Stack of cellular lamellae forms a silvered cortex to conceal the opaque organ in a transparent gastropod in epipelagic habitat.

Background: Gelatinous zooplankton in epipelagic environments often have highly transparent bodies to avoid detection by their visual predators and prey; however, the digestive systems are often exceptionally opaque even in these organisms. In a holoplanktonic gastropod, Pterotrachea coronata, the visceral nucleus is an opaque organ located at the posterior end of its alimentary system, but this organ has a mirrored surface to conceal its internal opaque tissue. Results: Our ultrastructural observation proved that the cortex of the visceral nucleus comprised a stack of thin cellular lamellae forming a Bragg reflector, and the thickness of lamellae (0.16 µm in average) and the spaces between the lamellae (0.1 µm in average) tended to become thinner toward inner lamellae. Based on the measured values, we built virtual models of the multilamellar layer comprising 50 lamellae and spaces, and the light reflection on the models was calculated using rigorous coupled wave analysis to evaluate their properties as reflectors. Our simulation supported the idea that the layer is a reflective tissue, and the thickness of the lamella/space must be chirped to reflect sunlight as white/silver light, mostly independent of the angle of incidence. Conclusions: In P. coronata, the cortex of the visceral nucleus comprised multicellular lamellae that form a chirped Bragg reflector. It is distinct in structure from the intracellular Bragg structures of common iridophores. This novel Bragg reflector demonstrates the diversity and convergent evolution of reflective tissue using reflectin-like proteins in Mollusca.

Innate immunity in the edible ascidian Halocynthia roretzi developing soft tunic syndrome: Hemolymph can eliminate the causative flagellates and discriminate allogeneic hemocytes.

The infection of the kinetoplastid flagellate Azumiobodo hoyamushi causes soft tunic syndrome that often results in mass mortality in the aquaculture of the edible ascidian Halocynthia roretzi. In the diseased ascidian individuals, the flagellates are exclusively found in the tunic matrix that entirely cover the epidermis, and never invade into internal tissues, such as a mantle. The present study for the first time demonstrated that the ascidian blood plasma and hemolymph have an activity to agglutinate and disintegrate the flagellates, suggesting the innate immunity protects the internal tissue from the invasion of A. hoyamushi. This activity is indifferent between the healthy and the diseased individuals. Allo-specific recognition and cytotoxic reaction among ascidian hemocytes, so-called contact reaction, occur among the individuals of healthy-healthy, healthy-diseased, and diseased-diseased combination, and therefore, the hemocytes from diseased individuals still retain the allo-reactivity. Moreover, the allo-reactive combinations are not changed under the presence of the flagellates, indicating the flagellates neither suppress nor induce the effector system of the contact reaction. These results suggest that the infection of A. hoyamushi does not impair the innate immunity in the ascidian hemolymph.

Prevalence, complete genome, and metabolic potentials of a phylogenetically novel cyanobacterial symbiont in the coral-killing sponge, Terpios hoshinota.

Terpios hoshinota is an aggressive, space-competing sponge that kills various stony corals. Outbreaks of this species have led to intense damage to coral reefs in many locations. Here, the first large-scale 16S rRNA gene survey across three oceans revealed that bacteria related to the taxa Prochloron, Endozoicomonas, SAR116, Ruegeria, and unclassified Proteobacteria were prevalent in T. hoshinota. A Prochloron-related bacterium was the most dominant and prevalent cyanobacterium in T. hoshinota. The complete genome of this uncultivated cyanobacterium and pigment analysis demonstrated that it has phycobiliproteins and lacks chlorophyll b, which is inconsistent with the definition of Prochloron. Furthermore, the cyanobacterium was phylogenetically distinct from Prochloron, strongly suggesting that it should be a sister taxon to Prochloron. Therefore, we proposed this symbiotic cyanobacterium as a novel species under the new genus Candidatus Paraprochloron terpiosi. Comparative genomic analyses revealed that 'Paraprochloron' and Prochloron exhibit distinct genomic features and DNA replication machinery. We also characterized the metabolic potentials of 'Paraprochloron terpiosi' in carbon and nitrogen cycling and propose a model for interactions between it and T. hoshinota. This study builds a foundation for the study of the T. hoshinota microbiome and paves the way for better understanding of ecosystems involving this coral-killing sponge.

