Ascidian Molgula Research Articles

First Pacific record of the north Atlantic ascidian Molgula citrina – bioinvasion or circumpolar distribution?

The small brooding ascidian Molgula citrina Alder and Hancock, 1848 has long been known as a common inhabitant of shallow waters on both sides of the north Atlantic on subtidal natural hard substrates and also as a fouler of floating docks. There are published records from the White Sea (NW Russia), but none from the north Pacific. In May and August 2008, a number of adult brooding Molgula sp. specimens were collected from floating docks at the small fishing village of Seldovia on the Kenai Peninsula at Kachemak Bay, Alaska. Morphologically these individuals exactly match Atlantic specimens of Molgula citrina in all characters that were examined. The unique constellation of characters for this species differentiates it from all other Molgula species, as noted by Van Name (1945). In addition, the full-length 18S and 28S rDNA sequences are identical for both the Alaska specimens and New England M. citrina. Is this a new invasion, or is M. citrina a northern circumpolar species that was somehow overlooked? If this new record indicates a natural distribution, why has it not been collected before? If it is a recent introduction, it is unlikely that M. citrina, an inhabitant of cold waters, could survive in sea chests of ships from the N. Atlantic arriving in Alaska via the Panama Canal, but the intriguing idea exists of transport from Atlantic to Pacific through the Northwest or Northeast Passages. As global warming diminishes the ice cover in this region, more and more ships are traversing the Northwest Passage across northern Canada as well as the NE Passage across northern Russia, representing significant new routes for anthropogenic transport of marine species. The tadpoles of this small brooder are retained for some time after hatching, resulting in an extremely short free larval life, but could survive as metamorphosed juveniles attached in sea chests or free-floating in ballast water. They have a wide temperature tolerance and once they metamorphose can live free-floating in sea water for some time. They become very sticky and will ultimately stick to whatever they contact. Thus they could conceivably live for many generations in sea chests and sustain a viable population from which to invade new habitats.

Occurrence Of Ascidian Molgula Sp. From The Coastal Waters Of Visakhapatnam, India

AbstractA specimen of solitary ascidian Molgula sp., collected from seaweeds growing at Thotlakonda beach, Visakhapatnam is reported for the first time from Visakhapatnam coast of Indian waters.

Occurrence Of Ascidian Molgula Sp. From The Coastal Waters Of Visakhapatnam, India

AbstractA specimen of solitary ascidian Molgula sp., collected from seaweeds growing at Thotlakonda beach, Visakhapatnam is reported for the first time from Visakhapatnam coast of Indian waters.

Effect of excretory-secretory products of some fouling organisms on settling and metamorphosis of the larvae of Styela rustica (Ascidiae)

The effect of the excretory-secretory products of some fouling animals on the settling and metamorphosis of larvae of the solitary ascidian Styela rustica was assessed. The substances secreted by the sponge Halichondria panicea stimulated settling of larvae, but concurrently blocked their metamorphosis. The excretory-secretory products of the mussel Mytilus edulis and the ascidian Molgula citrine did not affect settling of the S. rustica larvae but impeded their subsequent development. Water conditioned by the bivalve Hiatella arctica, stimulated settling and, apparently, metamorphosis of the larvae of S. rustica. The chemical substances produced by adult individuals of S. rustica facilitated settling of conspecific larvae but slightly delayed their metamorphosis.

Hair cells in an ascidian (Tunicata) and their evolution in chordates

In ascidians, mechanoreceptors of the oral area are involved in monitoring the incoming water flow. Sensory cells are represented by scattered, ciliated primary cells (sending their own axons to the cerebral ganglion) or secondary sensory cells (axonless cells forming afferent and efferent synapses with neurons, whose somata are located in the ganglion) of the coronal organ. Coronal cells have varying morphologies: in species of the Enterogona order, they are multiciliate, whereas those of Pleurogona possess an apical apparatus composed of one or two cilia accompanied by stereovilli, in some cases also graded in length. The coronal organ has been proposed as a homologue to the vertebrate octavo-lateralis system, because coronal cells resemble vertebrate hair cells for morphology, embryonic origin and arrangement. In the ascidian Molgula socialis (Pleurogona), we now describe the morphology of the coronal organ, which contains a few associated rows of sensory cells that run the whole length of the oral velum and the branched tentacles. Three kinds of sensory cells, accompanied by specialised supporting cells, are present. Comparisons between the coronal organ and other chordate mechanosensory structures suggest that hair cells originated in the common ancestor of chordates.

