Transmission of cyanobacterial symbionts during embryogenesis in the coral reef ascidians Trididemnum nubilum and T. clinides (Didemnidae, Ascidiacea, Chordata)

Vertical transmission of cyanobacterial symbionts occurs in didemnid ascidians harboring Prochloron as an obligate symbiont; the photosymbionts are transferred from the parental ascidian colony to the offspring in various ways depending on host species. Although several didemnids harbor non-Prochloron cyanobacteria in their tunics, few studies have reported the processes of vertical transmission in these didemnids. Here we describe the histological processes of the transmission of cyanobacteria in two didemnids, Trididemnum nubilum harboring Synechocystis and T. clinides harboring three cyanobacterial species. In both species, the photosymbionts in the tunic of the parent colony were apparently captured by the tunic cells of the host and transferred to the embryos brooded in the tunic. The symbiont cells were then incorporated into the inner tunic of the embryo. This mode of transmission is essentially the same as that of T. miniatum harboring Prochloron in the tunic, although there are some differences among species in the timing of the release of the symbionts from the tunic cells. We suggest that the similar modes of vertical transmission are an example of convergent evolution caused by constraints in the distribution patterns of symbiont cells in the host colony.     

Ultraviolet-light-absorbing tunic cells in didemnid ascidians hosting a symbiotic photo-oxygenic prokaryote,Prochloron

Coral reef invertebrates that host phototrophic symbionts are thought to protect themselves and their symbionts with mycosporine-like amino acids (MAAs)-UV-absorbing substances that act as sunscreens (Dunlap, W. C., and J. M. Shick, 1998. J. Phycol. 34: 418-430). However, the histological distribution of MAAs in the host tissues has not yet been visualized. We have localized the UV-absorbing substances in the tissues of two colonial didemnid ascidians-Lissoclinum patella and Diplosoma sp.-that contain the symbiotic photo-oxygenic prokaryote Prochloron sp. Cross-sections of unfixed tissue from these ascidians were examined by UV-light microscopy at 320 or 330 nm, wavelengths at which UV light is absorbed by MAAs. Within the tunic, the gelatinous integument of the colony, UV light was exclusively absorbed by a particular type of cell, the tunic bladder cell. Tunic bladder cells with strong UV absorption were denser in the upper tunic, which lies over a colony's zooids, than in the basal tunic underlying the zooid. In the upper tunic, those cells with strong UV absorption were most dense near the surface. The tunic bladder cell is highly vacuolated, and the vacuole contains strong acid, which destabilizes MAAs. Furthermore, the UV-absorbing portion of tunic bladder cells seemed to be cup-shaped, indicating that the MAAs are not localized in the vacuole, but in the cytoplasm. These results strongly suggest that didemnid ascidians accumulate MAAs in tunic bladder cells as a protection against UV radiation.     

Vertical transmission of photosymbionts in the colonial ascidian Didemnum molle: the larval tunic prevents symbionts from attaching to the anterior part of larvae

Morphological processes in the vertical transmission of photosymbionts were investigated in the Prochloron-bearing ascidian Didemnum molle. Prochloron cells were found exclusively in the common cloacal cavity of the colony, attached mainly to the tunic lining of the cavity wall. Oocytes were found in the abdominal region of each zooid, but no Prochloron cells were associated with this stage. During embryogenesis, embryos moved into the tunic core of the colony and were always separated from Prochloron cells in the cloacal cavity by the tunic matrix, until they hatched out from the tunic core. In swimming larvae, Prochloron cells covered the surface of the posterior half of the larval trunk, whereas a thin larval tunic layer covered the anterior half, where no Prochloron cells were found. The tunic of the posterior half of the larval trunk had many folds that enfolded the Prochloron cells and may be adhesive in order to acquire Prochloron cells from the mother colony. The thin larval tunic layer is probably not adhesive and protects the anterior half of the trunk from interference by Prochloron cells with sensory receptors and adhesive organs.     

