Botryllid Ascidians Research Articles

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.

Studies on Japanese Botryllid Ascidians. III. A new species of the genus Botryllus with a vivid colony color from the vicinity of Shimoda.

The morphology and life history of a new species of the genus Botryllus belonging to the family Botryllidae are described in detail. This ascidian was collected from the stony shore in the cove near Shimoda Marine Research Center, University of Tsukuba (Shimoda, Shizuoka Prefecture, Japan). The ascidian colony was easily distinguished from colonies of other botryllids because it was very thin and bright pink in color. The arrangement of ovary and testis in this ascidian was the same as that in other species of the genus Botryllus. This ascidian was prolific, with 1-5 embryos on each side of a zooid, and the embryos of this ascidian developed in the peribranchial cavity without any brooding organs as in Botryllus scalaris. We observed the processes and features of the allorecognition reaction in colony specificity and found that allorejection occurred after fusion of the vascular system between two incompatible colonies. This manner of allorejection is also shown in B. scalaris and Botryllus delicatus; however, the reaction speed of allorejection is faster than that of B. delicatus and similar to that of B. scalaris. These results indicate that this ascidian might be closely related to B. scalaris.

Colonial allorecognition, hemolytic rejection, and viviparity in botryllid ascidians.

Allorecognition is a fundamental system that animals use to maintain individuality. Although embryos are usually semiallogeneic with their mother, viviparous animals are required to allow these embryos to develop inside the maternal body, but must also eliminate an "invasion" by nonself. In colonial ascidians of the family Botryllidae, when two colonies are brought into contact at their growing edges, a hemolytic rejection reaction occurs between allogeneic colonies. Morula cells, a type of hemocyte, are the major effector cells in the hemolytic rejection. Morula cells infiltrate and aggregate where the two colonies make contact, and then discharge their vacuolar contents, which contain phenoloxidase and quinones. In viviparous botryllids, colonial contact at artificially cut surfaces always results in colonial fusion and establishment of a common vascular network even between allogeneic colonies in which the growing-edge contact results in rejection. This colonial fusion between incompatible colonies (surgical fusion) suggests that the allorecognition sites are not distributed in the vascular system in which the embryos are brooded. It is supposed that a common ancestor of the viviparous species lost the capacity for allorecognition in their vascular system to protect its embryos from alloreactivity, when it changed from ovoviviparous to viviparous in the course of evolution. The limited distribution of allorecognition sites would be a solution to the embryo-parent histoincompatibility in viviparity.

The colonial urochordate Botryllus schlosseri: from stem cells and natural tissue transplantation to issues in evolutionary ecology.

The urochordates, whose stem groups may have included the direct predecessors of the chordate line, serve as an excellent model group of organisms for a variety of scientific disciplines. One taxon, the botryllid ascidian, has emerged as the model system for studying allorecognition; this work has concentrated on the cosmopolitan species Botryllus schlosseri. Studies analyzing self-nonself recognition in this colonial marine organism point to three levels of allorecognition, each associated with different outcomes. The first level controls natural allogeneic rejections and fusions, in which blood-shared chimeras are formed. The second level leads to morphological resorption of partners within chimeras while the third allows the development of somatic and germ cell parasitic events. Recent studies on multi-chimeric entities formed in allogeneic fusions reveal evolutionary links between allorecognition, stem cell biology and ecology. Thus, the Botryllus system generates perspectives from different biological disciplines to yield a unique life history portrait.

Studies on Japanese botryllid ascidians. II. A new species of the genus Botryllus from the vicinity of Shimoda.

Morphology and life history of a new species of the genus Botryllus belonging to the family Botryllidae were described in detail. This ascidian was collected from the stony shore in the vicinity of Shimoda (Shizuoka prefecture, Japan). The arrangement of ovary and testis in this ascidian was the same as that in other species of the genus Botryllus, while the embryo developed in a brood pouch formed from the invagination of peribranchial epithelium, as in the other genus Botrylloides. The processes and features of the allorecognition reaction of this ascidian were observed. The reaction showed the same processes as that in the species of the genus Botrylloides. Therefore, this ascidian has both features of the two genera of the family Botryllidae, which strongly suggests the necessity of reconsidering on the classificatory criteria of botryllid ascidians.