How to Wear a Sandy Coat: Secretory Cells in the Dorsal Epidermis in the Sea Cucumber Holothuria atra (Echinodermata: Holothuroidea).

Holothuria atra is a black sea cucumber commonly found on the sandy bottom of Okinawan coral reefs. The body surface of H. atra is usually covered with sand; however, sand never covers the body of another black congener, Holothuria leucospilota, which is sympatrically distributed with H. atra. The epidermal structures were examined in these two species by means of transmission electron microscopy to determine how sand adheres to the surface of H. atra. While the epidermis was basically composed of support cells bearing microvilli and vacuolated cells probably corresponding to mucus cells, two types of granular cells, type 1 and 2, were also found at the tip of the tube feet. These granular cells were closely similar in structure to secretory cells that have been supposed to secrete adhesive substances in other holothurians. Type 1 granular cells were also found in the dorsal epidermis of H. atra but not in H. leucospilota. Therefore, adhesive secretion by type 1 granular cells probably enables the attachment of sand to the H. atra body.

Exumbrellar Surface of Jellyfish: A Comparative Fine Structure Study with Remarks on Surface Reflectance.

The exumbrellar surfaces of six pelagic cnidarians from three classes were ultra-structurally compared to reveal their structural diversity in relation to their gelatinous, transparent bodies. We examined two hydrozoans (Diphyes chamissonis and Colobonema sericeum), a cubozoan (Chironex yamaguchii), and three scyphozoans (Atolla vanhöffeni, Aurelia coerulea, and Mastigias papua). The exumbrellar surfaces of the mesoglea in D. chamissonis, Ch. yamaguchii, Au. coerulea, and M. papua were covered with a simple epidermis; the shapes of the epidermal cells were remarkably different among the species. The epidermal cells of Ch. yamaguchii and M. papua possessed an array of microvilli on the apical side. The array possibly reduced light reflectance and provided some other surface properties, as seen for the cuticular nipple array in tunicates, considering the length, width, and pitch of the microvilli. The reduction of light reflectance on the array of microvilli was supported by the simulation with rigorous coupled wave analysis (RCWA). Microvilli were sparse and did not form an array in metephyrae of Au. coerulea. The mesoglea matrix beneath the basal side of the epidermis was loose in all of the species. The exumbrellar side of the mesoglea was exposed only in the mesopelagic species, At. vanhöffeni and Co. sericeum, and electron-dense layer(s) covered the surface of the mesoglea. It is uncertain whether the exumbrellar epidermis is absent in these species or the epidermal cells are completely exfoliated during the sampling and handling processes. In the latter case, the electron-dense layer(s) on the mesoglea surface might originally underlie the epidermis.

Reacquisition of light-harvesting genes in a marine cyanobacterium confers a broader solar niche.

The evolution of phenotypic plasticity, i.e., the environmental induction of alternative phenotypes by the same genotype, can be an important mechanism of biological diversification.1,2 For example, an evolved increase in plasticity may promote ecological niche expansion as well as the innovation of novel traits;3 however, both the role of phenotypic plasticity in adaptive evolution and its underlying mechanisms are still poorly understood.4,5 Here, we report that the Chlorophyll d-producing marine cyanobacterium Acaryochloris marina strain MBIC11017 has evolved greater photosynthetic plasticity by reacquiring light-harvesting genes via horizontal gene transfer. The genes, which had been lost by the A. marina ancestor, are involved in the production and degradation of the light-harvesting phycobiliprotein phycocyanin. A. marina MBIC11017 exhibits a high degree of wavelength-dependence in phycocyanin production, and this ability enables it to grow with yellow and green light wavelengths that are inaccessible to other A. marina. Consequently, this strain has a broader solar niche than its close relatives. We discuss the role of horizontal gene transfer for regaining a lost phenotype in light of Dollo's Law6 that the loss of a complex trait is irreversible.

