Energetics of larval swimming and metamorphosis in four species of Bugula (Bryozoa)

The amount of energy available to larvae during swimming, location of a suitable recruitment site, and metamorphosis influences the length of time they can spend in the plankton. Energetic parameters such as swimming speed, oxygen consumption during swimming and metamorphosis, and elemental carbon and nitrogen content were measured for larvae of four species of bryozoans, Bugula neritina, B. simplex, B. stolonifera, and B. turrita. The larvae of these species are aplanktotrophic with a short free-swimming phase ranging from less than one hour to a maximum of about 36 hours. There is about a fivefold difference in larval volume among the four species, which scales linearly with elemental carbon content and, presumably, with the amount of endogenous reserves available for swimming and metamorphosis. Mean larval swimming speeds (in centimeters per second) were similar among species. Specific metabolic rate and larval size were inversely related. For larvae of a given species, respiration rates remained similar for swimming and metamorphosis; however, because metamorphosis lasts about twice as long as a maximal larval swimming phase, it was more energetically demanding. Larger larvae expended more energy to complete metamorphosis than did smaller larvae, but in terms of the percentage of larval energy reserves consumed, swimming and metamorphosis were more "expensive" for smaller larvae. A comparison of the energy expended during larval swimming calculated on the basis of oxygen consumption and on the basis of elemental carbon decrease suggests that larvae of Bugula spp. may not use significant amounts of dissolved organic material (DOM) to supplement their endogenous energy reserves.     

Proteomic analysis of larvae during development, attachment, and metamorphosis in the fouling barnacle, Balanus amphitrite

The barnacle, Balanus amphitrite, is one of the primary model organisms for rocky-shore ecology studies and biofouling research. This barnacle species has a complex life cycle during which the swimming nauplius molts six times and transforms into a cyprid stage. Cyprids must attach to a surface to metamorphose into a juvenile barnacle. To clarify the overall profile of protein expression during larval development and metamorphosis, 2-DE was used to compare the proteome of the nauplius, the swimming cyprid, the attached cyprid, and the metamorphosed cyprid. The proteome of the swimming cyprid was distinctly different from that of other life stages and had about 400 spots. The proteomes of the attached and metamorphosed cyprids were similar with respect to major proteins but had significantly lower numbers of spots compared to that of swimming larval stages. Obviously, synthesis of most proteins from swimming cyprids was switched off after attachment and metamorphosis. Our advanced MS analysis (MALDI-TOF/TOF MS/MS) allowed us to identify the proteins that were differentially and abundantly expressed in the swimming cyprid. These proteins included signal transduction proteins (adenylate cyclase and calmodulin) and juvenile hormone binding proteins. In summary, for the first time, we have analyzed the global protein expression pattern of fouling marine invertebrate larvae during metamorphosis. Our study provides new insights into the mechanisms of barnacle larval metamorphosis and also provides a foundation for exploring novel targets for antifouling treatments.     

The effect of butenolide on behavioral and morphological changes in two marine fouling species, the barnacle Balanus amphitrite and the bryozoan Bugula neritina

Butenolide [5-octylfuran-2(5H)-one] is a very promising antifouling compound. Here, the effects of butenolide on larval behavior and histology are compared in two major fouling organisms, viz. cypris larvae of Balanus amphitrite and swimming larvae of Bugula neritina. Butenolide diminished the positive phototactic behavior of B. amphitrite (EC50=0.82 μg ml(-1)) and B. neritina (EC50=3 μg ml(-1)). Its effect on the attachment of cyprids of B. amphitrite was influenced by temperature, and butenolide increased attachment of larvae of B. neritina to the bottom of the experimental wells. At concentrations of 4 μg ml(-1) and 10 μg ml(-1), butenolide decreased attachment of B. amphitrite and B. neritina, respectively, but the effects were reversible within a certain treatment time. Morphologically, butenolide inhibited the swelling of secretory granules and altered the rough endoplasmic reticulum (RER) in the cement gland of B. amphitrite cyprids. In B. neritina swimming larvae, butenolide reduced the number of secretory granules in the pyriform-glandular complex.     