Excitation energy relaxation in a symbiotic cyanobacterium, Prochloron didemni, occurring in coral-reef ascidians, and in a free-living cyanobacterium, Prochlorothrix hollandica

The marine cyanobacterium Prochloron is a unique photosynthetic organism that lives in obligate symbiosis with colonial ascidians. We compared Prochloron harbored in four different host species and cultured Prochlorothrix by means of spectroscopic measurements, including time-resolved fluorescence, to investigate host-induced differences in light-harvesting strategies between the cyanobacteria. The light-harvesting efficiency of photosystems including antenna Pcb, PS II-PS I connection, and pigment status, especially that of PS I Red Chls, were different among the four samples. We also discuss relationships between these observed characteristics and the light conditions, to which Prochloron cells are exposed, influenced by distribution pattern in the host colonies, presence or absence of tunic spicules, and microenvironments within the ascidians' habitat.     

Tunic cells in pyrosomes (Thaliacea, Urochordata): cell morphology, distribution, and motility

Abstract. Cellular components of the tunic were histologically examined in 3 pyrosome species representing all 3 genera of the order: Pyrosoma atlanticum, Pyrosomella verticillata , and Pyrostremma spinosum . Three cell types are distributed in the tunic. Tunic amebocytes, irregularly shaped and motile, often contain granules and/or phagosomes. Spherical tunic cells contain many round vesicles with eosinophilic and acidic materials. Tunic net cells form a cellular network in which their long filopodia connect with one another. The net cells are densely distributed just beneath the tunic surface lining the common cloacal cavity and may produce tension to maintain the colony shape. The presence of net cells suggests a phylogenetic relationship between pyrosomes and some aplousobranch ascidians. Test fibers are multicellular cords that run in the tunic and connect the zooids. In P. atlanticum , they are attached to they are attached to the epidermal cells of the zooids, and transverse cloacal muscles are attached to the other (proximal) side of the epidermal cells. The test fibers may mediate coordination of the zooids and control muscle contraction.     

Ascidian tunic cells: morphology and functional diversity of free cells outside the epidermis

Abstract. Tunic cells are free cells distributed in the tunic, the integumentary matrix of tunicates. In ascidians, various types of tunic cells have been described both in solitary and in colonial species. Many of them are functionally specialized and are related to the protection of the animal, such as phagocytosis to prevent infection, acid storage to avoid predation, and pigmentation to protect against solar radiation. While some tunic cells are known to play a role in colonial allorecognition, bioluminescence, and algal symbiosis, the functional roles of many cell types still remain to be determined. The composition of tunic-cell types varies among ascidian species, most likely reflecting the functional requirements of the tunic in each species. Although some cell types, e.g., tunic net cells and tunic bladder cells, are restricted to particular taxa of ascidians, tunic phagocytes are found in all known ascidians. Therefore, tunic phagocytes are hypothesized to be basal and shared with ancestral tunicates. In some ascidians, phagocytic cells are involved in other functions, such as pigmentation, intracellular photosymbiosis, and bioluminescence. These specialized phagocytic cells are hypothesized to be derived from tunic phagocytes, suggesting that tunic cells have a high potential to diversify and evolve a wide variety of cellular functions.     

Host specificity and phylogeography of the prochlorophyte Prochloron sp., an obligate symbiont in didemnid ascidians

Prochloron is an oxygenic photosynthetic bacterium that lives in obligate symbiosis with didemnid ascidians, such as Diplosoma spp., Lissoclinum spp. and Trididemnum spp. This study investigated the genetic diversity of the genus Prochloron by constructing a phylogenetic tree based on the 16S rRNA gene sequences of 27 isolates from 11 species of didemnid ascidians collected from Japan, Australia and the USA. The 27 isolates formed three phylogenetic groups: 22 of the samples were identified to be closely related members of Prochloron. Two samples, isolated from Trididemnum nubilum and Trididemnum clinides, were found to belong to the species Synechocystis trididemni, the closest relative of Prochloron. Three isolates formed a separate group from both Prochloron sp. and S. trididemni, potentially indicating a new symbiotic phylotype. Genomic polymorphism analysis, employing cyanobacterium-specific highly iterative palindrome 1 repeats, could not delineate the isolates further. For the Prochloron sp. isolates, the phylogenetic outcome was independent of host species and geographic origin of the sample indicating a low level of host specificity, low genetic variation within the taxon and possibly a lack of a host-symbiont relationship during reproductive dispersal. This study contributes significantly to the understanding of Prochloron diversity and phylogeny, and implications for the evolutionary relationship of prochlorophytes, cyanobacteria and chloroplasts are also discussed.     