Latitudinal Difference in the Species Richness of Photosymbiotic Ascidians Along the East Coast of Taiwan.

Some didemnid ascidians harbor cyanobacterial symbionts, and this is the only obligate photosymbiosis system known in chordates. These photosymbiotic ascidians are found only in tropical and subtropical waters, probably because the photosymbionts are vulnerable to low temperatures. We surveyed the photosymbiotic ascidian fauna along the east coast of Taiwan. The present and previous reports recorded 13 species in Taiwan, and the species richness and composition is different in five areas along the east coast. Along the middle-east, southeast, and south coasts, five or more species have been recorded, whereas only one species has been found along the northeast coast, and no species have been recorded on the north coast. This gap in the species richness is probably related to the Kuroshio Current, which travels from south to north along the east coast of Taiwan but changes to an easterly direction off the northeast coast. Increases in water temperature due to global warming could cause northward expansion of the distribution ranges of these ascidians in the future. Hence, the photosymbiotic ascidian fauna could be an environmental indicator in subtropical, shallow water, and the present study provides a basic dataset that illustrates the current status of photosymbiotic ascidians in Taiwan.

Wettability and Substrate Selection in the Larval Settlement of the Solitary Ascidian Phallusia philippinensis (Phlebobranchia: Ascidiidae).

For marine benthic animals, the selection of substrate by larvae is important for their survival, with early post-settlement mortality often being affected by the microenvironment where they settle. We tested the substrate preference of the larvae of the ascidian Phallusia philippinensis toward nine commercially available substrates. In the present assay, the larvae settled on one of four substrates for seven substrate combinations with different wettabilities; we counted the number of settled larvae on each of the four substrates, and Manly's selection indices were compared to determine the preference rank of each substrate. Larvae significantly preferred more hydrophobic and oleophilic substrates to hydrophilic and oleophobic ones. While it is uncertain how the larvae detect the properties of the substrate surface, they might be able to sense the physical force, such as stickiness and repellent force. Although a hydrophobic surface is not common in a natural marine environment, the use of hydrophobic materials (as flypaper-like tools) for ascidian larvae might help to prevent the settlement of non-indigenous ascidians in aquaculture facilities.

Seasonality and Longevity of the Functional Chloroplasts Retained by the Sacoglossan Sea Slug Plakobranchus ocellatus van Hasselt, 1824 Inhabiting A Subtropical Back Reef Off Okinawa-jima Island, Japan.

Plakobranchus ocellatus is a sacoglossan sea slug that feeds on multiple algal species and retains chloroplasts as kleptoplasts for several months. The seasonal differences in the photosynthetic properties of kleptoplasts were examined in sacoglossans collected from a subtropical back reef off of Okinawa-jima (26°21'55"N 127°44'10"E) in 2017-2018. The effective quantum yield of photosystem II in kleptoplasts indicated that stronger ambient light causes more stress in kleptoplasts. The maximum quantum yields (QY) at 20°C, 30°C, and 40°C indicated that kleptoplasts were more functional in photosynthesis in winter than in spring or summer, whereas kleptoplasts may have the highest tolerance to high temperatures in summer. In the long-starvation experiment (LSE), the relative ratio of body weight (relW) linearly decreased and the sacoglossans died within 2 months in the total dark condition, whereas in the LSE with illumination, the animals survived up to 5 months. The time course for the decrease in the relative ratio of the QY (relQY) in the LSE indicated that the photosynthetic function was almost normal for 2 months, regardless of the presence or absence of illumination, after which time relQY gradually decreased to zero. In the field, P. ocellatus continuously took up new kleptoplasts that have suitable properties of photosynthetic ability for each season. In a subtropical environment, in which water temperatures vary from below 20°C to above 30°C, seasonal changes could cause a temporary shortage of algal food and affect the photosynthetic activity of P. ocellatus kleptoplast. Our results, however, indicated the kleptoplasts of P. ocellatus functioned normally for several months and maintained the presence of this sacoglossan in a subtropical environment throughout the year.