Tissue-specific profile of DNA replication in the swimming larvae of Ciona intestinalis

The cell cycle is strictly regulated during development and its regulation is essential for organ formation and developmental timing. Here we observed the pattern of DNA replication in swimming larvae of an ascidian, Ciona intestinalis. Usually, Ciona swimming larvae obtain competence for metamorphosis at about 4-5 h after hatching, and these competent larvae initiate metamorphosis soon after they adhere to substrate with their papillae. In these larvae, three major tissues (epidermis, endoderm and mesenchyme) showed extensive DNA replication with distinct pattern and timing, suggesting tissue-specific cell cycle regulation. However, DNA replication did not continue in aged larvae which kept swimming for several days, suggesting that the cell cycle is arrested in these larvae at a certain time to prevent further growth of adult organ rudiments until the initiation of metamorphosis. Inhibition of the cell cycle by aphidicolin during the larval stage affects only the speed of metamorphosis, and not the formation of adult organ rudiments or the timing of the initiation of metamorphosis. However, after the completion of tail resorption, DNA replication is necessary for further metamorphic events. Our data showed that DNA synthesis in the larval trunk is not directly associated with the organization of adult organs, but it contributes to the speed of metamorphosis after settlement.     

Three‐dimensional fate mapping of larval tissues through metamorphosis in the glass sponge Oopsacas minuta

The tissue of glass sponges (Class Hexactinellida) is unique among metazoans in being largely syncytial, a state that arises during early embryogenesis when blastomeres fuse. In addition, hexactinellids are one of only two poriferan groups that already have clearly formed flagellated chambers as larvae. The fate of the larval chambers and of other tissues during metamorphosis is unknown. One species of hexactinellid, Oopsacas minuta, is found in submarine caves in the Mediterranean and is reproductive year round, which facilitates developmental studies; however, describing metamorphosis has been a challenge because the syncytial nature of the tissue makes it difficult to trace the fates using conventional cell tracking markers. We used three‐dimensional models to map the fate of larval tissues of O. minuta through metamorphosis and provide the first detailed account of larval tissue reorganization at metamorphosis of a glass sponge larva. Larvae settle on their anterior swimming pole or on one side. The multiciliated cells that formed a belt around the larva are discarded during the first stage of metamorphosis. We found that larval flagellated chambers are retained throughout metamorphosis and become the kernels of the first pumping chambers of the juvenile sponge. As larvae of O. minuta settle, larval chambers are enlarged by syncytial tissues containing yolk inclusions. Lipid inclusions at the basal attachment site gradually became smaller during the six weeks of our study. In O. minuta, the flagellated chambers that differentiate in the larva become the post‐metamorphic flagellated chambers, which corroborate the view that internalization of these chambers during embryogenesis is a process that resembles gastrulation processes in other animals.     

Identification of the aromatic L-amino acid decarboxylase (AADC) gene and its expression in the attachment and metamorphosis of the barnacle, Balanus amphitrite

Serotonin and dopamine are involved in the attachment and metamorphosis of cypris larvae of barnacles. Aromatic L-amino acid decarboxylase (AADC) gene, the product of which catalyzes the synthesis of serotonin and dopamine from L-5-hydroxytryptophan and L-3,4-dihydroxyphenylalanine, respectively, was characterized. A DNA clone containing part of an AADC sequence was obtained from the genomic DNA library of the barnacle, Balanus amphitrite. This clone had four putative exons consisting of 226 amino acids with an identity of 63.2% and a similarity of 92.1% with human AADC. Northern blot analysis showed that AADC mRNA was expressed at all stages of barnacles: naupliar larvae, cypris larvae and adult barnacles. Two inducers of larval attachment and metamorphosis; that is, serotonin and extract of adult barnacles, obviously increased the expression of AADC mRNA at an early cypris larval stage. These results suggest that intracellular biosynthesis of serotonin, or dopamine, or both is at least partly involved in the control of the attachment and metamorphosis of cypris larvae.     

Proteomic profiling of the silkworm skeletal muscle proteins during larval-pupal metamorphosis

The silkworm is a typical holometabolous insect going through drastic morphological changes upon metamorphosis from larvae to pupae. Comprehensive studies focusing on the changes help elucidate understanding of a biogenic mechanism. Here, we report the initial profile of the intersegmental muscle (ISM) proteins of the silkworm during larval-pupal metamorphosis. In total, 258 protein spots were resolved by two-dimensional gel electrophoresis (2-DE). Fifty-seven larval proteins were identified, where 3 proteins were exclusively detected in larval samples. Fifty-four other proteins were common in pupal samples. Of these, 12 proteins belonging to the contractile apparatus, metabolism, regulation, and signal transduction were altered in their contents during the metamorphosis from larvae to pupae. Three pupa-defective proteins were identified as isoforms of troponin I, followed by an immunoblotting validation. This data will be helpful in understanding the biochemistry of an insect ISM.     