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

The colonial ascidian Aplidium yamazii exhibited an allorejection reaction when two allogeneic colonies were brought into contact at their growing edges or at artificial cut surfaces. This species has no vascular network in the tunic, unlike the botryllid ascidians, which have a vascular network throughout the colony's common tunic. In the allorejection reaction induced by contact at the growing edges, some small, hard-packed tunic masses were formed at the contact points. Histological and electron microscopic investigation of these tunic masses revealed that they contained aggregates of tunic cells, with tunic phagocytes being the major cell type present. Some of the tunic phagocytes in these tunic masses appeared to be disintegrating. When allogeneic colonies were placed in contact at their artificial cut surfaces, the colonies partially fused, then separated. In this allorejection reaction, some loosely packed tunic masses remained in the gap between the two withdrawn colonies. These results strongly suggest that the tunic phagocytes are likely to be the major effector cells in the allorejection reaction. We also propose that the tunic phagocytes are not only the effector cells in the allorejection reaction but also bear the sites of allorecognition.     

Down under the tunic: bacterial biodiversity hotspots and widespread ammonia-oxidizing archaea in coral reef ascidians

Ascidians are ecologically important components of marine ecosystems yet the ascidian microbiota remains largely unexplored beyond a few model species. We used 16S rRNA gene tag pyrosequencing to provide a comprehensive characterization of microbial symbionts in the tunic of 42 Great Barrier Reef ascidian samples representing 25 species. Results revealed high bacterial biodiversity (3 217 unique operational taxonomic units (OTU_0.03) from 19 described and 14 candidate phyla) and the widespread occurrence of ammonia-oxidizing Thaumarchaeota in coral reef ascidians (24 of 25 host species). The ascidian microbiota was clearly differentiated from seawater microbial communities and included symbiont lineages shared with other invertebrate hosts as well as unique, ascidian-specific phylotypes. Several rare seawater microbes were markedly enriched (200–700 fold) in the ascidian tunic, suggesting that the rare biosphere of seawater may act as a conduit for horizontal symbiont transfer. However, most OTUs (71%) were rare and specific to single hosts and a significant correlation between host relatedness and symbiont community similarity was detected, indicating a high degree of host-specificity and potential role of vertical transmission in structuring these communities. We hypothesize that the complex ascidian microbiota revealed herein is maintained by the dynamic microenvironments within the ascidian tunic, offering optimal conditions for different metabolic pathways such as ample chemical substrate (ammonia-rich host waste) and physical habitat (high oxygen, low irradiance) for nitrification. Thus, ascidian hosts provide unique and fertile niches for diverse microorganisms and may represent an important and previously unrecognized habitat for nitrite/nitrate regeneration in coral reef ecosystems.     

Switching of metabolic-rate scaling between allometry and isometry in colonial ascidians

The metabolic rate and its scaling relationship to colony size were studied in the colonial ascidian Botrylloides simodensis. The colonial metabolic rate, measured by the oxygen consumption rate (V(O2) in millilitres of O(2) per hour) and the colony mass (wet weight M(w) in grams) showed the allometric relationship (V(O2) = 0.0412 M(w)(0.799). The power coefficient was statistically not different from 0.75, the value for unitary organisms. The size of the zooids and the tunic volume fraction in a colony were kept constant irrespective of the colonial size. These results, together with the two-dimensional colonial shape, excluded shape factors and colonial composition as possible causes of allometry. Botryllid ascidians show a takeover state in which all the zooids of the parent generation in a colony degenerate and zooids of a new generation develop in unison. The media for connection between zooids such as a common drainage system and connecting vessels to the common vascular system experienced reconstruction. The metabolic rate during the takeover state was halved and was directly proportional to the colonial mass. The scaling thus changed from being allometric to isometric. The alteration in the scaling that was associated with the loss of the connection between the zooids strongly support the hypothesis that the allometry was derived from mutual interaction among the zooids. The applicability of this hypothesis to unitary organisms is discussed.     

