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.     

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.     

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)     

Haemocytes and blastogenetic cycle in the colonial ascidian <Emphasis Type="Italic">Botryllus schlosseri</Emphasis>: a matter of life and death

A recurrent blastogenetic cycle characterizes colonies of the ascidian Botryllus schlosseri. This cycle starts when a new zooid generation opens its siphons and ends with take-over, when adult zooids cease filtering and are progressively resorbed and replaced by a new generation of buds, reaching functional maturity. During the generation change, massive apoptosis occurs in the colony, mainly in the tissues of old zooids. In the present study, we have investigated the behaviour of haemocytes during the colonial blastogenetic cycle, in terms of the occurrence of cell death and the expression of molecules involved in the induction of apoptosis. Our results indicate that, during take-over, caspase-3 activity in haemocyte lysates increases. In addition, about 20%–30% of haemocytes express phosphatidylserine on the outer leaflet of their plasma membrane, show DNA fragmentation and are immunopositive for caspase-3. Senescent cells are quickly ingested by circulating phagocytes that frequently, having once engulfed effete cells, in turn enter apoptosis. Dying cells and corpses are replaced by a new generation of cells that appear in the circulation during the generation change. This research was supported by the Italian M.I.U.R. (PRIN 2006)     

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.     

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.     

A morphological and immunohistochemical study of programmed cell death in Botryllus schlosseri (Tunicata,Ascidiacea)

The blastogenic cycle of the colonial ascidian Botryllus schlosseri concludes in a phase of selective cell and zooid death called takeover. Every week, all asexually derived parental zooids synchronously regress over a 30-h period and are replaced by a new generation. Here we document the sequential ultrastructural changes which accompany cell death during zooid degeneration. The principal mode of visceral cell death during takeover occurred by apoptosis, the majority of cells condensing and fragmenting into multiple membrane-bounded apoptotic bodies. Cytoplasmic organelles (mitochondria, basal bodies, striated rootlets) within apoptotic bodies retained ultrastructural integrity. Dying cells and fragments were then swiftly ingested by specialized blood macrophages or intraepithelial phagocytes and subsequently underwent secondary necrotic lysis. Certain organs (stomach, intestine) displayed a combination of necrotic and apoptotic changes. Lastly, the stomach, which demonstrated some of the earliest regressive changes, exhibited intense cytoplasmic immunostaining with a monoclonal antibody to ubiquitin at the onset of takeover. Affinity-purified rabbit antiserum against sodium dodecyl sulfate-denatured ubiquitin detected a characteristic 8.6-kDa mono-ubiquitin band by Western blot analysis. Collectively, these findings raise the possibility that cell death during takeover is a dynamic process which requires active participation of cells in their own destruction.     

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.     

Apoptosis and phosphatidylserine-mediated recognition during the take-over phase of the colonial life-cycle in the ascidian<Emphasis Type="Italic"> Botryllus schlosseri</Emphasis>

Colonies of the ascidian Botryllus schlosseri undergo recurrent generation changes in which adult zooids are gradually resorbed and replaced by new blastogenic generations. During these periods, known as take-over phases, programmed cell death, which, on the basis of morphological analysis is ascribed to apoptosis, occurs widely in zooid tissues. In the present report, we re-investigate cell death during the take-over process. Results confirm the occurrence of diffuse apoptosis, as evidenced by chromatin condensation, positivity to the TUNEL reaction and expression of phosphatidylserine on the outer leaflet of the plasma membrane. Apoptosis also occurs among haemocytes, and senescent blood cells are actively recognised and ingested by circulating professional phagocytes. Both phosphatidylserine and CD36, a component of the thrombospondin receptor, are involved in the recognition of apoptotic haemocytes, which fosters the idea that fundamental recognition mechanisms are well conserved throughout chordate evolution.     

Programmed cell death in vegetative development: apoptosis during the colonial life cycle of the ascidian Botryllus schlosseri

Programmed cell death (PCD) by apoptosis is a physiological mechanism by which cells are eliminated during embryonic and post-embryonic stages of animal life cycle. During asexual reproduction, the zooids of colonial ascidians originate from an assorted cell population instead of a single zygote, so that we assume that regulation of the equilibrium among proliferation, differentiation and cell death may follow different pathways in comparison to the embryonic development. Here we investigate the presence of apoptotic events throughout the blastogenetic life cycle of the colonial ascidian Botryllus schlosseri, by means of terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) coupled with histochemical and electron microscopy techniques. The occurrence of low levels of morphogenetic cell death suggests that, in contrast to what happens during sexual development (embryogenesis and metamorphosis), apoptosis does not play a pivotal role during asexual propagation in botryllid ascidian. Nevertheless, PCD emerges as a key force to regulate homeostasis in adult zooids and to shape and modulate the growth of the whole colony.     

