Cyclical generation and degeneration of organs in a colonial urochordate involves crosstalk between old and new: a model for development and regeneration

Botryllus schlosseri is a colonial marine urochordate in which all adult organisms (called zooids) in a colony die synchronously by apoptosis (programmed cell death) in cyclical fashion. During this death phase called takeover, cell corpses within the dying organism are engulfed by circulating phagocytic cells. The "old" zooids and their organs are resorbed within 24-36 h (programmed cell removal). This process coincides temporally with the growth of asexually derived primary buds, that harbor a small number of undifferentiated cells, into mature zooids containing functional organs and tissues with the same body plan as adult zooids from which they budded. Within these colonies, all zooids share a ramifying network of extracorporeal blood vessels embedded in a gelatinous tunic. The underlying mechanisms regulating programmed cell death and programmed cell removal in this organism are unknown. In this study, we extirpated buds or zooids from B. schlosseri colonies in order to investigate the interplay that exists between buds, zooids, and the vascular system during takeover. Our findings indicate that, in the complete absence of buds (budectomy), organs from adult zooids underwent programmed cell death but were markedly impaired in their ability to be resorbed despite engulfment of zooid-derived cell corpses by phagocytes. However, when buds were removed from only half of the flower-shaped systems of zooids in a colony (hemibudectomy), the budectomized zooids were completely resorbed within 36-48 h following onset of programmed cell death. Furthermore, if hemibudectomies were carried out by using small colonies, leaving only a single functional bud, zooids from the old generation were also resorbed, albeit delayed to 48-60 h following onset of programmed cell death. This bud eventually reached functional maturity, but grew significantly larger in size than any control zooid, and exhibited hyperplasia. This finding strongly suggested that components of the dying zooid viscera could be reutilized by the developing buds, possibly as part of a colony-wide recycling mechanism. In order to test this hypothesis, zooids were surgically removed (zooidectomy) at the onset of takeover, and bud growth was quantitatively determined. In these zooidectomized colonies, bud growth was severely curtailed. In most solitary, long-lived animals, organs and tissues are maintained by processes of continual death and removal of aging cells counterbalanced by regeneration with stem and progenitor cells. In the colonial tunicate B. schlosseri, the same kinds of processes ensure the longevity of the colony (an animal) by cycles of death and regeneration of its constituent zooids (also animals).     

A morphological study of nonrandom senescence in a colonial urochordate

Botryllus schlosseri is a clonally modular ascidian, in which individuals (zooids) have a finite life span that is intimately associated with a weekly budding process called blastogenesis. Every blastogenic cycle concludes with a synchronized phase of regression called takeover, during which all zooids in a colony die, primarily by apoptosis, and are replaced by a new generation of asexually derived zooids. We have previously documented that, in addition to this cyclical death phase, entire colonies undergo senescence during which all asexually derived individuals in a colony, buds and zooids, die in concert. In addition, when a specific parent colony (genet) is experimentally separated into a number of clonal replicates (ramets), ramets frequently undergo senescence simultaneously, indicating that mortality can manifest itself in nonrandom fashion. Here, we document a morphological portrait of senescence in laboratory-maintained colonies from Monterey Bay, California, that exhibit nonrandom mortality. Nonrandom senescence proceeded according to a series of characteristic changes within the colony over a period of about one week. These changes included systemic constriction and congestion of the vasculature accompanied by massive accumulation of pigment cells in the zooid body wall (mantle), blood vessels, and ampullae; gradual shrinkage of individual zooids; loss of colonial architecture, and ultimately death. At the ultrastructural level, individual cells exhibited changes typical of ischemic cell death, culminating in necrotic cell lysis rather than apoptosis. Collectively, these observations indicate that senescence is accompanied by unique morphological changes that occur systemically, and which are distinct from those occurring during takeover. We discuss our findings in relation to current experimental models of aging and the possible role of a humoral factor in bringing about the onset of senescence.     