Morphological consequences for multi-partner chimerism in Botrylloides, a colonial urochordate

Studies on multi-partner chimeras (MC) of the colonial urochordate Botryllus schlosseri have revealed that these chimeras form more stable and vigorous entities than bi-partner chimeras (BC). This outcome has been further studied here on another botryllid species. Botrylloides leachi subpopulation 1, by analysing the morphological consequences of BC, chimeras comprising three partners, tri-chimeras (TC) and chimeras comprising six partners each, hexa-chimeras (HC). For each chimerical type, five different genotypic combinations with three replicates per combination were established. Chimeras were observed for up to 10 months, at which point, all had died. While life spans of BC, TC and HC were the same, the average maximum sizes of HC were higher resulting from more than three times greater daily growth rate. BC and TC reached a maximal chimeric size at a much earlier age. Some morphological resorptions were also expressed differently in HC as compared to BC. When comparing chimerical parameters of bi- vs. multi-chimerism between Botryllus and Botrylloides, these two genera differ in nine out of 13 characteristics tested. However, it seems that irrespective to the species studied and the different characteristics expressed, MC in botryllid ascidians is a commonly developed phenomenon shaped by evolutionary pressures that interact on a ‘group’ level instead of each individual partner. MC reveal modified entities in which contradicting intraspecific interactions are alleviated.

Studies on the Structure of the Brooding Organs of Two Botryllid Ascidians, Botryllus delicatus and Botryllus sexiens

Abstract The formative processes of the brooding organs of two botryllid ascidians, Botryllus delicatus and Botryllus sexiens, were observed. The structure of these organs was also studied in detail histologically. The brooding organs of these two species were formed from the branchial sac, in contrast to the brooding organs of other botryllids, which are formed from the peribranchial wall. The structures of the brooding organs of B. delicatus and B. sexiens were different, but the processes of their formation were almost the same. As the maturation of the oocytes occurred, a part of the epithelium of the branchial sac extended toward the peribranchial epithelium covering the ovary. The extension of the branchial epithelium looked like a bowl and made a surrounding space with the peribranchial epithelium in a peribranchial cavity. The oocyte was ovulated into this space and fertilized there. After ovulation, the extension of the branchial epithelium formed a sac-like brooding organ in combination with the...

Studies on Japanese Botryllid Ascidians. I. A New Species of the Genus Botryllus from the Izu Islands

An investigation was made of the morphology and life history of an unidentified botryllid ascidian that was first collected from the stony shore of Shikine Island of the Izu Islands in Japan. This species has a very soft and transparent tunic, which is different from that of other botryllids. The arrangement of ovary and testis in this species is the same as those of other species of Botryllus. The embryo develops in the sac-like brooding organ formed in the peribranchial cavity of a zooid. Some colonial parts of significant size that include only the vascular system (i.e., without zooids) often extend from the colony margins, and in these parts vascular budding often occurs. Then, the processes and features of the allorecognition reaction of this ascidian were observed. Allorejection occurred after fusion of the vascular systems between two incompatible colonies. This feature is also observed in Botryllus scalaris, but it takes this ascidian much longer to initiate the allorejection reaction than B. scalaris after fusion of blood vessels between two incompatible colonies. It was concluded, on the basis of this study, that this ascidian should be designated as a new species belonging to the genus Botryllus.

A Comparison of Hemocytes and Their Phenoloxidase Activity among Botryllid Ascidians

The colony specificity of colonial animals involves allorejection reactions, which are the defense reactions for allogeneic tissues that occur naturally. In colonial botryllid ascidians, all of the species already studied have colony specificity, and their allorejection modes differ from one another. However, in most of these botryllids, morula cells (MCs) always participate in the allorejection reactions, and the prophenoloxidase (proPO) system of MCs is considered to contribute to the allorejection reaction. The present study was performed using five botryllids and Symplegma reptans, which is closely related to botryllids, in an effort to clarify the relationship between the modes of allorejection and the characteristics of MCs, such as the ratio of MCs to total hemocytes and the phenoloxidase (PO) activity levels in the MCs. The MCs of these six ascidians resembled one another morphologically and the MCs of all species showed PO activity. In Botryllus scalaris, PO activity was also found in granular leukocytes, but the level of activity was much lower than that in MCs. The PO of these species resembles one another, at least in terms of their sensitivity to inhibition by common inhibitors of the proPO system. The PO activity per fixed number of hemocytes varied among these ascidians. This variation was due to a difference in the ratio of MCs to total hemocytes and/or a difference in PO activity per MC. In most ascidians, except for B. scalaris, the rejection reaction area showed a higher level of PO activity than the fusion area of the syngeneic colonies. These results suggest that the characteristics of MCs including their PO activity are closely correlated with the mode of the allorejection reaction.

