Light induced larval release of a colonial ascidian

Larval release and photobehavior were studied in the colonial ascidian Polyandrocarpa zorritensis. The test hypothesis was that if larval release is induced by light, then larvae should be attracted to settlement areas where light is sufficient for larval release. Light induced larval release but the time course varied with light intensity. As the intensity of either sunlight or blue-green light decreased (1) the time until the beginning of larval release (latency) became longer, (2) the mean time of larval release increased, and (3) the time interval over which larvae were released increased. The threshold light intensity to induce larval release in blue-green light (8.75x10(12) photons cm(-2) s(-1)) was lower than that in sunlight (3.6x10(13) photons cm(-2) s(-1)). Light induced larval release was not affected by currents up to 15 cm s(-1). Larvae aggregate in light when given a choice between light and dark. This response did not vary with larval age. The lowest light intensity, at which larvae could distinguish between light and dark was 5.0x10(12) photons cm(-2) s(-1) in blue-green light and 2.9x10(14) photons cm(-2) s(-1) in sunlight. Thus, the hypothesis is supported because larvae are attracted to areas where light intensity is sufficient for larval release.     

Vasa expression in a colonial ascidian, Botrylloides violaceus

Evolution of solitary or colonial life histories in tunicates is accompanied by dramatic developmental changes that affect morphology and reproduction. We compared vasa expression in a solitary ascidian and a closely related colonial ascidian, in an effort to uncover developmental mechanisms important during the evolution of these contrasting life histories, including the ability to reproduce by budding. In this study, we explored the origin of germ cells in new buds developing by asexual reproduction in a colonial ascidian, Botrylloides violaceus and compared it to the source of germ cells in a solitary ascidian Boltenia villosa. We studied expression by in situ hybridization of vasa, a DEAD box RNA helicase gene found in germ cells across the metazoans. In B. villosa, bv-vasa mRNA was expressed in putative germ cells and oocytes of adult gonads, and was sequestered into a posterior lineage during embryogenesis. In mature colonies of the ascidian B. violaceus, bot-vasa mRNA was expressed in putative spermatogonia, in oocytes of zooids, and in some circulating cells in the zooids and differentiating buds. We propose that expression of vasa in cells other than gonadal germ cells of zooids in a colonial ascidian may serve as a source of germ-line stem cells in the colony.     

Remarkable longevity of dilute sperm in a free-spawning colonial ascidian

Many benthic marine invertebrates reproduce by releasing sperm into the sea (free-spawning), but the amount of time that sperm are viable after spawning may have different consequences for fertilization, depending on the type of free-spawner. In egg-broadcasting marine organisms, gamete age is usually assumed to be irrelevant because of the low probability of contact between dilute sperm and egg. However, direct dilution effects might be reduced in egg-brooding free-spawners that filter dilute sperm out of the water column, and sperm longevity may play a role in facilitating fertilization in these taxa. We investigated the effects of time, temperature, and mixing on the viability of naturally released sperm of the colonial ascidian Botryllus schlosseri. Our data indicate that B. schlosseri sperm have a functional life span that is considerably longer than those of the sperm of many other marine invertebrate taxa (half-life of approximately 16 to 26 h), are able to fertilize eggs at extremely low external sperm concentrations (ca. 10(1) sperm ml(-1)), and have a longevity that varies with temperature. It is possible that such prolonged sperm longevity may be achieved by reductions in motility, reactivation of quiescent sperm by chemical cues, or intermittent swimming.     

Telomerase deficiency in a colonial ascidian after prolonged asexual propagation

In organisms that propagate by agametic cloning, the parental body is the reproductive unit and fitness increases with clonal size, so that colonial metazoans, despite lack of experimental data, have been considered potentially immortal. Using asexual propagation rate as a measure of somatic performance, and telomerase activity and relative telomere length as molecular markers of senescence, old (7-12 years) asexual strains of a colonial ascidian, Diplosoma listerianum, were compared with their recent sexually produced progeny. We report for the first time evidence for long-term molecular senescence in asexual lineages of a metazoan, and that only passage between sexual generations provides total rejuvenation permitting indefinite propagation and growth. Thus, this colonial ascidian has not fully escaped ageing. The possibility of somatic replicative senescence also potentially helps to explain why metazoans, with the capacity for asexual propagation through agametic cloning, commonly undergo cycles of sexual reproduction in the wild.     