Complexity generated by iteration of hierarchical modules in bryozoa

Growth in colonial organisms by iteration of modules inherently provides for an increase in available morpho-ecospace relative to their solitary relatives. Therefore, the interpretation of the functional or evolutionary significance of complexity within groups that exhibit modular growth may need to be considered under criteria modified from those used to interpret complexity in solitary organisms. Primary modules, corresponding to individuals, are the fundamental building blocks of a colonial organism. Groups of primary modules commonly form a second-order modular unit, such as a branch, which may then be iterated to form a more complex colony. Aspects of overall colony form, along with their implications for ecology and evolution, are reflected in second-order modular (structural) units to a far greater degree than by primary modular units (zooids). A colony generated by modular growth can be classified by identifying its second-order modular (structural) unit and then by characterizing the nature and relationships of these iterated units within the colony. This approach to classifying modular growth habits provides a standardized terminology and allows for direct comparison of a suite of functionally analogous character states among taxa with specific parameters of their ecology.     

A new cheilostome bryozoan with gigantic zooids from the North-West Pacific

Gontarella gigantea gen. et sp. nov. is described from two stations, one in the Sea of Okhotsk and the second on the Pacific side of the Small Kuril Arc. This membraniporiform anascan cheilostome bryozoan has very large zooids, the largest known among extant sheet-like encrusting anascans. Comparative data on similar sheet-like cheilostomes gathered from the literature shows that the new species represents a conspicuous outlier in size, with the surface area of the zooid being approximately twice that of the next largest species. Skeletal evidence, including the lack of ovicells, indicates that G. gigantea belongs within the malacostegan family Electridae. The gigantic ancestrula suggests that the species has a cyphonautes larva about 1 mm in maximum dimension.     

Random amplified polymorphic DNA (RAPD) analysis reveals extensive natural chimerism in a marine protochordate

Random amplified polymorphic DNA (RAPD) analysis was applied to individual modules (zooids) of a colonial ascidian to investigate the presence and extent of chimerism, the parabiotic association of different genetic entities. The technique proved to be rapid and efficient for distinguishing different genotypes present in a colony, and revealed genetic mosaicism in wild material, as well as in laboratory cultures following planned fusion. Approximately one-third of colonies in the natural population studied possessed multiple genotypes, presumably as the result of fusion of different colonies. Furthermore, individual zooids of different genetic origin often intermingled after colony fusion, spreading each genotype throughout a larger total area.     

Cephalodiscus reproductive biology (Pterobranchia,Hemichordata)

Sexually mature adults and embryos and larvae of Cephalodiscus nigrescens and C. gracilis were studied by light and electron microscopy. Contrary to claims in the literature, individual coenecial cavities are inhabited by colonies of up to 15 joined zooids and not by single individuals, which is important for the interpretation of the mode of life of the related fossil group the graptolites. Some aspects of the reproductive apparatus and reproduction in Cephalodiscus are reported. The ultrastructure of the spermatozoon is described for the first time. Coelom formation is by schizocoely. The structure of the larva at several developmental stages is illustrated. Not all fertilised eggs are destined to become motile larvae and some develop into zooids omitting the motile stage. The lumen of the oviduct is much larger than previously supposed. Spermatozoa are shed into the cavity of the coenecium. It is proposed that fertilisation takes place within the coenecium. The ultrastructure of the enigmatic black ‘Comma Body’ is described and a reproductive function is proposed. Budding takes place from a base common to several zooids. This base probably also serves as an attachment foot. Large masses of yolk have been discovered within the coelom of some zooids and muscle stalks. It is inconceivable that a colony of Cephalodiscus nigrescens could survive unless it spent most of its life outside the coenecium.     