Analysis of large scale expression sequenced tags (ESTs) from the anural ascidian, Molgula tectiformis

Anural ascidians show embryogenesis during which tail formation does not take place. This mode of development is a derived character acquired several times independently in ascidian evolution. We identified approximately 20,000 each ESTs (i. e. 10,000 clones each were sequenced from both 5′ and 3′ ends) of adult gonads, cleaving-embryos, gastrulae/neurulae, embryos before hatching, and hatched larvae of the anural ascidian Molgula tectiformis, in order to comprehensively investigate the molecular mechanism of tailless evolution. Analyses of these ESTs showed that in this species, (1) the expression of embryonic/larval muscle structural genes which are expressed abundantly during embryogenesis of the urodele ascidian Ciona intestinalis, is suppressed; (2) genes that encode proteins with no similarity to known proteins of other organisms are abundantly expressed; (3) genes that show similarity with those up-regulated at metamorphosis in urodele ascidians are up-regulated within several hours after hatching; and (4) 15 of 35 putative orthologues of the downstream components of Brachyury, a key transcription factor for ascidian notochord formation, were found in the ESTs, even though differentiation of notochord is suppressed in this species. We discuss these remarkable results that allow insight into the molecular mechanism(s) responsible for the anural mode of ascidian development.

Effects of small-scale disturbance on invasion success in marine communities

Introductions of non-indigenous species have resulted in many ecological problems including the reduction of biodiversity, decline of commercially important species and alteration of ecosystems. The link between disturbance and invasion potential has rarely been studied in the marine environment where dominance hierarchies, dynamics of larval supply, and resource acquisition may differ greatly from terrestrial systems. In this study, hard substrate marine communities in Long Island Sound, USA were used to assess the effect of disturbance on resident species and recent invaders, ascidian growth form (i.e. colonial and solitary growth form), and the dominant species-specific responses within the community. Community age was an additional factor considered through manipulation of 5-wk old assemblages and 1-yr old assemblages. Disturbance treatments, exposing primary substrate, were characterized by frequency (single, biweekly, monthly) and magnitude (20%, 48%, 80%) of disturbance. In communities of different ages, disturbance frequency had a significant positive effect on space occupation of recent invaders and a significant negative effect on resident species. In the 5-wk community, magnitude of disturbance also had a significant effect. Disturbance also had a significant effect on ascidian growth form; colonial species occupied more primary space than controls in response to increased disturbance frequency and magnitude. In contrast, solitary species occupied significantly less space than controls. Species-specific responses were similar regardless of community age. The non-native colonial ascidian Diplosoma listerianum responded positively to increased disturbance frequency and magnitude, and occupied more primary space in treatments than in controls. The resident solitary ascidian Molgula manhattensis responded negatively to increased disturbance frequency and magnitude, and occupied less primary space in treatments than in controls. Small-scale biological disturbances, by creating space, may facilitate the success of invasive species and colonial organisms in the development of subtidal hard substrate communities.

Ultraviolet irradiation of eggs and blastomere isolation experiments suggest that gastrulation in the direct developing ascidian, Molgula pacifica, requires localized cytoplasmic determinants in the egg and cell signaling beginning at the two-cell stage.

Gastrulation in the maximum direct developing ascidian Molgula pacifica is highly modified compared with commonly studied "model" ascidians in that endoderm cells situated in the vegetal pole region do not undergo typical invagination and due to the absence of a typical blastopore the involution of mesoderm cells is highly modified. At the gastrula stage, embryos are comprised of a central cluster of large yolky cells that are surrounded by a single layer of ectoderm cells in which there is only a slight indication of an inward movement of cells at the vegetal pole. As a consequence, these embryos do not form an archenteron. In the present study, ultraviolet (UV) irradiation of fertilized eggs tested the possibility that cortical cytoplasmic factors are required for gastrulation, and blastomere isolation experiments tested the possibility that cell signaling beginning at the two-cell stage may be required for the development of the gastrula. Irradiation of unoriented fertilized eggs with UV light resulted in late cleavage stage embryos that failed to undergo gastrulation. When blastomeres were isolated from two-cell embryos, they developed into late cleavage stage embryos; however, they did not undergo gastrulation and subsequently develop into juveniles. These results suggest that cytoplasmic factors required for gastrulation are localized in the egg cortex, but in contrast to previously studied indirect developers, these factors are not exclusively localized in the vegetal pole region at the first stage of ooplasmic segregation. Furthermore, the inability of embryos derived from blastomeres isolated at the two-cell stage to undergo gastrulation and develop into juveniles suggests that important cell signaling begins as early as the two-cell stage in M. pacifica. These results are discussed in terms of the evolution of maximum direct development in ascidians.