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.     

Ammonia-oxidizing Crenarchaeota and nitrification inside the tissue of a colonial ascidian

Marine Crenarchaeota represent an abundant component of the oceanic microbiota that play an important role in the global nitrogen cycle. Here we report the association of the colonial ascidian Cystodytes dellechiajei with putative ammonia-oxidizing Crenarchaeota that could actively be involved in nitrification inside the animal tissue. As shown by 16S rRNA gene analysis, the ascidian-associated Crenarchaeota were phylogenetically related to Nitrosopumilus maritimus, the first marine archaeon isolated in pure culture that grows chemolithoautotrophically oxidizing ammonia to nitrite aerobically. Catalysed reporter deposition (CARD)-FISH revealed that the Crenarchaeota were specifically located inside the tunic tissue of the colony, where moreover the expression of amoA gene was detected. The amoA gene encodes the alpha-subunit of ammonia monooxygenase, which is involved in the first step of nitrification, the oxidation of ammonia to nitrite. Sequencing of amoA gene showed that they were phylogenetically related to amoA genes of N. maritimus and other putative ammonia-oxidizing marine Crenarchaeota. In order to track the suspected nitrification activity inside the ascidian colony under in vivo conditions, microsensor profiles were measured through the tunic tissue. Net NO(x) production was detected in the tunic layer 1200-1800 microm with rates of 58-90 nmol cm(-3) h(-1). Oxygen and pH microsensor profiles showed that the layer of net NO(x) production coincided with O(2) concentrations of 103-116 microM and pH value of 5.2. Together, molecular and microsensor data indicate that Crenarchaeota could oxidize ammonia to nitrite aerobically, and thus be involved in nitrification inside the ascidian tissue.     

Relationships among hemocytes, tunic cells, germ cells, and accessory cells in the colonial ascidian Botryllus schlosseri

Monoclonal antibodies were raised against hemocytes of the colonial ascidian Botryllus schlosseri as possible tools to study hemocyte differentiation. In this species, blood cells are involved in various biological functions, such as immunosurveillance, encapsulation of foreign bodies, metal accumulation, and allorecognition. The latter process drives the fusion or rejection of contacting colonies, according to whether they do or do not share at least one allele at the fusibility/histocompatibility (Fu/HC) locus. Hemocytes take part in the rejection reaction, which suggests that they express molecules, coded by the Fu/HC locus, on their surface. A homozygous colony at the Fu/HC locus was used to produce the antibodies, which were screened by immunocytochemistry on hemocyte monolayers, immunohistochemistry on colony paraffin sections, and immunoblotting on colony homogenates. Here, we report on one of the obtained antibodies (1D8), which recognized a surface epitope on hemocytes of the donor colony and other colonies, apparently in a manner specific to the Fu/HC genotype. It also labeled a single 80-kDa band in colony homogenates. In addition, it specifically recognized tunic cells, germ cells, and their accessory cells. These results strengthen the assumption of a close relationship among these types of cells and blood cells, and suggest a close relationship among the above cells, probably deriving from undifferentiated blood cells.     

Increased inter-colony fusion rates are associated with reduced COI haplotype diversity in an invasive colonial ascidian Didemnum vexillum

Considerable progress in our understanding of the population genetic changes associated with biological invasions has been made over the past decade. Using selectively neutral loci, it has been established that reductions in genetic diversity, reflecting founder effects, have occurred during the establishment of some invasive populations. However, some colonial organisms may actually gain an ecological advantage from reduced genetic diversity because of the associated reduction in inter-colony conflict. Here we report population genetic analyses, along with colony fusion experiments, for a highly invasive colonial ascidian, Didemnum vexillum. Analyses based on mitochondrial cytochrome oxidase I (COI) partial coding sequences revealed two distinct D. vexillum clades. One COI clade appears to be restricted to the probable native region (i.e., north-west Pacific Ocean), while the other clade is present in widely dispersed temperate coastal waters around the world. This clade structure was supported by 18S ribosomal DNA (rDNA) sequence data, which revealed a one base-pair difference between the two clades. Recently established populations of D. vexillum in New Zealand displayed greatly reduced COI genetic diversity when compared with D. vexillum in Japan. In association with this reduction in genetic diversity was a significantly higher inter-colony fusion rate between randomly paired New Zealand D. vexillum colonies (80%, standard deviation ±18%) when compared with colonies found in Japan (27%, standard deviation ±15%). The results of this study add to growing evidence that for colonial organisms reductions in population level genetic diversity may alter colony interaction dynamics and enhance the invasive potential of newly colonizing species.     

