scattered in tentacles, calyx, and buds, but absent in stalk and foot (Fig. 1D–E). Pigment disappears after fixation. Total length (from basal attachment of foot to uppermost part of tentacle membrane) 461–1226 μm. Calyx length (from lower end of stomach to uppermost part of tentacle membrane) 245–461 μm. Stalk 0.83–1.76 times as long as the calyx. Tentacles number 14 or 15 at the bud stage and increase after liberation from parent, but never exceed 18 (Fig. 4). Lateral sense organ on calyx absent. No conspicuous appendage anywhere on body. Transparent granular cells (about 10–15 μm in diameter) scattered on calyx, tentacles, and stalk (Figs. 1D, 3C–F), and the number of cells varies among individuals. Granular cells also present in buds (Fig. 3I) but distributed more densely than in adults. Foot with foot gland, as well as foot groove lined with accessory cells, present in both adult and buds (Figs. 2A–E, 3G–H). Foot size (from frontal end to posterior tip) about 160 μm in both adults and large buds. Bean-shaped foot gland about 90 μm long (Fig. 3G–H). Entire body free of covering detritus. Grown bud resembles parent in general shape, but is smaller in size (Figs. 2A–B, 3A).
Buds emerge from paired lateral pockets of the calyx at the level of the upper half of the stomach. Buds are attached to parent’s calyx by the tip of the foot (Fig. 2A). Large buds have 14 to 15 tentacles (Fig. 4). Up to four buds occur simultaneously on a single parent; only one bud enlarges at a time. Sexual reproduction was not observed in our specimens.
This species was found on Plakortis sp. on the slope of a coral reef at a depth of 10–15 m (Fig. 1A). Plakortis sp. is a common sponge at this site, and the loxosomatids were often found on it at high densities (Fig. 1A–C), although still many Plakortis sp. at the same locality were free of loxosomatids.
Sponges are a major host for loxosomatids (Nielsen, 1964); to date, 16 species, all of which belong to the genus Loxosomella, have been found on various sponges. Many of these species have a conspicuous lateral lobe on the foot that is absent in the new species. Among the 16 Loxosomella inhabiting sponges, four (L. bocki, Franzén, 1966; L. cochlear (Schmidt, 1876); L. museriensis Bobin, 1968; and L. vivipara, Nielsen, 1966a) lack the lateral lobe on the foot. Loxosomella plakorticola is distinguished from these four as follows. Loxosomella bocki produces “internal buds” with deep budding pockets that completely cover early-stage buds. The pockets of L. plakorticola are not as deep, and even small buds are visible externally (Figs. 2A, 3B). Loxosomella cochlear has only eight tentacles in the adult stage (Schmidt, 1876). Large specimens of L. museriensis have a very short stalk and the lobe on the lower, lateral part of calyx; these also differ from L. plakorticola in having conspicuous lateral sense organs in the calyx (Bobin, 1968). In general appearance, L. plakorticola most resembles L. vivipara, whose large cells in the calyx are possibly homologous to the granular cells found in the former species. However, the tentacles of L. vivipara number only 12 to 16. Moreover, some individuals of L. vivipara have a unique rounded structure on the back side of the calyx that is composed of more than 20 cells and a central hollow (Nielsen, 1966a). This structure was not observed in L. plakorticola.
Specimens of L. plakorticola sp. nov. remain attached to sponges even after fixation. When being removed from the sponge by needle or by jet current, specimens usually retain some sponge tissue in the foot groove (Fig. 5A), a longitudinal crevice on the underside of the slug-like foot (Fig. 5B).
In a semi-sagittal section, the foot gland in the basal part of the stalk is elongated downward, nearly to the frontal end of the foot groove. Thick muscle fibers run from the backside of the foot gland toward the posterior tip of the foot (Fig. 5C). In another semi-sagittal section, a row of groove accessory cells (GACs) runs just above the foot groove (Fig. 5D). The foot gland is composed of several gland cells; at the opening of the gland, long gland cells are compactly aggregated, and each cell faces the opening of the multi-ciliated cell surface (Fig. 5E). Each gland cell contains many secretory granules (about 0.5 μm in diameter) and much rough ER (Fig. 5F).
A cross-section shows that the foot groove is nearly half the depth of the foot (Fig. 6A–B). Two pairs of GACs are positioned in the upper lateral area of the groove (Fig. 6B). Each GAC has a large nucleus, but the cytoplasm contains no secretory granules. There are muscle fibers beside the groove as well as in the upper and lateral sides of the foot (Fig. 6A–B). The groove wall is covered by microvilli of epidermal cells in the upper half and a cuticle layer in the lower half. The microvilli seem to hold the sponge tissue (Fig. 6B).
