Inhibition of settlement and metamorphosis of the ascidian Herdmania curvata by non-geniculate coralline algae

The surfaces of non-geniculate coralline algae (NCA) are known to induce the settlement and metamorphosis of disparate marine taxa. In this study we investigate the responsiveness of larvae of Herdmania curvata (Ascidiacea: Stolidobranchia) to three species of NCA (Neo-goniolithon brassica-florida, Hydrolithon onkodes, and Lithothamnium prolifer) that cohabit the slope and crest of Heron Reef, Great Barrier Reef. H. curvata larvae were first exposed to these NCA at or within 2 h of hatching, which is 1 to 2 h prior to attaining competence, and then cultured continuously with the NCA for 12 to 14 h. Rates of settlement and metamorphosis of H, curvata cultured in laboratory chambers in the presence of the different NCA were significantly lower than spontaneous rates in seawater. The limited settlement in treatments containing NCA were confined entirely to the chamber periphery, and settlement never occurred on the surface of the NCA. The inhibitory effect was dose-dependent and was stronger in H. brassica-florida and H. onkodes than in L. prolifer. Larvae that did not settle in treatments with NCA had rounded anterior trunks and, in extreme cases, kinked tails with rounded and dissociated tail muscle cells. In some individuals, we observed the anterior chemosensory papillae being sloughed off the larval body. Morphological analysis of trunk ectodermal and mesenchymal nuclei of larvae cultured in the presence of the NCA revealed that general necrotic cell death was occurring. Importantly, H. curvata larvae that were exposed to NCA could not subsequently be induced to metamorphose in KCl-elevated seawater, whereas larvae not exposed to NCA metamorphosed at high rates in KCl-elevated seawater.

Larval activity levels and delayed metamorphosis affect post-larval performance in the colonial, ascidian Diplosoma listerianum

It is becoming widely recognized that extending the larval period of marine invertebrates, especially of species with non-feeding larvae, can affect post-larval performance. As these carry-over effects are presumed to be caused by the depletion of larval energy reserves, we predicted that the level of larval activity would also affect post-larval performance. This prediction was tested with the cosmopolitan colonial ascidian Diplosoma listerianum in field experiments in southern Australia. Diplosoma larvae, brooded in the parent colony, are competent to settle immediately after spawning, and they remain competent to metamorphose for > 15 h. Some larvae were induced to metamorphose 0 to 6 h after release, whilst others were induced to swim actively by alternating light and dark periods for up to 3 h prior to metamorphosis. Juvenile colonies were then transplanted to a subtidal field site in Port Phillip Bay and left to grow for up to 3 wk. Extending the larval period and increasing the amount of swimming both produced carry-over effects on post-larval performance. Colonies survived at different rates among experiments, but larval experience did not affect survival rates. Delays in metamorphosis and increased swimming activity did, however, reduce colony growth rates dramatically, resulting in 50% fewer zooids per colony. Moreover, such colonies produced initial zooids with smaller feeding structures, with the width of branchial baskets reduced by 10 to 15%. These differences in branchial basket size persisted and were still apparent in newly budded zooids 3 wk after metamorphosis. Our results suggest that, for D. listerianum, larval maintenance, swimming, and metamorphosis all use energy from a common pool, and increases in the allocation to maintenance or swimming come at the expense of post-larval performance.

The relative energetic costs of the larval period, larval swimming and metamorphosis for the ascidian Diplosoma listerianum

Variation in larval quality has been shown to strongly affect the post-metamorphic performance of a wide range of marine invertebrate species. Extending the larval period of non-feeding larvae strongly affects post-metamorphic survival and growth in a range of species. These 'carry-over' effects are assumed to be due to changes in larval energetic reserves but direct tests are surprisingly rare. Here, we examine the energetic costs ( relative to the costs of metamorphosis) of extending the larval period of the colonial ascidian Diplosoma listerianum. We also manipulated larval activity levels and compared the energy consumption rates of swimming larvae and inactive larvae. Larval swimming was, energetically, very costly relative to either metamorphosis or merely extending the larval period. At least 25% of the larval energetic reserves are available for larval swimming but metamorphosis was relatively inexpensive in this species and larval reserves can be used for post-metamorphic growth. The carry-over effects previously observed in this species appear to be nutritionally mediated and even short (< 3 h) periods of larval swimming can significantly deplete larval energy reserves.