Phylogenetic diversity of bacteria associated with ascidians in Eel Pond (Woods Hole, Massachusetts, USA)

Recent studies have revealed that many marine invertebrates are closely associated with diverse microorganisms, frequently resulting in the production of compounds of biomedical interest. Thus far, ascidians have not been widely examined for the presence of bacterial associations, although the production of secondary metabolites is well documented. In the present study, we examined the gonad of Molgula manhattensis and the tunic surfaces of Botryllus schlosseri, Didemnum sp., and Botrylloides violaceus for the presence of associated bacteria. These ascidians are common inhabitants of the coastal ocean of Cape Cod, Massachusetts. We used denaturing gradient gel electrophoresis (DGGE) as well as cloning and sequencing of 16S rDNA to analyze the microbial communities. There is a strong evidence that spiroplasma-like bacteria colonize the gonad of the solitary ascidian M. manhattensis. The bacteria might be vertically transmitted and may be involved in the production of secondary metabolites that deter predators of the ascidians. The bacterial communities found on the tunic surfaces of the colonial ascidians were found to be more diverse. However, in all cases the bacterial communities were predominated by alpha-proteobacteria. Alpha-proteobacteria related to the obtained sequences have been identified as symbionts in a variety of hosts, suggesting a specific role for these bacteria. However, based on our data it is difficult to differentiate between persistently and only transiently associated bacteria. Overall, this study demonstrates that ascidian species are associated with diverse bacterial populations. Future studies will aim to elucidate the precise relationships between bacteria and ascidians and to identify bioactive compounds that might be produced as a result of these relationships.     

Soft tunic syndrome in the edible ascidian Halocynthia roretzi is caused by a kinetoplastid protist

An etiological study was conducted to clarify whether the flagellate-like cells found in histological preparations of the tunic of diseased Halocynthia roretzi (Drasche) were the causative agent of soft tunic syndrome in this ascidian. When pieces of softened diseased tunic were incubated overnight in sterile seawater, live flagellated cells, which were actively swimming in the seawater, were observed in 47 out of 61 diseased ascidians (77%), but not in moribund or abnormal individuals with normal tunics (n = 36) nor in healthy animals (n = 19). The flagellate was morphologically very similar to those observed in histological sections of the diseased tunic. By contrast, flagellates were not found in tunic pieces of healthy, moribund, and abnormal individuals that did not exhibit softening of the tunic. Light and electron microscopy revealed that the flagellate has polykinetoplastic mitochondria with discoidal cristae. The cytomorphologies of the flagellate were the same as those of the flagellate-like cells in the diseased tunic. We cultured the flagellate from the softened tunic in vitro and confirmed that the tunics of healthy ascidians, which were immersion-challenged with suspensions of the subcultured flagellates, became softened 17 d after exposure, including the final 12 d in aerated, running seawater. The occurrence of flagellates was also confirmed by incubating pieces of soft tunic from experimentally infected animals in seawater overnight. These results indicate that the flagellate is the causative agent of soft tunic syndrome.     