'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.     

Colony Growth Pattern in Electra pilosa (Linnaeus) and Comparable Encrusting Cheilostome Bryozoans

Colony growth pattern is described in E. pilosa, an abundant cheilostome bryozoan commonly found as an epiphyte of Laminaria. Each zooid has 4 potential budding loci—one distal, two lateral and one proximal. The ancestrula buds daughter zooids from all of these loci; the two lateral buds appear first, followed by the distal bud and, after a long delay, the proximal bud. The laterally budded zooids curve inwards as they grow to form a triad with their distally budded sibling zooid. ‘Mature’ multiserial colonies growing on flat substrata consist of a series of radially diverging sectors. Each sector has an axis, generally of 3 parallel rows of zooids, flanked by wings consisting of rows of zooids originating as lateral buds from the section axis which infills the area between the axes. Occasional colonies occur with uniserial or semiuniserial growth patterns. These resemble colonies of the obligatory uniserial species Pyripora catenularia and poorly fed colonies of the related Conopeum tenuissimum, which is normally multiserial like E. pilosa. The ‘composite multiserial’ colonies of E. pilosa differ in several respects from ‘unitary multiserial’ colonies characteristic of most sheet-like cheilostomes, including the well-known Membranipora membranacea. Composite and unitary multiserial growth patterns may have evolved independently from uniserial ancestors.     

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.     

Multizooidal Budding in Parasmittina trispinosa (Johnston) (Bryozoa,Cheilostomata)

Two principally different wall types occur in the bryozoan colony: Exterior walls delimiting the super-individual, the colony, against its surroundings and interior walls dividing the body cavity of the colony thus defined into units which develop into sub-individuals, the zooids. In the gymnolaemate bryozoans generally, whether uniserial or multiserial, the longitudinal zooid walls are exterior, the transverse (proximal and distal) zooid walls interior ones. The radiating zooid rows grow apically to form “tubes” each surrounded by exterior walls but subdivided by interior (transverse) walls. The stenolaemate bryozoans show a contrasting mode of growth in which the colony swells in the distal direction to form one confluent cavity surrounded by an exterior wall but internally subdivided into zooids by interior walls. In the otherwise typical gymnolaemate Parasmittina trispinosa the growing edge is composed of a series of “giant buds” each surrounded by exterior walls on its lateral, frontal, basal and distal sides and forming an undifferentiated chamber usually 2–3 times as broad and 3 or more times as long as the final zooid. Its lumen is subdivided by interior walls into zooids 2–3, occasionally 4, in breadth. This type of zooid formation is therefore similar to the “common bud” or, better-named, “multizooidal budding” characteristic of the stenoleamates but has certainly evolved independently as a special modification of the usual gymnolaemate budding.     

Cell proliferation and growth inZoothamnium niveum (Oligohymenophora, Peritrichida) — Thiotrophic bacteria symbiosis

Chemolithoautotrophic, sulphide-oxidizing (thiotrophic) symbioses represent spectacular adaptations to fluctuating environmental gradients and survival is often accomplished when growth is fuelled by sufficient nourishment through the symbionts leading to fast cell proliferation. Here we show 5′-bromo-2′ deoxyuridine (BrdU) pulse labelling of vegetative growingZoothamnium niveum, a colonial ciliate obligately associated with thiotrophic ectosymbionts, and demonstrate age related growth profiles in three heteromorphic host cell types. At the colony’s apex, a large top terminal zooid performed high proliferation activity, which decreased significantly with increasing colony age but was still present in old colonies indicating that this cell possesses lifelong cell division potential. In contrast, terminal branch zooids proliferated independent of colony age but appeared to be limited by their cell division capacity predetermined by branch size, thus leading to the strict, feather-shaped colony form. Appearance of labelled terminal branch zooids allowed us to distinguish a highly proliferating apical colony region from an almost inactive, senescent basal region. In macrozooids attached to the colony, extensive BrdU labelling suggests that DNA synthesis occurs in preparation for a new generation. As motile swarmers, the macrozooids seem to be arrested in the cell cycle and mitosis and cell division occur when the swarmer settles and transforms into a top terminal zooid buildingup a new colony.     