The effects of ambient flow velocity,colony size,and upstream colonies on the feeding success of Bryozoa. II. Conopeum reticulum (Linnaeus), an encrusting species

The effects of ambient flow velocity, colony size, and the presence of an actively-feeding colony upstream on the feeding success of the encrusting bryozoan Conopeum reticulum (Linnaeus) were studied. Zooids from both large and small colonies showed a reduction in feeding as flow velocity increased, however, the reduction in feeding was less for zooids from large colonies except at very fast ambient flow velocities. The greater pumping capacity of large colonies may result in a relatively greater per zooid feeding success from moving water. The presence of an actively-feeding colony upstream was found to enhance the feeding of zooids on downstream colonies. Diversion of flowing water by actively-feeding colonies upstream may account for the observed enhancement of feeding by zooids on colonies downstream.The results from this study on an encrusting species are compared with results from a previous study on feeding from flow by an arborescent bryozoan, and the feeding performances of these two colony types are related to their respective flow microhabitats.     

Does polymorphism predict physiological connectedness? A test using two encrusting bryozoans

A colonial lifestyle necessitates communication between colony members to coordinate functions and enable resource sharing through physiological integration. Colonial integration is predicted to increase with both the size of the colony and the level of specialization (polymorphism). In modular colonies, although integration might be reflected in structural characteristics such as module spacing or branching patterns, physiological integration is fundamentally dependent on the level of connectedness between modules. In cheilostome bryozoans, funicular tissue links adjacent zooids through pores within zooid walls and is the most likely means of nutrient transport within colonies. We sought to determine whether the relative numbers of pores (septulae) and pore plates (septal chambers) per zooid differed across colony regions in a monomorphic species, Watersipora subtorquata, and one showing some polymorphism, Mucropetraliella ellerii. Within each species, the morphology of pore plates corresponded to functional predictions based on their position within the zooid, and connection numbers per zooid increased with colony size. Contrary to expectations, however, the more complex species, M. ellerii, had significantly fewer porous connections per zooid than W. subtorquata. Physiological connectedness was therefore not predicted by simple assessment of polymorphism in these species and may not be sufficient to infer colonial integration in related taxa.     

Response of a Sphagnum bog plant community to elevated CO2 and N supply

The factors determining herbaceous canopy architecture are poorlyunderstood, especially in natural and semi-natural plant communities. Inthis study, we tested three main hypotheses: (1) the structure of herbaceouscanopies can be explained by the vertical distribution of functional groupsdefined by leaf width and the presence/absence of leaves on upright stem;(2) the degree of canopy stratification is greater in habitats that experiencelower spatial heterogeneity in the supply of light (i.e., grasslands as opposedto forest herb layers); and (3) there is significant variation among specieswithin a growth-form, with respect to their vertical position in thecanopy. We used plant foliage height distribution data from 14 grassland and 13forest herbaceous communities to test these hypotheses. A general linear mixedmodel was applied to specify the proportions of total variance in the foliageheight, accounted for by the fixed effects of plants' basicgrowth-form properties (growth-form) and community type(forest/grassland), and by the random effects of sampling site, samplingpoint, and individual species. We were also interested in the correlation ofthedegree of the stratification with various community characteristics(productivity, other canopy properties, species richness, variation ofspecies' traits) and light availability. There was some evidence ofoverall canopy stratification according toplant growth-form, since plants with leafy stem were locatedsignificantly higher. However, such a pattern of two more or less distinctlayers (grasses + upright forbs and rosette forbs) occurred withconsistency only in grasslands (greater homogeneity in light). Thebetween-species variation within a growth-form was a highlysignificant predictor of canopy vertical structure in the 27 communities. Theproportion of total observed variance, explainable throughspecies-specific effects, was comparable to that caused bybetween-site differences. The effect of community horizontal pattern wasless obvious, but still significant. The site by site analysis revealed thatthe degree to which horizontalpatchiness explained variation in vertical canopy structure was negativelyrelated to the relative importance of species-specific effects, showingthat small between-species differences lead to a more obviouswithin-community horizontal pattern, and vice versa. The upper bound ofthe degree of foliage stratification, according to growth-form, wasrelated to the variability of species light requirements and to relative (tocommunity pool size) richness, indicating that certain aspects of canopyarchitecture might be explained through community species composition anddiversity pattern.     