Behavior of Hemocytes in the Allorejection Reaction in Two Compound Ascidians, Botryllus scalaris and Symplegma reptans.

In botryllid ascidians, the type of allorejection reaction differs among species. Comparative studies of these different reactions contribute to our understanding of the allorecognition and nonself-rejection system. We studied the morphology of hemocyte behavior during allorejection reactions in two species, Botryllus scalaris and Symplegma reptans, which stand at important points in botryllid phylogeny. In B. scalaris, phagocytes mediated hemocyte aggregation, resulting in interruption of blood flow just after vascular fusion of incompatible colonies. Although previous studies indicate that morula cells (MCs) play a central role in the rejection reaction, the MCs of B. scalaris did not participate in the rejection reaction. Colonies of S. reptans showed two types of allorejection reaction that started at different points in the process of vascular fusion between two colonies. In both types of rejection reaction, the MCs played a central role and behaved similarly to those of all botryllids except B. scalaris and a botryllid from Israel. These observations suggest that the differences in hemocyte behavior and allorecognition site observed in this study reflect the variation in allorejection reactions among botryllids.

Phenoloxidase activity of blood cells in botryllid ascidians

Phenoloxidase activity of blood cells in botryllid ascidians

Characteristics of allogeneic resorption in Botrylloides from the Mediterranean coast of Israel

Some of the characteristics of allogeneic resorption have been revealed in studies of chimeras of two Botrylloides subpopulations (SP1 and SP3) inhabiting the Mediterranean coast of Israel. Both Botrylloides SPs exhibited the same outcomes, which differ significantly from the resorption documented in the well-studied botryllid ascidian, Botryllus schlosseri: 1. Resorption in Botrylloides chimeras was usually a very fast process starting 1 to 26 days after fusion and terminating in <9 days thereafter. 2. Resorption occurs at any blastogenic stage. 3. Resorption is complete, including zooids, buds, blood vessels and ampullae. 4. Either one of the genotypes can be resorbed in multipair chimeras of any specific combination. S. Resorption starts simultaneously in all zooids of the partner to be resorbed. 6. Time for resorption and directionality were not affected by the size ratios between the partners (0.3–1.8). As in B. schlosseri, other phenomena (disconnection, chimeric death and retreat growth) interrupt resorption in ca. 60% of the chimeras, under laboratory conditions. We propose that external stress conditions also have a significant role in a nonimmunological expression of resorption in Botrylloides.

Adult rafting versus larval swimming: dispersal and recruitment of a botryllid ascidian on eelgrass

The importance of larval dispersal in determining the distribution and abundance of benthic marine organisms is well recognized; however, the contribution of post-larval dispersal has not been measured. I compared the dispersal of swimming larvae with that of rafting colonies in a population of compound ascidians, Botrylloides sp., living attached to leaves of the eelgrass Zostera marina in Tomales Bay, California, USA in 1990–1992. Colonies rafting on broken eelgrass traveled over 200 times farther and had comparable recruitment success relative to swimming larvae. The recruitment of rafting colonies into new habitats was facilitated by the ability of thesecolonial animals to grow asexually onto surrounding substrata. Rafting colonies brooded larvae that were subsequently relased after the colony settled into a new habitat. These results suggest that colonization of new habitats can occur by post-larvae as well as larvae, and that long-range dispersal by species with short-lived larvae may occur by post-larval rafting.