Tunic cell populations during fusion events in the colonial ascidian Didemnum vexillum (Tunicata)

We documented changes in the abundance and distribution patterns of tunic cells involved in the allorecognition response of the colonial aplousobranch Didemnum vexillum, whose zooids do not share a common vascular system. A histological examination of the fusion zone of isogeneic (CIAs) and allogeneic (CAAs) fused colony assays revealed that tunic cuticles were rapidly regenerated. The underlying tunic matrix fused readily in all assays and controls. We identified four different types of tunic cells. Phagocytic cells represented the most abundant cell type in allogeneic fusions, followed by morula cells. These cells were more abundant at the immediate fusion junction than at 120 μm or 240 μm from the junction, most likely because they mediate the allorecognition reaction. Elongated filopodial cells also were present, although only at very low abundances, and a layer of bladder cells was located immediately below the cuticle. Our results provide quantitative evidence for the involvement of tunic cells in the allorecognition response of a highly invasive ascidian.     

Immuno- and histochemistry characteristics of morula and test cells in three ascidian species

One of the hypotheses suggests that test cells play a part in a larval tunic formation like morula cells in adult ascidians. It was shown that the antibodies against morula cell proteins of 26 and 48 kDa of the ascidian Styela rustica react on the paraffin sections with both the granules of morula cells and test cells of ascidians S. rustica and Boltenia echinata. Among the test cell proteins of S. rustica SDS-electrophoresis revealed at least 5 major proteins but no one with the molecular mass of 26 and 48 kDa and none of them react with the antibodies. At the same time AB26 bind the proteins with similar molecular masses in blood cells and in the probe containing test cells--27 and 28 kDa, correspondingly,--of ascidian Molgula citrina. Comparative histochemical analysis of morula and test cells of these three ascidian species was carried out. There are a lot of acid polysaccharides combined with proteins in test cells whereas morula cells contain mainly positively charged proteins. Thus it could be supposed that degree of manifestation of antigens might be different in the conditions of immunoblot and immunohistochemical analysis. The hypothesis of the similarity in morula and test cells functions and their interrelationship is discussed.     

Convergent evolution of the vertical transmission mode of the cyanobacterial obligate symbiont Prochloron distributed in the tunic of colonial ascidians

In chordates, obligate photosynthetic symbiosis has been reported exclusively in some colonial ascidians of the family Didemnidae. The vertical transmission of the symbionts is crucial in establishing the obligate symbiosis between the cyanobacteria and the host ascidians. The results of comparative surveys on the morphological processes of cyanobacterial transmission suggest the occurrence of convergent evolution of the vertical transmission in the host species harboring symbionts in the cloacal cavity. In Trididemnum species harboring cyanobacterial cells in the tunic, the symbiont cells are transported by the tunic cells to the tunic of embryos brooded in the tunic of the parent colony. The present study examined whether the mode of symbiont transmission is the same in host species harboring the symbionts in the tunic, regardless of host genera, or whether non-Trididemnum hosts have a different vertical transmission mode. Our results showed that the vertical transmission process in Lissoclinum midui was almost the same as in the Trididemnum species, supporting the occurrence of convergent evolution in the two distinct didemnid genera, that is, Trididemnum and Lissoclinum. High plasticity of the embryogenic process in didemnid ascidians may be important in developing the mechanism of vertical transmission; this assumption may also explain why the obligate cyanobacterial symbiosis has been exclusively established in didemnid ascidians among chordates.     

A comparative study of the non-acidic chemically mediated antifoulant properties of three sympatric species of ascidians associated with seagrass habitats