BS-cadherin in the colonial urochordate Botryllus schlosseri: one protein, many functions

Botryllus schlosseri is a colonial urochordate composed of coexisting modules of three asexually derived generations, the zooids and two cohorts of buds, each at disparate developmental stage. Functional zooids are replaced weekly by the older generation of buds through a highly synchronized developmental cycle called blastogenesis (which is, in turn, divided into four major stages, A to D). In this study, we examined the mode of expression of BS-cadherin, a 130-kDa transmembrane protein isolated from this species, during blastogenesis. BS-Cadherin is expressed extensively in internal organs of developing buds, embryos, ampullae and, briefly, in the digestive system of zooids at early blastogenic stage D (in contrast to low mRNA expression at this stage). In vitro trypsin assays on single-cell suspensions prepared from blastogenic stage D zooids, confirmed that BS-cadherin protein is expressed on cell surfaces and is, therefore, functional. BS-Cadherin expression is also upregulated in response to various stress conditions, such as oxidative stress, injury and allorecognition. It plays an important role in colony morphogenesis, because siRNA knockdown during D/A blastogenic transition causes chaotic colonial structures and disrupts oocytes homing onto their bud niches. These results reveal that BS-cadherin protein functions are exerted through a specific spatiotemporal pattern and fluctuating expression levels, in both development/regular homeostasis and in response to various stress conditions.     

Developmental pattern of colonies in the polystyelid ascidian,Polyandrocarpa misakiensis

Summary

The growth pattern of zooids formed asexually by budding was studied in the colonial ascidian, Polyandrocarpa misakiensis. Each colony started from a blas- tozooid (the first generation) on the glass plate in two series of experiments. To evaluate the growth of colonies, lineage of all the zooids of three successive generations was traced on photographs which were taken once a week. The zooids of the first generation produced many buds from any basal margin of the zooidal body, and those of the second generation produced a small number of buds mainly from anterior parts of the zooidal body. The zooids of the second generation produced by early budding of mother zooids were clearly more prolific than those produced by late budding. Circular colonies which developed around a zooid of the first generation consisted of stratified zones of successive generations. Each zone was composed of two subzones; the outer one mainly containing early-produced zooids, and the inner one mainly containing late-produced zooids. The zooids in the marginal area of colony are early-produced ones from generation to generation. The seawater temperature may influence the growth of zooids and/or the frequency of budding.     

Evolution of erect helical colony form in the Bryozoa: phylogenetic, functional, and ecological factors

Erect helical colony forms have evolved at least six separate times within the Bryozoa, but only among those in which branches are composed of a single layer of feeding zooids. The four best known genera with helical colony forms evolved independently, and each occupied different benthic marine environments, achieved different growth habits, and utilized different aspects of an array of functional potentials resulting from the radially symmetrical colonies. Examination of the distribution of these four genera ( Archimedes , Bugula , Crisidmonea , and Retiflustra ) within a theoretical morphospace of hypothetical helical colony form reveals that each occupies its own characteristic region of morphospace, broadly overlapping in some dimensions but separated in others. The genera differ markedly in the branching densities within their filtration-sheet whorls, reflecting their phylogenetic legacies rather than constructional or functional constraints associated with helical growth. In contrast, all tend toward helices in which the radiating whorls of the unilaminate branches are held at an average of 50–60° to the central axis of the colony, and this may reflect a common functional optimum associated with the cilia-driven, auto-generated currents within the helix. The region of morphospace characterized by high values of surface area – i.e. helical geometries with branches orientated at very low angles to the central axis, and with very closely spaced whorls along the axis – is entirely empty of bryozoans, and these geometries apparently represent functionally unrealistic colony forms.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 80 , 235–260.     

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.     