Growing or reproducing in a temperate sea: optimization of resource allocation in a colonial ascidian

Relatively little is known about the life cycles of ascidians in temperate seas. Here, we investigated the biological cycle of the colonial ascidian Didemnum fulgens, a dominant species in some shallow localities of the NW Mediterranean Sea. Growth rates and frequencies of fission/fusion events were calculated over a period of 13 months, and the reproductive cycle determined after 32 months of observation. For analyses of reproduction, zooids were dissected in the laboratory and classified into five reproductive categories; these data were used to calculate a maturity index. For growth analyses, underwater photographs of marked colonies were used to estimate the surface area of D. fulgens colonies, calculate monthly growth rates, and document fusion and fission events. Clear seasonal patterns in reproduction and growth were observed, with distinct periods of investment into each function. Gonad maturation started in winter and larval release occurred in early summer, just before maximal sea temperatures were reached. After reproducing, colonies shrank and aestivated during the warmer summer months. Growth occurred during the cooler months, with maximal and minimal growth rates observed in winter and summer, respectively. Fusions and fissions occurred year‐round, although fissions were more frequent in fall (coincident with high growth rates) and fusions in spring (coincident with reproduction). These results add to the mounting evidence that ascidian life cycles in temperate seas are characterized by a trade‐off between investment in reproduction and growth, triggered by seasonal temperature shifts and constrained by resource availability during summer.     

Muscle differentiation in a colonial ascidian: organisation, gene expression and evolutionary considerations

Background  

Ascidians are tunicates, the taxon recently proposed as sister group to the vertebrates. They possess a chordate-like swimming larva, which metamorphoses into a sessile adult. Several ascidian species form colonies of clonal individuals by asexual reproduction. During their life cycle, ascidians present three muscle types: striated in larval tail, striated in the heart, and unstriated in the adult body-wall.     

Pattern of cell proliferation during budding in the colonial ascidian Diplosoma listerianum

Many invertebrates reproduce asexually by budding, but morphogenesis and the role of cell proliferation in this diverse and nonconserved regeneration-like process are generally poorly understood and particularly little investigated in didemnid ascidians. We here analyzed cell proliferation patterns and telomerase activity during budding in the colonial didemnid ascidian Diplosoma listerianum, with special focus on the thoracic bud where a new brain develops de novo. To help define developmental stages of the thoracic bud, the distribution of acetylated tubulin was also examined. We found extensive cell proliferation in both the thoracic and abdominal buds of D. listerianum as well as higher telomerase activity in bud tissue compared to adult tissues. In the parent adult, proliferation was found in various tissues, but was especially intense in the adult esophagus and epicardial structures that protrude into the proliferating and developing buds, confirming these tissues as the primary source of the cells that form the buds. The neural complex in the thoracic bud forms from a hollow tube that appears to separate into the neural gland and the cerebral ganglion. Whereas most of the bud undergoes proliferation, including the hollow tube and the neural gland, the cerebral ganglion shows little or no proliferation. Pulse-chase labeling experiments indicate that the ganglion, as well as the myocardium, in adult zooids are instead composed of postmitotic cells.     

Stem cells in asexual reproduction of the colonial ascidian <Emphasis Type="Italic">Botryllus tubaratus</Emphasis> (Tunicata: Ascidiacea)

Histological, cytochemical and ultrastructure research on the budding of the colonial ascidian Botryllus tuberatus aimed at searching for stem cells was performed. A dense mass of undifferentiated cells and the connection of the outer epidermal and inner atrial epithelia were revealed for the first time in the early buds of B. tuberatus. Undifferentiated cells revealed in the early buds and vascular system of the colony had morphological features of the stem and primary germ cells of metazoans. Intensive expression of alkaline phosphatase, the cytochemical marker of embryonal stem and primary germ cells of vertebrate animals, was revealed in developing buds and in some cells of the hematocyte population. Based on the literature and the author’s data it is hypothesized that the self-renewing pool of stem cells of the colonial ascidian B. tuberatus is the cellular basis of its reproductive strategy, including both sexual and asexual reproduction.     