The present study investigated aspects of the antifoulant properties of three sympatric species of ascidians found in seagrass habitats of the Gulf of Mexico, Southern Atlantic Ocean, and Caribbean. Field observations in Saint Joseph Bay, Florida indicate that all three species are common and that the tunic of the solitary ascidian Molgula occidentalis is often heavily fouled, while the outer surfaces of both the colonial ascidians Amaroucium stellatum and Botryllus planus are free of fouling organisms. Antifoulant activities of a suite of increasing hydrophilic organic extracts prepared from the tunic of M. occidentalis and whole colonies of A. stellatum and B. planus were measured using both sympatric microbial (bacteria) and macroinvertebrate (cyprid larvae of Balanus amphitrite) fouling organisms in laboratory bioassays. In addition, field antifoulant assays were conducted by combining organic extracts with controlled-release resin and subsequently coating this material on to acrylic rods deployed in the field for a 72 h period. Extracts of the tunic of M. occidentalis generally did not inhibit bacterial growth. The exception was the methanol extract, which inhibited growth in one of the six marine bacteria tested. Moreover, only the highest concentrations of hexane and methanol tunic extracts tested prevented attachment of cyprid larvae. Field assays revealed no antifoulant activity on rods coated with resin containing extracts of M. occidentalis. Inhibition of both microbial growth and cyprid settlement were much more pronounced in whole-organism extracts of the two colonial ascidians. Most potent were the aqueous methanol extracts of colonies of B. planus and A. stellatum which inhibited growth in five of the six marine bacteria tested. In addition, hydrophilic and lipophilic extracts of the colonial ascidians significantly inhibited attachment of cyprid larvae, in many instances across a wide range of extract concentrations. Field antifoulant assays indicated that extracts of both colonial ascidians inhibited settlement of bryozoans and barnacles. The findings indicate that the colonial ascidians B. planus and A. stellatum possess chemical antifoulant properties. In contrast, the solitary ascidian M. occidentalis appears to either tolerate fouling or possess other non-chemical mechanisms to cope with the risks associated with epibiont overgrowth.     

Microbial community associated with the colonial ascidian Cystodytes dellechiajei

The ascidian Cystodytes dellechiajei (Della Valle, 1877) (phylum Chordata, class Ascidiacea, family Polycitoridae) is a colonial tunicate that inhabits benthic rock environments in the Atlantic, Pacific and Indian Oceans, as well as the Mediterranean Sea. Its life cycle has two phases, the adult sessile colony and the free-living larva. Both adult zooids and larvae are surrounded by a protective tunic that contains several eukaryotic cell lines, is composed mainly of acidic mucopolysacharides associated with collagen and elastin-like proteins, and is covered by a thin cuticle. The microbiota associated with the tunic tissues of adult colonies and larva of C. dellechiajei has been examined by optical, confocal and electron microscopy and by fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), and 16S rRNA gene clone library analysis. Microscopy analyses indicated the presence inside the tunic, both for the adult and the larva, of a dense community of Bacteria while only the external surface of colony cuticle was colonized by diatoms, rodophyte algae and prokaryotic-like epiphytes. Transmission electron microscopy showed tunic eukaryotic cells that were engulfing and lysing bacteria. 16S rRNA gene analyses (DGGE and clone libraries) and FISH indicated that the community inside the tunic tissues of the adults and larvae was dominated by Alphaproteobacteria. Bacteria belonging to the phyla Gammaproteobacteria and Bacteroidetes were also detected in the adults. Many of the 16S rRNA gene sequences in the tunic tissues were related to known aerobic anoxygenic phototrophs (AAP), like Roseobacter sp. and Erythrobacter sp. In order to check whether the gene pufM, coding for the M subunit of the reaction centre complex of aerobic anoxygenic photosynthesis, was being expressed inside the ascidian tissues, two libraries, one for an adult colony and one for larva, of cDNA from the expressed pufM gene were also constructed. The sequences most frequently (64% for colony and 67% for larva) retrieved from these libraries presented > 90% aa identity with the pufM gene product of the Roseobacter-like group, a cluster of AAP widely detected in marine planktonic environments.     