Measurement of Tunic Hardness in an Edible Ascidian, Halocynthia roretzi, with Remarks on Soft Tunic Syndrome.

The infection caused by a kinetoplastid flagellate, Azumiobodo hoyamushi, in an ascidian, Halocynthia roretzi, results in softening of the tunic, and finally death. This disease is usually recognized using palpation of the softening tunic, and A. hoyamushi infection is detectable using microscopy or PCR amplification of specific gene fragments. The present study is the first quantitative evaluation of the symptoms of soft tunic syndrome by measuring the amount of bending (bending) and the peak force required to pierce the tunic (force). There was a strong correlation between bending and force. Correlation analyses among other parameters (ascidian total weight, tunic thickness, and tunic water content) indicated that larger ascidians had harder and thicker tunics with a higher water content. As compared to the tunic of healthy individuals, softened tunic was thinner and had lower water content. Infected tunics thus possibly lose water and become softer and thinner. Mechanisms for maintaining the appropriate water level content may be crucial for preventing tunic softening.

Tunic extract of the host ascidian attracts the causal agent of soft tunic syndrome, Azumiobodo hoyamushi (Kinetoplastea: Neobodonida).

Azumiobodo hoyamushi, a kinetoplastid flagellate, is the causative agent of soft tunic syndrome, an infectious disease of the edible ascidian Halocynthia roretzi. The flagellate is thought to invade the tunic matrix via a damaged area of the tunic on the siphon wall. We hypothesized that the flagellate locates the tunic entry site by a chemotactic response to soluble substances diffused from the host ascidians. To investigate this hypothesis, we examined whether the flagellate shows a chemotactic response to tissue extracts (tunic and other tissues) from the host ascidian H. roretzi. We tested extracts from 5 tissues as well as hemolymph. Only the tunic extract showed significant positive chemotactic activity, and the activity decreased with increasing dilution. Furthermore, autoclaved tunic extract, extracts from diseased individuals, and extract from the styelid ascidian Styela clava also had chemotactic activity, although the activities were lower than that of tunic extract from healthy H. roretzi. Ultrafiltration of the tunic extract through a 3 kDa cutoff membrane completely abrogated the activity; the ultrafiltration retentate still showed activity. Thus, the soluble factors that attract the flagellate are present exclusively in the tunic extract, and the chemotactic factors are larger than 3 kDa. Our experiments also suggested that the tunic extract contains both heat-stable and heat-labile factors. We conclude that the flagellate locates the tunic entry site by chemotaxis toward soluble factors that diffuse from a damaged area of the tunic on the siphon wall.

Lomanoticola nishiharai n. sp., a New Species of Copepod Parasitic on the Facelinid Nudibranch, Sakuraeolis enosimensis (Baba, 1930), from the Seto Inland Sea, Western Japan, Including Histological Observations of the Female Lateral Body Process.

Splanchnotrophidae Norman and Scott, 1906 is a family of parasitic copepods that infest nudibranchian and sacoglossan sea slugs. In this study, a new species of splanchnotrophid copepod, Lomanoticola nishiharai n. sp., is described based on specimens of both sexes collected from the facelinid nudibranch, Sakuraeolis enosimensis (Baba, 1930), in the Seto Inland Sea off Hiroshima, central Japan. It represents the third species of Lomanoticola Scott and Scott, 1895 and is characterized by the following female characters: the cephalosome distinctly protruded; the second and third lateral processes on the body originated from same bases; the caudal rami bears a seta V which almost same as long as the rami. Ultrastructural observations revealed that the cuticular surface of the lateral process on the female body is covered with numerous protuberances that may have protective functions against the cellular immune system of the host.