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.     

Phenotypic plasticity of reproductive effort in a colonial ascidian,Botryllus schlosseri

Phenotypic plasticity is the capability of a genotype to produce different phenotypes in different environments. Previous studies have indicated phenotypic variability in asexual, male, and female reproduction in Botryllus schlosseri, a hermaphroditic, colonial ascidian, but not explicitly tested for genotype by environment interactions that indicate genetic variation in plastic responses. Consequently, clones derived from an estuarine population were deployed at their native site and a warmer, higher productivity site 10 km up-river. Male reproduction was assayed by testis size, female reproduction by the number of eggs produced, and asexual reproduction by colony growth rate. To test for ontogenetic effects, data were collected from two different generations of zooids born in the field. Analyses of variance indicated plasticity in asexual and female reproduction during the first zooid generation and plasticity in all three traits during the third zooid generation. Reaction norms varied significantly among genotypes in direction and magnitude for asexual reproduction at both times, implying that selection on asexual reproduction is weak. Sperm production during the third zooid generation was significantly lower at the nonnative site, but there was no genotype by environment interaction. The reaction norms for female reproduction varied significantly among genotypes in direction and magnitude during the first zooid generation, but only varied in magnitude during the third generation, with egg production being higher in all genotypes at the nonnative site. Comparisons of weighted frequency distributions between sites demonstrated that differences in egg production in the third generation were due to increases in the proportion of reproductive zooids within a colony. The greater emphasis on female reproduction at a site associated with higher food availability and temperature, and the greater emphasis on male reproduction at a colder, food-limited site, supports predictions from sex allocation theory.     

Unconventional signalling in tunicates

The zooids in colonial tunicates do not appear to be directly interconnected by nerves, but this has not prevented the evolution of coordinated behaviour in several groups. In Botryllus and other colonial styelid asci‐dians the endothelium lining the blood vessels is excitable and transmits action potentials from cell to cell via gap junctions. These signals mediate protective contractions of the zooids and synchronize contractions of the vascular ampullae. In didemnid ascidians such as Diplosoma a network of myocytes in the tunic serves to transmit excitation and to cause contractions of the cloacal apertures. Individual zooids of Pyrosoma protect themselves by closing their siphons and arresting their branchial cilia when stimulated. At the same time a flash of light is emitted. Neighbouring zooids sense the flash with their photoreceptors and respond in turn with protective responses and light emission. Protective responses thus spread by photic signalling and propagate from zooid to zooid through the colony in a saltatory manner. In chains of Salpafusifortnis, changes in the direction and/or speed of swimming are transmitted from zooid to zooid via adhesion plaques. When a zooid is stimulated, its body‐wall epithelium conducts action potentials to the plaque connecting it to the next zooid, exciting receptor neurons in that zooid. These receptors have sensory processes that bridge the gap between the two zooids. The sensory neurons so excited in the second zooid conduct impulses to the brain where they alter the motor output pattern, and at the same time generate epithelial action potentials that travel to the next zooid in line, where the same thing happens.

It is not clear why these unconventional signalling methods have evolved but the tunic may be an inhospitable environment for nerves, making conventional nervous links impossible.     

Delayed insemination results in embryo mortality in a brooding ascidian

We explored the effects of temporal variation in sperm availability on fertilization and subsequent larval development in the colonial ascidian Botryllus schlosseri, a brooding hermaphrodite that has a sexual cycle linked to an asexual zooid replacement cycle. We developed a method to quantify the timing of events early in this cycle, and then isolated colonies before the start of the cycle and inseminated them at various times. Colony-wide fertilization levels (assayed by early cleavage) increased from zero to 100% during the period when the siphons of a new generation of zooids were first opening, and remained high for 24 h before slowly declining over the next 48 h. Because embryos are brooded until just before the zooids degenerate at the end of a cycle, delayed fertilization might also affect whether embryos can complete development within the cycle. Consequently, we also determined the effect of delayed insemination on successful embryo development through larval release and metamorphosis. When fertilization was delayed beyond the completion of siphon opening, there was an exponential decline in the percentage of eggs that ultimately produced a metamorphosed larva at the end of the cycle. Thus, even though the majority of oocytes can be fertilized when insemination is delayed for up to 48 h, the resulting embryos cannot complete development before the brooding zooids degenerate.