'Cup cell disease' in the colonial tunicate Botryllus schlosseri

A new progressive, fatal disease called 'cup cell disease' was characterized in ex situ cultures of Botryllus schlosseri, a colonial tunicate. The disease originated as a few dark spots growing within zooids. The infected colonies then started to deteriorate, morphologically diagnosed by ampullar retraction, lethargic blood circulation and by a swollen and soft tunic matrix. In later stages of the disease, developed buds were also affected. Many large black dots were scattered within the tunic matrix, and zooids were transformed to opaque, dilated, sac-like structures, signaling impending death. Colonies were infected periodically, even without direct tissue contact. The time course from first appearance to colony death ranged between 30 and 45 d. Histological studies, in vitro culturing of blood cells and blood smears revealed the existence of numerous cup-like cells (up to 4.8 microm diameter on average) with a yellowish cell wall and transparent cytoplasm that was not stained by various dyes (except azocarmine-G). Cells were refractive under bright-field illumination and revealed a flattened wall with flanges, characteristic of species of the phylum Haplosporidia. Cup cells aggregated in blood vessels and in internal parts of zooids and buds and were phagocytosed by blood cells. In a single case, plasmodia-like structures were found only in the tunic matrix of an infected colony. This is the first record in botryllid ascidians of an infectious lethal disease associated with haplosporidian protists.     

An evaluation of chemical and physical defenses against fish predation in a suite of seagrass-associated ascidians

The palatability of two solitary and three colonial species of ascidians commonly found in sub-tropical seagrass meadows was evaluated using the abundant, sympatric, omnivorous pinfish Lagodon rhomboides as a model predator. Bite-sized pieces of fresh tissues of both solitary and one of the three colonial ascidian species were unpalatable to fish. Lipophilic and hydrophilic extracts of the three unpalatable species did not cause feeding deterrence indicating that secondary metabolites are not responsible for the lack of palatability. Distaplia bermudensis, the one colonial ascidian that was unpalatable to fish, had a highly acidified outer tunic (pH = 1.5). We tested the ability of acidified agar food pellets (pH = 1.5) to deter pinfish and found that the fish readily ingested acidified pellets. The toughness of the tunic of all five ascidian species was evaluated by measuring the Force (N) required to penetrate the tunic using a penetrometer. Tunic toughness is likely to explain the lack of palatability of the solitary ascidians Styela plicata and Molgula occidentalis as their tunics required a force of > 34 N to penetrate. Tunic toughness may be a particularly effective adaptation for ascidian defense in seagrass habitats where fish with strong crushing jaws, such as those that commonly occur in coral reef systems, are rare.     

Not like Botryllus: indiscriminate post-metamorphic fusion in a compound ascidian

Fusion trials between metamorphs of the aplousobranch compound ascidian Diplosoma listerianum indicated that chimera formation was not dependent on relatedness. Similar, high rates of union were observed between full siblings, half siblings, unrelated individuals from the same population, and individuals from two geographically distant localities. This is in contrast to the well-studied ascidian genus Botryllus, in which a highly polymorphic allorecognition system governing the fusion–non-fusion reaction (colony specificity) largely limits fusion to close relatives. Fusion in Botryllus establishes a vascular chimera throughout which stem cells may circulate, promoting cell lineage competition between the fusion partners. The restriction of fusion to close kin in Botryllus is thought to reduce the inclusive fitness costs of competitive interactions between cell lineages within the chimera. In contrast to Botryllus, modules (zooids) of a D. listerianum colony are not interlinked by blood vessels, seemingly precluding the exchange of stem cells. The apparent absence of strict colony specificity in D. listerianum is thus in keeping with the predictions of the Botryllus model for the maintenance of allorecognition polymorphism. However, colony specificity has been reported in other species of aplousobranch ascidian that also lack a common vascular system. In these, the threat of migrating blood-borne stem cells cannot be responsible for the presence of colony specificity. One possibility, requiring experimental investigation, is that stem cells could perhaps migrate between zooids by another route, such as through the matrix of the colonial tunic. Even in the absence of stem cell exchange, cheating on the costs of colony maintenance and defence could also produce selective forces favouring colony specificity. In compound ascidians, this could involve unequal contribution to extrazooidal structures, principally the tunic and related tissues. This consideration seems potentially relevant to the lack of discrimination during fusion in D. listerianum, since extrazooidal somatic investment in this species appears minimal, severely limiting the scope for this other form of cheating. The various possible modes of exploitative interaction between fused colonies are not mutually exclusive, and offer fundamentally similar explanations for colony specificity. If none of them can be shown to occur in non-botryllid species possessing colony specificity, the generality of the Botryllus model may require re-evaluation.