A Comparative Study of the Non-acidic Chemically Mediated Antifoulant Properties of Three Sympatric Species of Ascidians Associated with Seagrass Habitats

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

The introduction of the ascidian Molgula manhattensis (De Kay,1843) into Peter the Great Bay (Sea of Japan).

The occurrence of the solitary ascidian Molgula manhattensis (De Kay, 1843) in fouling communities of test plates immersed in the Golden Horn Inlet (Vladivostok port) and in the Rynda Inlet (Russky Is.) of Peter the Great Bay, Sea of Japan, was recorded in 1999. This species became dominant in the fouling community after 4 months of immersion of the test plates.

A multigene locus containing the Manx and bobcat genes is required for development of chordate features in the ascidian tadpole larva.

The Manx gene is required for the development of the tail and other chordate features in the ascidian tadpole larva. To determine the structure of the Manx gene, we isolated and sequenced genomic clones from the tailed ascidian Molgula oculata. The Manx gene contains 9 exons and encodes both major and minor Manx mRNAs, which differ in the length of their 5' untranslated regions. The coding region of the single-copy bobcat gene, which encodes a DEAD-box RNA helicase, is embedded within the first Manx intron. The organization of the bobcat and Manx transcription units was determined by comparing genomic and cDNA clones. The Manx-bobcat gene locus has an unusual organization in which a non-coding first exon is alternatively spliced at the 5' end of two different mRNAs. The bobcat and Manx genes are expressed coordinately during oogenesis and embryogenesis, but not during spermatogenesis, in which bobcat mRNA accumulates independently of Manx mRNA. Similar to Manx, zygotic bobcat transcripts accumulate in the embryonic primordia responsible for generating chordate features, including the dorsal neural tube and notochord, are downregulated during embryogenesis in the tailless species Molgula occulta and are upregulated in M. occulta X M. oculata hybrids, which restore these chordate features. Antisense experiments indicate that zygotic bobcat expression is required for development of the same suite of chordate features as Manx. The results show that the Manx-bobcat gene complex has a role in the development of chordate features in ascidian tadpole larvae.

Benthic faunal associations on soft substrates at Potter Cove, King George Island, Antarctica

Associations of benthic invertebrates from Potter Cove (Antarctica) were defined from photo-transects. Density, percentage cover, species richness S′, diversity index H′, evenness index J′ and mean-size estimations were studied in relation to water depth down to 30 m. A clear bathymetric pattern was evident, with two different communities at 15 and 30 m, and a transition area between 20 and 25 m. At 15 m we observed a small number of taxa (nine in total), a high percentage of bare substratum (95%), and the dominant species were pennatulids and the bivalve Laternula elliptica. From 20 to 30 m the dominant species was the ascidian Molgula pedunculata and there was a slight increase in S′, H′ and J′, as well as in the mean size of individuals, especially in M. pedunculata, while the proportion of bare substratum showed a constant decrease with depth. Using multivariate analyses, three faunal assemblages related to depth were defined and a strong association of some species, mainly predators and opportunistic necrophages, with M. pedunculata was revealed. Ice impact (icebergs and anchor ice) seems to be the major regulating factor of benthic assemblages in shallow waters.

Direct Development in the Ascidian Molgula retortiformis (Verrill, 1871).