Surgical fusion between incompatible colonies of the compound ascidian, Botrylloides fuscus

In the compound ascidian Botrylloides fuscus, allorecognition responses were observed at the colonial margin (natural growing edge) as necrotic reactions in the subcuticular region of the facing tunic. However, artificial cut surface contacts between incompatible colonies resulted in the formation of chimeras with common vascular systems, termed “surgical fusion.” The occurrence of surgical fusion in B. fuscus contrasts with rejection occurring under the same conditions in other botryllids. Together the two observations suggest that self-nonself recognition may be a specialized function of tunic cells and their precursors in the blood, and the fusion (vascular interconnection) is a different process than rejection with different effectors and recognition systems. In all pairwise combinations among 12 and 14 colonies, more than 60% of combinations resulted in fusion when colonies were assayed by growing edge contact. This rate of fusible combinations is much higher than those of the other botryllid ascidians previously studied.

Allorecognition in compound ascidians.

In botryllids (colonial ascidians), there are two types of allorecognition: colony specificity and colony resorption. Colony specificity is manifested by fusion and rejection between two conspecific colonies. The genetic basis for this colony specificity resides in a single highly polymorphic gene locus (fusibility locus) with codominantly expressed alleles. Two colonies with no alleles in common at this locus reject each other, whereas colonies sharing at least one allele at the fusibility locus fuse and form a chimera. That is, in colony specificity, self components are distinguished from nonself components, and failure to recognize self induces rapid rejection reactions. The process of rejection in colony specificity is not uniform among all botryllid ascidians. Colony resorption can occur after the establishment of fusion between two colonies. Zooids of one partner of a chimera are resorbed more than 1 week after fusion, or, by amputation of fused blood vessels, the chimera becomes separated into the two original colonies. Colony resorption is also controlled mainly by the fusibility locus. It usually occurs in a chimera between two colonies sharing only one allele at this locus. In colony resorption, nonself determinants are recognized and chronic rejection reactions are induced resembling MHC-dependent graft rejection. Based on these findings, the fusibility locus of botryllids seems to be very similar to the MHC of vertebrates. Considering that vertebrates evolved directly from ascidians, it is likely that the fusibility locus is an ancestral form of the vertebrate MHC.

A Colonial Invertebrate Species that Displays a Hierarchy of Allorecognition Responses.

When two colonies of the compound ascidian Botryllus schlosseri come into contact with each other, they either fuse or reject. This allorecognition is governed genetically by multiple, codominantly expressed alleles at a single, highly polymorphic haplotype called the fusibility/histocompatibility (Fu/HC) locus. Two colonies sharing one or both alleles at this locus can fuse via their extracorporeal tunic blood vessels. Thereafter, in laboratory studies, one partner in the chimera is usually resorbed. The direction of resorption appears to be inherited, as multiple subclones of asexually-derived individuals from colony A always resorb paired subclones from colony B, independent of laboratory conditions or colony age. We established 121 pairs of chimeric partners by fusions of relatives from four generations within a pedigree, all homozygotes (AA line) at their Fu/HC haplotype. This was carried out by self- and defined-crosses done in the laboratory on two outbred founder colonies (each AB at the fusibility locus) which were taken from the field. We found that the resorption phenomenon is characterized by a linear hierarchy within each generation of colonies, which is expressed by the existence of at least 5 intermediate groups. However, the time for resorption did not correlate with the position in the hierarchy. Analysis of resorption hierarchies between different generations revealed that mother colonies always resorbed their self crossed offspring. More interesting, colonies low in the hierarchy within a specific generation reproducibly resorbed the self crossed offspring of a superior kin. Chimeras between defined-crossed offspring of different generations revealed nontransitive types of hierarchies which were correlated with the relative position of each colony in the linear hierarchy established for the colonies within each generation. We propose that colony resorption in colonial botryllid ascidians is controlled by several allorecognition elements that determine a resorption hierarchy.