Loss and gain of the juvenile rudiment and metamorphic competence during starvation and feeding of bryozoan larvae

SUMMARY In many animals, larval structures and juvenile rudiments develop independently. One advantage of this independence is that juvenile rudiments can be expended as a nutrient reserve or for energy conservation. When bryozoan cyphonautes larvae were starved, structures required for settlement and metamorphosis shrank. When the larvae were again fed, these structures grew back. Starvation reduced the size of both the internal sac, a rudiment of postlarval juvenile structures, and the pyriform organ, which functions in sensing and crawling on the substratum at settlement. In contrast, starvation affected neither the size of the larval shell nor the lengths of the ciliary bands used in swimming and feeding. Starved larvae that had reduced the pyriform organ and internal sac did not metamorphose in response to stimuli from a laminarian alga. The laminarian alga did stimulate metamorphosis of the same larvae after renewed feeding, when the larvae had regrown these structures. Thus starved larvae expended body parts needed for settlement and metamorphosis when food was scarce while retaining structures for feeding, swimming, and defense. Starved larvae thereby retained the capacity to regrow structures needed for settlement and metamorphosis when they again encountered food. Advantages from expendable juvenile rudiments may enhance selection for their being developmentally distinct from structures for larval swimming and feeding.     

Hydrolysis and synthesis of substrate proteins for cathepsin L in the brain basement membranes of Sarcophaga during metamorphosis

Previously, we identified two proteins with molecular masses of 200 and 210 kDa in basement membranes of Sarcophaga imaginal discs as substrates for cathepsin L [Homma, K. and Natori, S. (1996) Eur. J. Biochem. 240, 443-447]. Here we demonstrated that the same proteins were also present in the basement membranes of larval brains. These proteins were suggested to be digested by cathepsin L secreted from the larval brains in response to 20-HE. From the behavior of these proteins during metamorphosis, we concluded that the basement membranes of larval brains are degraded at the early pupal stage and synthesized again at the late pupal stage, coinciding with the timing of brain remodeling that takes place during metamorphosis. Possibly, the transient disappearance of the basement membranes makes brain remodeling easier, and cathepsin L is suggested to play a crucial role in the degradation of the basement membranes.     

Changes in the proteome and phosphoproteome expression in the bryozoan Bugula neritina larvae in response to the antifouling agent butenolide

Larval attachment and metamorphosis, commonly referred to as larval settlement, of marine sessile invertebrates can be triggered or blocked by chemical cues and affected by changes in overall protein expression pattern and phosphorylation dynamics. This study focuses on the effects of butenolide, an effective larval settlement inhibitor, on larval settlement at the proteome level in the bryozoan Bugula neritina. Liquid‐phase IEF sample prefractionation combined with 2‐DE and MALDI‐TOF MS was used to identify the differentially expressed proteins. Substantial changes occurred both in protein abundance and in phosphorylation status during larval settlement and when settling larvae were challenged with butenolide. The proteins that responded to treatment were identified as structural proteins, molecular chaperones, mitochondrial peptidases and calcium‐binding proteins. Compared with our earlier results, both genistein and butenolide inhibited larval settlement of B. neritina primarily by changes in protein abundance and the phosphorylation status of proteins but have different protein targets in the same species. Clearly, to design potent antifouling compounds and to understand the mode of action of compounds, more studies on the effects of different compounds on proteome and phosphoproteome of different larval species are required.     

Characterization and expression of calmodulin gene during larval settlement and metamorphosis of the polychaete Hydroides elegans