Induction of metamorphosis with potassium ions requires development of competence and an anterior signalling centre in the ascidian Herdmania momus

Increased Kt concentration in seawater induces metamorphosis in the ascidian Herdmania momus. Larvae cultivated at 24 degrees C exhibit highest rates of metamorphosis when treated with 40 mM KCl-elevated seawater at 21 degrees C. At 24 degrees C, H. momus larvae develop competence to respond to KCl-seawater and initiate metamorphosis approximately 3 h after hatching. Larval trunks and tails separated from the anterior papillae region, but maintained in a common tunic at a distance of greater than 60 mu m, do not undergo metamorphosis when treated with KCl-seawater; normal muscle degradation does not occur in separated tails while ampullae develop from papillae-containing anterior fragments. Normal programmed degradation of myofibrils occurs when posterior fragments are fused to papillae-containing anterior fragments. These data indicate that H. momus settlement and metamorphosis only occurs when larvae have attained competence, and suggest that an anterior signalling centre is stimulated to release a factor that induces metamorphosis.

Muscle-specific regulation of tropomyosin gene expression and myofibrillogenesis differs among muscle systems examined at metamorphosis of the gastropod Haliotis rufescens

The spatial and temporal association of muscle-specific tropomyosin gene expression, and myofibril assembly and degradation during metamorphosis is analyzed in the gastropod mollusc. Haliotis rufescens. Metamorphosis of tile planktonic larva to the benthic juvenile includes rearrangement and atrophy of specific larval muscles, and biogenesis of the new juvenile muscle system. The major muscle of the larva - the larval retractor muscle - reorganizes at metamorphosis, with two suites of cells having different fates. The ventral cells degenerate, while the dorsal cells become part of the developing juvenile mantle musculature. Prior to these changes in myofibrillar structure, tropomyosin mRNA prevalence declines until undetectable in the ventral cells, while increasing markedly in the dorsal cells. In the foot muscle and right shell muscle, tropomyosin mRNA levels remain relatively stable, even trough myofibril content increases. In a population of median mesoderm cells destined to form de novo the major muscle of the juvenile and adult (the columellar muscle), tropomyosin expression is initiated at 45 h after induction of metamorphosis. Myofibrillar filamentous actin is not detected in these cells until about 7 days later. Given that patterns of tropomyosin mRNA accumulation in relation to myofibril assembly and disassembly differ significantly among the four major muscle systems examined, we suggest that different regulatory mechanisms, probably operating at both transcriptional and post-transcriptional levels, control the biogenesis and atrophy of different larval and postlarval muscles at metamorphosis.

A Mox homeobox gene in the gastropod mollusc Haliotis rufescens is differentially expressed during larval morphogenesis and metamorphosis

We have isolated a homeobox-containing cDNA from the gastropod mollusc Haliotis rufescens that is most similar to members of the Mox homeobox gene class, The derived Haliotis homeodomain sequence is 85% identical to mouse and frog Mox-2 homeodomains and 88.9% identical to the partial cnidarian cnox5-Hm homeodomain. Quantitative reverse transcription-polymerase chain reaction analysis of mRNA accumulation reveals that this gene, called HruMox, is expressed in the larva, but not in the early embryo, Transcripts are most prevalent during larval morphogenesis from trochophore to veliger. There are also transient increases in transcript prevalence 1 and 3 days after the intitiation of metamorphosis from veliger to juvenile. The identification of a molluscan Mox homeobox gene that is more closely related to vertebrate genes than other protostome (e.g. Drosophila) genes suggests the Mox class of homeobox genes may consist of several different families that have been conserved through evolution, (C) 1997 Federation of European Biochemical Societies.

The metamorphosis of lambda-fold 4-wheel systems into lambda-fold 4-cycle systems

A 4-wheel is a simple graph on 5 vertices with 8 edges, formed by taking a 4-cycle and joining a fifth vertex (the centre of the 4-wheel) to each of the other four vertices. A lambda -fold 4-wheel system of order n is an edge-disjoint decomposition of the complete multigraph lambdaK(n) into 4-wheels. Here, with five isolated possible exceptions when lambda = 2, we give necessary and sufficient conditions for a lambda -fold 4-wheel system of order n to be transformed into a lambda -fold Ccyde system of order n by removing the centre vertex from each 4-wheel, and its four adjacent edges (retaining the 4-cycle wheel rim), and reassembling these edges adjacent to wheel centres into 4-cycles.