The cellular features of the ascidian Molgula retortiformis (Verrill, 1871), a direct developing species, were investigated with the aid of transmission electron microscopy, histochemistry, and immunocytochemistry. Developmental comparisons between direct and indirect developing ascidians will further our understanding of how developmental processes evolve. M. retortiformis eggs are surrounded by a follicular envelope comprising a layer of outer follicle cells attached to an acellular chorion. The cytoplasm of M. retortiformis eggs contains large quantities of yolk and glycogen. Immediately after hatching, at day 2.5 of development, the cells constituting a juvenile exhibited similar ultrastructural features, except that the larger, deeper cells contained more yolk and glycogen than the epidermal cells. Differentiated muscle cells were absent in newly hatched M. retortiformis juveniles, and acetylcholinesterase (AChE) activity was not detected. Immunocytochemistry experiments using a vertebrate intermediate filament antibody (NN18) support the idea that the failure of newly hatched M. retortiformis juveniles to develop muscle cells may be due to the absence of a factor localized in the egg myoplasm. This paper concludes with a discussion of the "substrate hypothesis" and the evolution of ascidian direct development.

Reproductive ecology of the ascidiansMolgula CitrinaAlder & Hancock 1848 andAplidium Glabrum(verrill 1871) from the gulf of maine, U.S.A.

Abstract We examined long-term patterns of sexual reproduction in the solitary ascidian Molgula citrina, and both sexual reproduction and colony growth in the colonial ascidian Aplidium glabrum from the shallow subtidal zone in the Gulf of Maine, U.S.A.. Ascidians of each species were collected regularly from two sites over a three-year period, then examined in the laboratory to determine size and fecundity, and, for the colonial species, density of individuals (zooids) in colonies. Reproductive characteristics were compared between ascidian species and between sites. In both species, sexual reproduction was highly seasonal, exhibiting marked peaks from late spring throughout summer. Periods of peak asexual reproduction in A. glabrum generally occurred when ascidians were not reproducing sexually. Fecundity of M. citrina ( # larvae per gram of parent ascidian) during peak periods of sexual reproduction differed between sites. Colonies of A. glabrum also differed between sites in several characteristics: #zooids per gram, #larvae per zooid, #larvae per colony, and #larvae per gram. To determine whether size constrained brood capacity, we examined the allometric relationship between fecundity (also zooid number for the colonial species) and weight, and compared this relationship between sites within species. Fecundity of M. citrina exhibited an isometric relationship with body weight, and this relationship did not differ between two populations. Colony-wide fecundity in A. glabrum increased isometrically with colony weight, but fecundity of individual zooids was independent of colony weight, Bothtypes of fecundity relationships in A. glabrum differed between sites. Zooid number increased allometrically with colony weight, with larger colonies containing disproportionately-fewer zooids than smaller colonies.

Heterochronic expression of an adult muscle actin gene during ascidian larval development

Adultation is a heterochronic mode of development in which adult tissues and organs differentiate precociously during the larval phase. We have investigated the expression of an adult muscle actin gene during adultation in the ascidian Molgula citrina. Ascidians contain multiple muscle actin genes which are expressed in the larva, the adult, or during both phases of the life cycle. In ascidian species with conventional larval development, the larval mesenchyme cells, which are believed to be progenitors of the adult mesoderm, remain undifferentiated and do not express the muscle actin genes. In M. citrina, the mesenchyme cells differentiate precociously during larval development, suggesting a role in adultation. An adult muscle actin gene from M. citrina was obtained by screening a mantle cDNA library with a probe containing the coding region of SpMA1, a Styela plicata adult muscle actin gene. The screen yielded a cDNA clone designated McMA1, which contained virtually the complete coding and 3' noncoding regions of a muscle actin gene. The deduced McMA1 and SpMA1 proteins exhibit 97% identity in amino acid sequence and may be encoded by homologous genes. The McMA1 gene is expressed in juveniles and adults, but not in larval tail muscle cells, suggesting that it is an adult muscle actin gene. In situ hybridization with a 3' non-coding region probe was used to determine whether the McMA1 gene is expressed during adultation in M. citrina. McMA1 mRNA was first detected exclusively in the mesenchyme cells during the late tailbud stage and continued to accumulate in these cells during their migration into the future body cavity and heart primordium in the hatched larva. The McMA1 transcripts persisted in mesenchyme cells after larval metamorphosis. It is concluded that an adult muscle actin gene shows a heterochronic shift of expression into the larval phase during adultation in M. citrina.