The cuticle layer covers all outer surfaces of the foot (Fig. 6C–F). This layer consists of the microvilli of the epidermal cells, a fibrous matrix that runs parallel to the epidermal surface, and an outer cuticle cap covering the tip of each microvillus. The microvilli are sometimes branched, but each tip is covered by a cuticle cap, and the caps uniformly cover the cell surface. The cuticle layer has a similar structure everywhere on the foot surface, but is thinner (about 0.79–1.20 μm) on the upper (Fig. 6C) and lateral (Fig. 6D) surfaces than on the underside of the foot (Fig. 6E) and in the groove wall (Fig. 6F; about 1.48–1.83 μm). Moreover, the microvilli are rather straight and the matrix fibers are denser on the upper and lateral surfaces compared to the underside and groove wall. The cuticle layer ends in the middle of the foot groove, and the upper part of the groove wall is covered with the long “naked” microvilli of the epidermal cells (Fig. 6F).
This is the first report of a commensal loxosomatid from the Ryukyu Archipelago; all species reported to date from this area have been non-commensal (Iseto, 2001, 2002, 2003, 2005, 2006). Previous surveys used attachment panels (mainly glass slides), which typically collect only non-commensal species. More surveys of host candidate animals such as sponges, polychaetes, and bryozoans are needed to further outline the loxosomatid fauna in this region.
The host specificity of loxosomatids inhabiting sponges is not very strict, as some loxosomatids species have been found on several different sponges. For example, L. vivipara has been reported on Ircinia fasciculata, Tedania ignis, Aaptos aaptos, Chondrosia collectrix, and Chondrilla nucula (see Nielsen, 1966a); and Loxosomella tethyae (Salensky, 1877) has been reported on Tethya sp., Stylotella spp., and Microciona prolifera (see Nielsen, 1964, 1966b). Loxosomella plakorticola may be found on other sponges, or other loxosomatids may be associated with Plakortis sp. No loxosomatid species associated with sponges have ever been found on any other animal group or non-living substrata; these species associate exclusively with sponges. This indicates that loxosomatid species have an obligatory relationship to the host sponge and may be specially adapted to living on sponges. For example, because sponges are often toxic, loxosomatids may have acquired tolerance to these chemicals. Also, because the foot is the only structure in direct contact with the sponge, it may be adapted to attach to or grab onto the sponge surface. The foot was examined histologically and ultrastructurally in this study. Loxosomella plakorticola has a slug-like foot that is characteristic of Loxosomella and Loxocorone species (Iseto, 2002). Observations of this type of foot (e.g., Schmidt, 1876; Franzén, 1966; Nielsen and Jespersen, 1997) are basically consistent with the foot structure of L. plakorticola, in that they describe a foot gland, foot groove, and so-called accessory gland cells along this groove. However, the accessory gland cells of L. plakorticola lack secretory granules, which clearly indicates that these cells are not gland cells. The accessory gland cell in Loxosomella elegans also lacks secretion granules (Fig. 18 in Nielsen and Jespersen, 1997). Whereas, these cells have long been called gland cells (Schmidt, 1876), we propose to call them “groove accessory cells” (GACs).
Our observations reveal that the foot is covered by a common cuticle layer except on the upper half of the foot groove, which is covered by naked microvilli (Fig. 6B, F). The area lacking cuticle is probably the most flexible part of the foot, and sponge tissue appears to be held in this area (Fig. 6B). Several longitudinal muscles run along the foot groove (Figs. 5C, 6A, B), indicating how L. plakorticola sp. nov. grasps the sponge surface. When the longitudinal muscles contract, the cross-section of the foot increases. Because the cuticle layer is thick and rigid except along the foot groove, the lateral walls of the foot groove are pushed due to the expansion of the cross-section (Fig. 7). The muscle contraction decreases the width of the foot groove, and the foot pinches the sponge surface (Fig. 7). At present, it is not clear that this way of attaching to the host is common among loxosomatids on sponges or those associated with soft-bodied hosts.
The foot structure is a very important feature discriminating loxosomatid genera; examples are the disc-shaped foot in Loxosoma, slug-like foot in Loxosomella and Loxocorone (cf. Iseto, 2002), the foot with a pair of “terminal wings” in Loxomitra (Nielsen, 1966b; Iseto, 2002), and the small swelling with several gland cells in Loxomespilon (cf. Bobin and Prenant, 1953). However, few studies have investigated the histology and ultrastructure of these feet. Future comparative surveys on foot structure will enable researchers to describe not only the function but also the taxonomy and phylogeny of loxosomatid entoprocts.
We thank Dr. Y. Ise (University of Tokyo) for generic identification of the host sponge and Prof. M. Yokota (University of the Ryukyus) for valuable advice on nomenclature. This study was partly supported by the Fujiwara Foundation and the 21st Century COE Program of the University of the Ryukyus.
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