Phylogeography and speciation of colour morphs in the colonial ascidian Pseudodistoma crucigaster

Variation in pigmentation is common in marine invertebrates, although few studies have shown the existence of genetic differentiation of chromatic forms in these organisms. We studied the genetic structure of a colonial ascidian with populations of different colour morphs in the northwestern Mediterranean. A fragment of the c oxidase subunit 1 (COI) mitochondrial gene was sequenced in seven populations of Pseudodistoma crucigaster belonging to three different colour morphs (orange, yellow and grey). Maximum likelihood analyses showed two well-supported clades separating the orange morph from the yellow-grey morphotypes. Genetic divergence between these clades was 2.12%, and gamma(ST) values between populations of the two clades were high (average 0.936), pointing to genetic isolation. Nested clade and coalescence analyses suggest that a past fragmentation event may explain the phylogeographical origin of these two clades. Non-neutral mtDNA evolution is observed in our data when comparing the two clades, showing a significant excess of nonsynonymous polymorphism within the yellow-grey morphotype using the McDonald-Kreitman test, which is interpreted as further support of reproductive isolation. We conclude that the two clades might represent separate species. We compare the population genetic differentiation found with that estimated for other colonial and solitary ascidian species, and relate it to larval dispersal capabilities and other life-history traits.     

Fertilization in an egg-brooding colonial ascidian does not vary with population density

The possibility that free-spawning marine organisms may be subject to fertilization failure at low population density (due to the effects of sperm dilution) has sparked much interest, but these effects have been demonstrated only in a few species that broadcast their eggs. Some egg-brooding species may overcome dilution effects by filtering low concentrations of sperm from seawater and fertilizing eggs throughout an extended period of time. We examined the effects of population density and size on fertilization in Botryllus schlosseri, a hermaphroditic colonial ascidian that free-spawns sperm, but broods eggs. We experimentally manipulated the size and density of mating groups and surveyed fertilization levels in natural populations that varied in density. Fertilization was not affected by variation in population size or density in either the experimental or natural populations. Near the end of the reproductive season, some eggs may have been fertilized too late to complete development, suggesting a temporal form of sperm limitation that has not been considered in other systems. We also detected greater variability in fertilization levels at lower population density. Nevertheless, these results suggest that caution must be used in extrapolating reported density effects on fertilization to all taxa of free-spawners; density effects may be reduced in brooders that have efficient sperm collection mechanisms.     

Comparative morphology of the stolonic vessel in a didemnid ascidian and some related tissues in colonial ascidians

The stolonic vessel is a tubular projection of the epidermis from the anterior part of the abdomen in the didemnid ascidians, and the vessel has been supposed to be closely related to the stolons, vascular appendages, and the posterior ends of the abdomen in other aplousobranch ascidians. We compared the morphology of the stolonic vessels of Diplosoma virens with similar or related tissue in other colonial ascidians, e.g. stolons of Clavelina, vascular appendages of Distaplia and Eudistoma, tunic vesicle of Aplidium, and vascular ampullae of Botrylloides. The epidermis of the stolonic vessel is composed of cuboidal cells in lateral wall and columnar cells at the distal tip of the vessel. The cuboidal cells have microvilli that probably anchor the stolonic vessel to the tunic. The columnar cells contain round granules that may concern with the secretion of some tunic components. The secretion of the granules, however, could not observed in this study. The stolonic vessel of D. virens is similar in morphology to the vascular ampullae of Botrylloides and the tunic vesicle of Aplidium rather than the other tissue examined here. Since the cell morphology is supposed to reflect its function but not the phylogenetic relationship, the present study could not provide conclusive evidences to prove the homology and the phylogenetic relationship among the tubular, epidermal projections in the colonial ascidians.