Suppression of programmed cell death regulates the cyclical degeneration of organs in a colonial urochordate

The survival of animal tissues and organs is controlled through both activation and suppression of programmed cell death. In the colonial urochordate Botryllus schlosseri, the entire parental generation of zooids in a colony synchronously dies every week as the asexually derived generation of buds reaches functional maturity. This process, called takeover, involves massive programmed cell death (PCD) of zooid organs via apoptosis followed by programmed removal of cell corpses by blood phagocytes within approximately 1 day. We have previously reported that developing buds in conjunction with circulating phagocytes are key effectors of zooid resorption and macromolecular recycling during takeover, and as such engineer the reconstitution of a functional asexual generation every week [Lauzon, R.J., Ishizuka, K.J., Weissman, I.L., 2002. Cyclical generation and degeneration of organs in a colonial urochordate involves crosstalk between old and new: a model for development and regeneration. Dev. Biol. 249, 333-348]. Here, we demonstrate that zooid lifespan during cyclic blastogenesis is regulated by two independent signals: a bud-independent signal that activates zooid PCD and a bud-dependent, survival signal that acts in short-range fashion via the colonial vasculature. As zooids represent a transient, mass-produced commodity during Botryllus asexual development, PCD regulation in this animal via both activation and suppression enables it to remove and recycle its constituent zooids earlier when intra-colony resources are low, while maintaining the functional filter-feeding state when resources are adequate. We propose that this crosstalk mechanism between bud and parent optimizes survival of a B. schlosseri colony with each round of cyclic blastogenesis.     

Determination of polarity and bilateral asymmetry in palleal and vascular buds of the ascidian Botryllus schlosseri.

Reversal of the bilateral asymmetry of the zooids was induced in a series of colonies of Botryllus schlosseri. Palleal buds from colonies with normal or reversed bilateral asymmetry were isolated in the early stages from the parental zooids and cultured in the vascularized tunic of the same colony or of another colony with opposite asymmetry. Vascular budding was induced in colonies with either type of asymmetry.The bud polarity was shown to depend on the vascularization; the test vessel entering the isolated palleal bud always causes the entrance point to become the posterior end of the developing zooid. On the contrary, the bilateral asymmetric type is predetermined in the bud primordium; the isolated palleal buds develop the type of asymmetry of their parents, even when grafted in the test of a colony with opposite asymmetry. Since the same was also true of the vascular buds, it is concluded that the information for the kind of bilateral asymmetry to be developed is conveyed by the epidermal envelope of the bud. The epidermis of the parental zooids influences the palleal buds, whereas the wall of the test vessels, epidermal extrusions of the zooids, influences the vascular buds.     

Organization and Evolution of Animal Colonies

Based on the theory of organization and evolution of colonies in an extinct group of hemichordate graptolites (Urbanek, 1960, 1990) the relationship between the events in the late astogeny of bryozoan colonies and their somatic and reproductive cycle is proposed. The bryozoan colonies with simple organization are compared with graptoloid colonies and their structure is interpreted within the framework of the morphogen gradient theory. A morphogen produced by the founder-zooid (oozooid) diffuses along the long axis of the colony and controls the phenotypic expression of the size and structure of zooids. Evolutionary changes in the graptoloid colonies involve introduction of new characters and their spreading is also accompanied by gradient changes of their manifestation. Evolutionary mechanisms in bryozoan colonies are considered in terms of penetrance and expressivity of genes. In contrast to graptolites, many bryozoan colonies display multiple zones of astogenetic changes and repetitions.     