Factors necessary for restoring an evolutionary change in an anural ascidian embryo

The ascidian Molgula oculata has a tailed (or urodele) larva, whereas Molgula occulta develops directly via a tailless (or anural) embryo. Interspecific hybrid embryos produced by fertilizing M. occulta eggs with M. oculata sperm ( M. occulta × M. oculata hybrids) can develop urodele larval structures, including a brain pigment cell and a short tail containing a small notochord. Development of larval features differs in individual M. occulta clutches: some eggs develop into hybrids with both a brain pigment cell and a tail, some into hybrids with either a brain pigment cell or a tail, and others into hybrids without urodele features. The expression of a 58-kDa protein (p58), which is present in eggs and embryos of urodele ascidians but lacking in those of most anural species, also varies in expression between different clutches of M. occulta eggs. Western blot and immunofluorescence studies show that p58 expression is correlated with the ability of hybrid embryos to express urodele features. For example, clutches of M. occulta eggs containing relatively high levels of p58 produce many hybrids with both a brain pigment cell and a tail. Differential expression of p58 occurs during oogenesis in M. occulta individuals: p58 is found at similar levels in previtellogenic oocytes, but in some animals it disappears during vitellogenesis, while in others it persists throughout oogenesis and is present in mature eggs. When M. occulta eggs are extracted with Triton X-100, p58 remains in the detergent-insoluble fraction, suggesting that it is associated with the cytoskeleton. In most unfertilized M. occulta eggs, p58 is uniformly distributed, but after fertilization it is localized in the uncleaved zygote and then concentrated in embryonic ectoderm, notochord, and muscle lineage cells. Despite containing high levels of p58, gynogenetic hybrid embryos, produced by fertilizing M. occulta eggs with uv-irradiated M. oculata sperm, develop into hybrids without a brain pigment cell or a tail. The results suggest that both a functional paternal genome and p58 are necessary for restoration of larval features in M. occulta × M. oculata hybrids. The cytoskeletal complex containing p58 may mediate the localization of key maternal factors in the egg or may be involved in cellular interactions during embryogenesis which are responsible for development of urodele cell fates.

Ultrastructural and histochemical study of anural development in the ascidian Molgula pacifica (Huntsman)

Tadpole development is eliminated in the life cycle of the ascidian Molgula pacifica. The elimination of a tailed larva is termed anural development, in contrast to urodele development which is exhibited by most ascidian species. In the present study, transmission electron microscopy and histochemistry were used to gain a better understanding of anural development in M. pacifica. The fine structure of M. pacifica oocytes and fertilized eggs was similar to urodele oocytes and eggs, except that a perivitelline space and test cells were absent. M. pacifica embryos exhibited the typical cleavage pattern of urodele embryos. Gastrulation was initiated at the vegetal pole, as in urodeles, and occurred at the same time as in two urodele species (Molgula manhattensis and Pyura haustor). However, changes in cell shapes and cell movements of the vegetal pole cells that participate in gastrulation were highly modified compared to commonly studied ascidians. The changes in shapes and movements of the vegetal pole cells were minimal and resulted in embryos having a very small archenteron and blastopore. The presence of large, yolky cells in the interior of the embryo likely restricted vegetal cell movements. Two ultrastructurally distinct types of epidermal cells were evident at the gastrula stage. When gastrulae were manually dechorionated from their surrounding mucous-follicular envelope layers, the embryos were already surrounded by a thin tunic. When day 1 juveniles in the process of hatching were sectioned along the anterior-posterior axis, regional differences in cell types were evident. Differentiated muscle cells in the posterior region were not evident. Day 1 M. pacifica juveniles, anural-developing M. provisionalis juveniles and tadpoles from three urodele species were tested for their abilities to express AchE activity. The highest levels of AchE activity were detected in the larval tail muscle cells of urodeles, low levels of activity were detected in the posterior region of M. provisionalis juveniles, whereas M. pacifica juveniles did not exhibit AchE activity. The results are discussed in terms of evolutionary mechanisms responsible for anural development in ascidians.