In vitro culture of blood cells from the colonial protochordateBotryllus schlosseri

Primary cultures of circulatory blood cells from the colonial tunicateBotryllus schlosseri were cultivated in 96-well plates for up to 3 mo. in a medium based on Dulbecco’s modified Eagle’s medium, supplemented with salts to the botryllid ascidian hemolymph osmolarity, HEPES buffer,l-glutamin, fetal bovine serum, and antibiotics. Intercellular bridges between granular pigment cells were established within 24 h. The viability of these cells decreased slowly, and most died within 1 mo. without any sign of cell proliferation. Other cell types remained in an arrested state and were subjected to a weekly medium exchange. Spontaneous cell proliferation was randomly recorded in 6 to 10% of the wells from 2 wk to 1 mo. This proliferation was followed by the formation of masses of cell clumps, from which uniform hemocytes (5 µm, lymphocytelike cells) migrated peripherally. Stress conditions, which included longer intervals between medium exchanges and partial medium replacement, increased the probability of cell proliferation. From each proliferating primary culture, we successfully performed up to 10 plating cycles over a period of 15 wk, during which the cells differentiate in size but are uniformly structured. This produced the firstBotryllus lymphocytelike cell line. From this stage, cell numbers remained constant for up to 6 mo. without increase in cell number. Several mitogenic factors were employed on primary cultures.Botryllus and sea cucumber hemolymphs and mixed interleukins were found to augment significantly proliferation of at least one specific cell size, whereas cells were not markedly responsive to lectins (Concavalin A, wheat germ agglutinin,Ulex europaeus agglutinin), insulin, and retinoic acid. The results are discussed with respect to future efforts in the development of tunicate blood cell cultures.

Serum Amyloid P Component (SAP)‐Like Protein From Botryllid Ascidians Provides a Clue to Amyloid Function

The HA-1 lectin isolated from Botrylloides leachii has an amino acid composition similar to that of mammalian serum amyloid protein (SAP). SAP is a universal component of mammalian amyloid deposits. Like SAP, HA-1 has a disc ultrastructure, and antibody to HA-1 binds both (a) to amyloidlike fibers deposited between rejected Botrylloides colonies and (b) to cerebral amyloid deposits in Alzheimer's disease brains. Deposition of protochordate amyloid within rejection sites and surrounding fouling organisms implies that these fibers function as barriers to allogeneic and infectious challenge. Similarly, mammalian amyloid may also function to contain inflammatory lesions and to limit the spread of certain infections. Pathological amyloidotic conditions in humans, such as Alzheimer's disease, may result from unregulated expression of this primitive encapsulation response.

A New Type of the Manifestation of Colony Specificity in the Compound Ascidian,Botrylloides violaceusOka

A new type of colony specificity (= allogeneic recognition) is shown for Botrylloides violaceus. All botryllid ascidians previously studied for colony specificity show allo-recognition reactions, manifested as fusion or nonfusion (rejection), both at the colonial margin (= growing edge) and at the cut surface. By contrast allorecognition in Botrylloides violaceus is absent at the cut surface, but present at the growing edge. Juxtaposition of cut surfaces resulted in fusion of the colonies regardless of origin, while juxtaposition of natural growing edges resulted in fusion or rejection, according to the genetic combination of colonies. Similar results occurred among the sibling colonies derived from the same mother colony, in which pairs tunic necrosis was observed in areas where the two colonies partially fused. These features of allogeneic rejection in B. violaceus were very similar to those of "nonfusion" in Botrylloides simodensis. In inter-specific combinations between B. violaceus and B. simodensis, a remarkable necrotic reaction was observed in the zone of contact when two colonies touched at their cut surfaces. When brought into contact at their growing edges, they resulted in "nonfusion" without a particular reaction.

Pattern development in palleal buds of botryllid ascidians: Relation between parent and its buds in their body axes.

In order to determine how the zooid pattern (normal or reversed bilateral asymmetry) is transmitted through asexual reproduction in botryllid ascidians, the axial relationship between a zooid and its palleal buds was observed in Symplegmo reptans, Metandrocarpa uedai, Polyandrocarpa misakiensis, and P. stolonifera. The bud axes developed in harmony with the parent axes, with both normal and reversed body patterns, so that (1) when buds are given off from the right (left) side of the parent, their distal side becomes the right (left) in the future zooids (coincidence of the anteroposterior axis), (2) the free surface of the developing buds, like the parent, becomes dorsal (the coincidence of dorsoventral axis), and (3) the heart (gut loop), which is an organ representing the bilateral asymmetry, arises on the distal side of the developing buds, when the buds are derived from the side where the parent heart (gut loop) was situated (coincidence of the transverse axis). This shows that the bud develops as if it knew the position on the parent lateral wall from which it has arisen, a phenomenon referred to as the parental lateral effect.