Studies on Japanese botryllid ascidians. IV. A new species of the genus Botryllus with a unique colony shape, from the vicinity of Shimoda

The morphology and life history of a strange and unidentified botryllid ascidian were investigated. This ascidian was first collected from the stony shore of Ebisu Island in Shimoda, a city on Izu peninsula in central Japan. Unlike other botryllid ascidians, whose colonies are flat and smooth, this ascidian's colonies are rugged. In each colony, zooids are arranged into several oval systems, each of which has a thick part containing zooids and very thin parts that do not. The arrangement of ovary and testis in this species is the same as in other species of the genus Botryllus; the ovary is situated anterior to the testis. The embryo of this ascidian develops in the peribranchial cavity of its mother zooid without any brooding organs, as is the case with Botryllus scalaris and Botryllus puniceus. Meanwhile, the results of cut colony assay experiments did not show the existence of colony specificity in this ascidian. Even when two syngeneic colonies were brought into contact at their growing edges, none fused together. On the other hand, when two colonies were brought into contact with each other at their cut surfaces, they always fused into a single colony, regardless of their origin. Therefore, this species may be the only species that lacks colony specificity among the botryllids studied so far.     

Functional analysis of fenestellid bryozoan colonies

True colonies are more than aggregated individuals, they are integrated operational units which can be explained in functional terms. At the same time each constituent individual is fully functional. Paleobiological hypotheses about colonies must withstand testing on both individual and colonial levels. We illustrate our argument by analysis of fenestellid bryozoans. We propose that ciliary feeding currents swept through the fenestrules from frontal side to reverse, with the fenestrules as operational filtering units manned by co-operating zooids. We reconstruct viable current systems applicable to most fenestellids. For the amazing spiral structure of Archimedes we suggest a downward current spiralling about the central axis, since at each 'deck' the fenestrules filtered water downwards.     

Botryllus schlosseri (Tunicata) whole colony irradiation: do senescent zooid resorption and immunological resorption involve similar recognition events?

The colonial tunicate Botryllus schlosseri undergoes cyclic blastogenesis where feeding zooids are senescened and resorbed and a new generation of zooids takes over the colony. When non-identical colonies come into direct contact, they either reject each other or fuse. Fusion is usually followed by the resorption of one of the partners in the chimera (immunological resorption). The striking morphological similarities between the two resorption phenomena suggest that both may involve tissue destruction following self-nonself recognition events. Here we attempt to modify these two events by whole colony gamma irradiation assays. Three sets of experiments were performed: 1) different doses of whole colony irradiation for determination of irradiation effects (110 colonies, up to 8,000 rads); 2) pairs of irradiated-nonirradiated isografts of clonal replicates for the potential of reconstruction of the irradiated partners (23 pairs); 3) chimeras of irradiated-nonirradiated partners for analysis of resorption hierarchy. Mortality increased with the irradiation dose. All colonies exposed to more than 5,000 rads died within 19 days, while no colony died below 2,000 rads. The average mortality periods, in days, for doses of 6,000-8,000, 5,000, and 2,500-4,000 rads were 14.4 +/- 3.1 (n = 24), 19.8 +/- 6.0 (n = 15), and 19.6 + 5.1 (n = 22), respectively. Younger colonies (3-6 months old) may survive radiation better than older ones (more than 13 months). Many morphological alterations were recorded in irradiated colonies: ampullar contraction and/or dilation, accumulation of pigment cells within ampullae, abnormal bleeding from blood vessels, sluggish blood circulation, necrotic zones, reduction in bud number, and irregularities in zooid and system structures. With doses of 3,000-4,000 rads and above, irradiation arrested the formation of new buds and interrupted normal takeover, turning the colony into a chaotic bulk of vessels, buds, and zooid segments. Death supervened after a period of up to 1 month of poor condition, which was also characterized by loss of organization in systems. In isografts of irradiated-nonirradiated parts, the normal subclone resorbed all zooids and buds of the irradiated one within less than 1 week, even if it was up to 13 times smaller, without showing any sign of harmful effects. Thus, the irradiated subclone is not reconstituted by sharing blood circulation with a syngeneic part. Under 2,000 rads some of the irradiated zooids within this type of union started to regenerate, and at 1,000 rads no resorption was recorded, even though the number of zooids decreased in the irradiated part.(ABSTRACT TRUNCATED AT 400 WORDS)     

'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.