Ecological regulation of development: Induction of marine invertebrate metamorphosis

In the marine environment a wide range of invertebrates have a pelagobenthic lifecycle that includes planktonic larval and benthic adult phases. Transition between these morphologically and ecologically distinct phases typically occurs when the developmentally competent larva comes into contact with a species-specific environmental cue. This cue acts as a morphogenetic signal that induces the completion of the postlarval/juvenile/adult developmental program at metamorphosis. The development of competence often occurs hours to days after the larva is morphologically mature. In the non-feeding - lecithotrophic - larvae of the ascidian Herdmania curvata and the gastropod mollusc Haliotis asinina, gene expression patterns in pre-competent and competent stages are markedly different, reflecting the different developmental states of these larval stages. For example, the expression of Hemps, an EGF-like signalling peptide required for the induction of Herdmania metamorphosis, increases in competent larvae. Induction of settlement and metamorphosis results in further changes in developmental gene expression, which apparently is necessary for the complete transformation of the larval body plan into the adult form.

Embryogenesis and metamorphosis in a haplosclerid demosponge: gastrulation and transdifferentiation of larval ciliated cells to choanocytes

Early development and metamorphosis of Reniera sp., a haplosclerid demosponge, have been examined to determine how gastrulation occurs in this species, and whether there is an inversion of the primary germ layers at metamorphosis. Embryogenesis occurs by unequal cleavage of blastomeres to form a solid blastula consisting micro- and macromeres; multipolar migration of the micromeres to the surface of the embryo results in a bi-layered embryo and is interpreted as gastrulation. Polarity of the embryo is determined by the movement of pigment-containing micromeres to one pole of the embryo; this pole later becomes the posterior pole of the swimming larva. The bi-layered larva has a fully differentiated monociliated outer cell layer, and a solid interior of various cell types surrounded by dense collagen. The pigmented cells at the posterior pole give rise to long cilia that are capable of responding to environmental stimuli. Larvae settle on their anterior pole. Fluorescent labeling of the monociliated outer cell layer with a cell-lineage marker (CMFDA) demonstrates that the monociliated cells resorb their cilia, migrate inwards, and transdifferentiate into the choanocytes of the juvenile sponge, and into other amoeboid cells. The development of the flagellated choanocytes and other cells in the juvenile from the monociliated outer layer of this sponge's larva is interpreted as the dedifferentiation of fully differentiated larval cells-a process seen during the metamorphosis of other ciliated invertebrate larvae-not as inversion of the primary germ layers. These results suggest that the sequences of development in this haplosclerid demosponge are not very different than those observed in many cnidarians.

A sponge allelochemical induces ascidian settlement but inhibits metamorphosis

Secondary metabolites synthesised by sessile invertebrates appear to play a role in creating and maintaining space on hard substrata by repelling competitors. In this study, we investigated the responses of the larvae of the ascidian Herdmania curvata to haliclonacyclamine A (HA), the major component of a suite of cytotoxic alkaloids extracted from the sponge Haliclona sp. 628. Both Haliclona sp. 628 and Herdmania curvata inhabit the crest and slope of Heron Island Reef. High rates of settlement were induced in competent H. curvata larvae by a range of concentrations of HA, all lower than that naturally occurring in the sponge. HA did not induce precompetent larvae to settle. Although early metamorphosis of HA-induced larvae was normal, larvae exposed to all but the lowest concentration of HA were developmentally arrested after completion of tail resorption, at about 4 h after the initiation of metamorphosis. These postlarvae underwent extensive cellular necrosis within 24 h. We also demonstrate that the addition of a transcriptional inhibitor, actinomycin D, to larvae also causes inhibition of metamorphosis after tail resorption is completed. Analyses of incorporation of radiolabelled nucleotides to measure levels of transcription during normal development and after the addition of the transcriptional inhibitor indicate that there is a significant burst of transcriptional activity just after tail resorption is completed. Despite inhibiting metamorphosis at the same stage as actinomycin D, HA increases initial rates of RNA synthesis after induction of metamorphosis in a manner similar to that observed in normal postlarvae until the onset of cellular necrosis. We conclude that HA initially induces H. curvata larvae to settle and progress through early metamorphosis possibly by engaging the same pathway as other artificial and environmental cues but subsequently inhibits completion of metamorphosis, resulting in death of the postlarvae. Since HA does not affect overall transcription rates, it appears to disrupt another important developmental process during early metamorphosis.

The metamorphosis of lambda-fold K-3,K-3-designs into lambda-fold 6-cycle systems

In this paper necessary and sufficient conditions are given for the metamorphosis of a lambda-fold K-3,K-3-design of order n into a lambda-fold 6-cycle system of order n, by retaining one 6-cycle subgraph from each copy of K-3,K-3, and then rearranging the set of all the remaining edges, three from each K-3,K-3, into further 6-cycles so that the result is a lambda-fold 6-cycle system.