Physical properties of the tunic in the pinkish-brown salp Pegea confoederata (Tunicata: Thaliacea).

BACKGROUND: Invisibility in the water column is a crucial strategy for gelatinous zooplanktons in avoiding detection by visual predators, especially for animals distributed in the euphotic zone during the daytime; i.e., surface dwellers that do not undergo diel vertical migration. Salps, a member of the subphylum Tunicata (Urochordata), usually have a transparent body that is entirely covered with a cellulosic matrix, called the tunic. Some non-migrator species are known to exhibit a nano-scale nipple array on the tunic surface. However, the physical properties of the salp tunic has been poorly investigated, except for Thetys vagina, in which the tunic was expected to show low reflectance based on the refractive index of the tunic. Pegea confoederata is a non-vertical migrant salp showing pinkish-brown body. We measured the hardness, water content, absorption spectra, and refractive index of its tunic to evaluate its fragility and visibility. RESULTS: There are nipple-like protuberances about 80 nm high on the surface of the tunic in P. confoederata. The tunic is very soft; the maximum force to pierce the tunic with a steel rod (1 mm diameter) was < 1 N. The water content of the tunic was > 95%. The absorption spectra of the tunic had no prominent peaks in the wavelength range of 280-800 nm, indicating the tunic is nearly transparent. The difference in refractive indices between tunic and seawater was estimated as 0.002-0.015 at 589 nm. Rigorous coupled wave analyses (RCWA) of light reflection based on 3-dimensional models supported an anti-reflective effect of the nipple array on the tunic surface, which was estimated to vary slightly depending on the forms and the arrangement patterns of nipple-like protuberances in an array. CONCLUSIONS: The tunic of P. confoederata is very soft and contains more water than those of sessile tunicates (ascidians). Based on the refractive index of the tunic, light reflection is expected to be very low, making this salp's tunic barely visible in water column. Our results suggest that the nipple array may produce an anti-reflective effect.

Analysis of genes encoding high-antigenicity polypeptides in three serotypes of Miamiensis avidus.

The ciliate Miamiensis avidus causes scuticociliatosis in Japanese flounder Paralichthys olivaceus. We previously reported three serotypes of this ciliate distinguishable by serotype-specific antigenic polypeptides (serotype I, 30kDa; serotype II, 38kDa; serotype III, 34kDa). In this study, we determined the localization site of the serotype-specific polypeptides in the ciliate and determined the genes encoding the polypeptides, using the isolates IyoI (serotype I), Nakajima (serotype II), and Mie0301 (serotype III). SDS-PAGE and immunoblot analysis of cilia, membrane proteins, and cytoskeletal elements of the ciliates revealed that the polypeptides were abundant in the former two. Scanning electron microscopy of ciliates immobilized by homologous antiserum showed morphological changes in the cilia. These evidences suggested that the polypeptides were ciliary membrane immobilization antigens. The ciliary genes identified showed low identity scores-<51.5% between serotypes. To differentiate the serotypes, we designed serotype-specific PCR primer sets based on the DNA sequences. The PCR-based serotyping results were completely consistent with conventional serotyping methods (immobilization assay and immunoblot analysis). Twenty of 21 isolates were classified as either serotype I or II, and one isolate was undistinguishable. The combination of species-specific PCR previously reported and three serotype-specific PCR could be useful for identifying, serotyping, and surveillance for occurrences of new serotypes of M. avidus.

Cytoplasmic UV-R Absorption in an Integumentary Matrix (tunic) of Photosymbiotic Ascidian Colonies.