An evolutionary change in the muscle lineage of an anural ascidian embryo is restored by interspecific hybridization with a urodele ascidian

Anural ascidians do not develop into a conventional tailed larva with differentiated muscle cells, however, embryos of some anural ascidian species retain the ability to express acetylcholinesterase (AChE) in a vestigial muscle cell lineage. This study examines the number of AChE-positive cells that develop in the anural ascidian Molgula occulta relative to that in the closely related urodele (tailed) species, Molgula oculata. Histochemical assays showed that M. oculata embryos develop 36 to 38 AChE-positive cells, consistent with the number of tail muscle cells expressed in other urodele ascidians. In contrast, M. occulta embryos develop a mean of only 20 AChE-positive cells in their vestigial muscle lineage. Cleavage-arrested embryos of the anural species express AChE only in B-line blastomeres, showing that the vestigial muscle lineage cells are derived from the primary muscle lineage. Less than the expected number of AChE-positive B-line cells develop in cleavage-arrested anural embryos, however, implying that the allocation of primary muscle lineage cells is decreased. Eggs of the anural species can be fertilized with sperm of the urodele species resulting in the development of some larvae that contain a short tail and/or a brain melanocyte, specific features of urodele larvae. The typical urodele number of AChE-positive cells is restored in some of these hybrid embryos. Both primary and secondary muscle lineages are restored because cleavage-arrested hybrid embryos develop more AChE-positive cells in the B-line blastomeres and supernumerary AChE-positive cells in the A-line blastomeres. Hybrid embryos that develop the urodele complement of AChE-positive cells also form a tail and/or a brain melanocyte showing that restoration of muscle lineage cells is coupled to the development of other urodele features. AChE expression occurred in anural embryos with disorganized or dissociated blastomeres, indicating that AChE expression is determined autonomously. It is concluded that an evolutionary change in the allocation of larval muscle lineage cells occurs during development of the anural ascidian M. occulta which can be restored by interspecific hybridization with the urodele ascidian M. oculata.

Larval behavior, settlement preference, and induction of metamorphosis in the temperate solitary ascidian Molgula citrina Alder & Hancock

Tadpole larvae of the solitary ascidian Molgula citrina Alder & Hancock were examined in behavioral and settlement bioassays to test their reactions to several physical and biotic cues known to influence habitat choice in larvae of other ascidian species. These assays revealed that M. citrina larvae, though previously reported to lack photoreception, are nonetheless strongly influenced by lighting cues when sampling potential substrata. Larval responses to other settling larvae, adult conspecifics, a congener, and an heterospecific superior competitor were also tested. Larvae settled randomly with respect to each other both in the dark and under ambient light conditions, suggesting that the gregarious settlement exhibited by larvae of many benthic invertebrates does not occur in this species. In bioassays testing the metamorphosis-inducing potential of homogenized tunic from adult ascidians, M. citrina larvae responded differentially to the three ascidian species examined. Percent metamorphosis in a population of M. citrina larvae decreased when exposed to conspecific tunic material, but increased in the presence of tunic homogenate from the heterospecific competitor. Settlement responses of M. citrina larvae to light cues and to water-borne chemicals may influence adult distributions in this ascidian species.

Interspecific hybridization between an anural and urodele ascidian: Differential expression of urodele features suggests multiple mechanisms control anural development

Anural development in the ascidian Molgula occulta was examined using tissue-specific markers and interspecific hybridization. Unlike most ascidians, which develop into a swimming tadpole larva (urodele development), M. occulta eggs develop into a tailless slug-like larva (anural development) which metamorphoses into an adult. M. occulta embryos show conventional early cleavage patterns, gastrulation, and neurulation, but then diverge from the urodele developmental mode during larval morphogenesis. M. occulta larvae do not contain a pigmented sensory cell in their brain or form a tail with differentiated notochord and muscle cells. As shown by in situ hybridization with cloned probes and analysis of in vitro translation products, M. occulta embryos do not accumulate high levels of α actin or myosin heavy chain mRNA. In contrast, acetylcholinesterase is expressed in muscle lineage cells, indicating that various muscle cell features are differentially suppressed. M. occulta embryos also lack tyrosinase activity, suggesting that suppression of brain pigment cell differentiation occurs at an early step in development. M. occulta eggs fertilized with sperm from Molgula oculata (a closely related urodele species) develop into hybrid larvae exhibiting some of the missing urodele features. Some hybrid embryos develop tyrosinase activity and differentiate a brain pigment cell and a short row of notochord cells, and form a short tail. These urodele features appeared together or separately in different hybrid embryos suggesting that they develop by independent mechanisms. In contrast, α actin and myosin heavy chain mRNA accumulation was not enhanced in hybrid embryos. These results suggest that multiple mechanisms control anural development.