Noburu Sensui and Euichi Hirose (2018) In didemnid ascidians with cyanobacterial symbionts, the tunic has a specific peak absorbing ultraviolet radiation (UV-R) due to the presence of ultraviolet (UV)-absorbing compounds, which probably include mycosporine-like amino acids (MAAs). The UV-R absorbing tunic is supposed to protect the symbionts in the common cloacal cavity of the host colony. The histological distribution of UV-R absorption in the tunic was examined using a UV light microscope equipped with a digital camera, from which the low-pass filter of the UV-sensitive image sensor was removed. The cell peripheries of tunic bladder cells and cell-like objects were visualized with the trans-illumination of UV light, indicating UV-R absorption at that site. In contrast, tunic matrix and vacuolar content of tunic bladder cells appeared to lack of UV-R absorption, allowing damaging wavelengths to penetrate. Accordingly, UV-absorbing compounds are expected to be contained in the cytoplasmic matrix of tunic bladder cells and possibly other types of tunic cells.

Measurement of refractive indices of tunicates' tunics: light reflection of the transparent integuments in an ascidian Rhopalaea sp. and a salp Thetys vagina.

BACKGROUND: Tunic is a cellulosic, integumentary matrix found in tunicates (Subphylum Tunicata or Urochordata). The tunics of some ascidian species and pelagic tunicates, such as salps, are nearly transparent, which is useful in predator avoidance. Transparent materials can be detected visually using light reflected from their surfaces, with the different refractive indices between two media, i.e., tunic and seawater, being the measure of reflectance. A larger difference in refractive indices thus provides a larger measure of reflectance. RESULTS: We measured the refractive indices of the transparent tunic of Thetys vagina (salp: Thaliacea) and Rhopalaea sp. (ascidian: Ascidiacea) using an Abbe refractometer and an ellipsometer to estimate the light reflection at the tunic surface and evaluate the anti-reflection effect of the nipple array structure on the tunic surface of T. vagina. At D-line light (λ = 589 nm), the refractive indices of the tunics were 0.002-0.004 greater than seawater in the measurements by Abbe refractometer, and 0.02-0.03 greater than seawater in the measurements by ellipsometer. The refractive indices of tunics were slightly higher than that of seawater. According to the simulation of light reflection based on rigorous coupled wave analysis (RCWA), light at a large angle of incidence will be completely reflected from a surface when its refractive indices are smaller than seawater. Therefore, the refractive index of integument is important for enabling transparent organisms to remain invisible in the water column. CONCLUSION: In order to minimize reflectance, the refractive index should be similar to, but never smaller than, that of the surrounding seawater. The simulation also indicated that the presence or absence of a nipple array does not cause significant difference in reflectance on the surface. The nipple array on the tunic of the diurnal salp may have another function, such as bubble repellence, other than anti-reflection.

Seasonal Occurrence of Gastropterids (Gastropoda: Cephalaspidea) and Their Habitat Selection in a Subtropical Back-reef on Okinawajima Island (Ryukyu Archipelago, Japan).

Daisuke Tanamura and Euichi Hirose (2017) Gastropterids are generally small sea slugs with vivid colors. In a shallow back-reef at Zanpa (Okinawajima Island, Ryukyu Archipelago), ve gastropterid species were observed during route censuses (0-2.6 m in depth, 400 m in length) conducted from November 2011 to June 2014 (89 times, 32 months): Sagaminopteron ornatum, Siphopteron brunneomarginatum, Si. citrinum, Si. flavum, and Si. tigrinum. Among them, Si. tigrinum was observed only in 2013, but the other four species were observed every spring during the survey. When the route was divided into four zones based on depth and dominant substrata, each species was mainly found in particular zones. In laboratory experiments for substrate selection, Si. flavum significantly preferred natural sand and rubble to flat glass-bottoms, glass beads, dried sand, or bleached rubble, suggesting that substrate material is a potential key for habitat selection in this species. Although a larger number of individuals preferred natural sand from their principle habitat to natural sand from a deeper zone where this species was rarely observed, no significant difference was found in the preference by binomial test.

Does the kinorhynch have a hydrophobic body surface? Measurement of the wettability of a meiobenthic metazoan.

The body surface of aquatic invertebrates is generally thought to be hydrophilic to prevent the attachment of air bubbles. In contrast, some interstitial invertebrates, such as kinorhynchs and some crustaceans, have a hydrophobic body surface: they are often trapped at the water surface when the sediment in which they reside is mixed with air and water. Here, we directly measured the wettability of the body surface of the kinorhynch Echinoderes komatsui, using a microscopic contact angle meter. The intact body surface of live specimens was not hydrophobic, but the anterior part was less hydrophilic. Furthermore, washing with seawater significantly decreased the wettability of the body surface, but a hydrophilic surface was recovered after a 1 h incubation in seawater. We believe that the hydrophobic cuticle of the kinorhynch has a hydrophilic coat that is readily exfoliated by disturbance. Ultrastructural observations supported the presence of a mucus-like coating on the cuticle. Regulation of wettability is crucial to survival in shallow, fluctuating habitats for microscopic organisms and may also contribute to expansion of the dispersal range of these animals.

Population Dynamics of the Sea Slug Plakobranchus ocellatus (Opisthobranch: Sacoglossa: Elysioidea) on a Subtropical Coral Reef off Okinawa-jima Island, Ryukyu Archipelago, Japan.

Daisuke Tanamura and Euichi Hirose (2016) Plakobranchus ocellatus is a sacoglossan sea slug that can retain functional chloroplast from its algal food. This species feed on multiple species of siphonous green algae and can survive several months without food by utilizing retained chloroplasts in its digestive gland (kleptoplasty). While the population dynamics of opisthobranchs are often influenced by the seasonal fluctuation of the abundance of food resources, the fluctuation of food availability would not be a crucial factor to restrict the occurrence of P. ocellatus. We monitored the population density of P. ocellatus for 20 months on a subtropical coral reef where the water temperature fluctuated from 17°C to 32°C, in order to examine whether the population density, distribution pattern of individuals, and size distribution of P. ocellatus are stable or seasonally change. The present results showed that P. ocellatus appeared all year round in the study site, while the population density changed seasonally. The population density decreased in cold (≤ 21°C) and hot (≥ 27°C) periods, and densities in the months of intermediate temperature range (< 21°C, > 25°C) were significantly higher than the densities in other months (Student's t-test, P < 0.0001). Accordingly, population density is probably influenced by water temperature. Morisita's Iδ indicated that the sea slugs were distributed in random patterns (13 months) or clumped patterns (7 months). Our field observations indicated that the sea slugs do not feed in daytime, and probably feed at night. Whereas P. ocellatus individuals of less than 10 mm were rarely recorded in the monitoring area, a decrease of the average body length and increase in population density in April - May suggest active recruitment of small individuals in this period.

Cellulose is not degraded in the tunic of the edible ascidian Halocynthia roretzi contracting soft tunic syndrome.

Soft tunic syndrome is a fatal disease in the edible ascidian Halocynthia roretzi, causing serious damage to ascidian aquaculture in Korea and Japan. In diseased individuals, the tunic, an integumentary extracellular matrix of ascidians, softens and eventually tears. This is an infectious disease caused by the kinetoplastid flagellate Azumiobodo hoyamushi. However, the mechanism of tunic softening remains unknown. Because cellulose fibrils are the main component of the tunic, we compared the contents and structures of cellulose in healthy and diseased tunics by means of biochemical quantification and X-ray diffractometry. Unexpectedly, the cellulose contents and structures of cellulose microfibrils were almost the same regardless of the presence or absence of the disease. Therefore, it is unlikely that thinning of the microfibrils occurred in the softened tunic, because digestion should have resulted in decreases in crystallinity index and crystallite size. Moreover, cellulase was not detected in pure cultures of A. hoyamushi in biochemical and expressed sequence tag analyses. These results indicate that cellulose degradation does not occur in the softened tunic.