The polychaete Hydroides elegans (Serpulidae, Lophotrochozoa) is a problematic marine fouling organism in most tropical and subtropical coastal environment. Competent larvae of H. elegans undergo the transition from the swimming larval stage to the sessile juvenile stage with substantial morphological, physiological, and behavior changes. This transition is often referred to as larval settlement and metamorphosis. In this study, we examined the possible involvement of calmodulin (CaM) - a multifunctional calcium metabolism regulator, in the larval settlement and metamorphosis of H. elegans. A full-length CaM cDNA was successfully cloned from H. elegans (He-CaM) and it contained an open reading frame of 450 bp, encoding 149 amino acid residues. It was highly expressed in 12h post-metamorphic juveniles, and remained high in adults. In situ hybridization conducted in competent larvae and juveniles revealed that He-CaM gene was continuously expressed in the putative growth zones, branchial rudiments, and collar region, suggesting that He-CaM might be involved in tissue differentiation and development. Our subsequent bioassay revealed that the CaM inhibitor W7 could effectively inhibit larval settlement and metamorphosis, and cause some morphological defects of unsettled larvae. In conclusion, our results revealed that CaM has important functions in the larval settlement and metamorphosis of H. elegans.     

Ascidians as excellent chordate models for studying the development of the nervous system during embryogenesis and metamorphosis

The swimming larvae of the chordate ascidians possess a dorsal hollowed central nervous system (CNS), which is homologous to that of vertebrates. Despite the homology, the ascidian CNS consists of a countable number of cells. The simple nervous system of ascidians provides an excellent experimental system to study the developmental mechanisms of the chordate nervous system. The neural fate of the cells consisting of the ascidian CNS is determined in both autonomous and non-autonomous fashion during the cleavage stage. The ascidian neural plate performs the morphogenetic movement of neural tube closure that resembles that in vertebrate neural tube formation. Following neurulation, the CNS is separated into five distinct regions, whose homology with the regions of vertebrate CNS has been discussed. Following their larval stage, ascidians undergo a metamorphosis and become sessile adults. The metamorphosis is completed quickly, and therefore the metamorphosis of ascidians is a good experimental system to observe the reorganization of the CNS during metamorphosis. A recent study has shown that the major parts of the larval CNS remain after the metamorphosis to form the adult CNS. In contrast to such a conserved manner of CNS reorganization, most larval neurons disappear during metamorphosis. The larval glial cells in the CNS are the major source for the formation of the adult CNS, and some of the glial cells produce adult neurons.     

Ultrastructure of the mushroom body: digestion during metamorphosis of Utterbackia imbecillis (Bivalvia: Unionidae)

Abstract. Larvae of the freshwater mussel Utterbackia imbecillis metamorphose to juveniles either during their attachment to a host fish, or in vitro in a culture medium. This transformation includes degeneration of larval structures and development of the juvenile morphology. Early in metamorphosis the cells comprising the larval mantle enlarge and project into the mantle cavity, forming a structure referred to as the mushroom body. Its cells, which are ultrastructurally very similar to digestive cells of adult bivalves, are involved in pinocytosis or phagocytosis of the larval adductor muscle and of tissue from the host fish that is enclosed between the larval shells. Ingested material is passed from pinosomes to heterophagosomes which in turn fuse with heterolysosomes, where final degradation of ingested material occurs. Acid phosphatase activity was detected in heterophagosomes and heterolysosomes of all animals examined. In larvae that metamorphosed in vitro , the apical cytoplasm of the cells of the mushroom body, and the extracellular spaces among them, also exhibited acid phosphatase activity. Larvae reared on a host fish accumulated substantial deposits of lipids and glycogen within larval mantle cells during metamorphosis, whereas larvae reared in vitro did not. The larval mantle cells which constitute the mushroom body appear to be the primary sites of intracellular digestion of the larval adductor muscle and host tissue during metamorphosis.     

Presence of thyroid hormones in ascidian larvae and their involvement in metamorphosis

In this study we investigated the presence and localization of thyroxine in Ciona intestinalis larvae and its involvement in metamorphosis. To date, the mechanisms regulating the metamorphosis of ascidians remain largely unknown. In vivo treatment of swimming larvae with exogenous L-thyroxine and thiourea, and in vitro experiments utilizing high performance liquid chromatography, radioimmunoassay, and immunoperoxidase staining demonstrate the presence of thyroxine at the larval stage. This suggests that this hormone may participate in the control of metamorphosis and thus play a different role from that observed in adults.     

Expression of calmodulin and myosin light chain kinase during larval settlement of the Barnacle Balanus amphitrite

Barnacles are one of the most common organisms in intertidal areas. Their life cycle includes seven free-swimming larval stages and sessile juvenile and adult stages. The transition from the swimming to the sessile stages, referred to as larval settlement, is crucial for their survivor success and subsequent population distribution. In this study, we focused on the involvement of calmodulin (CaM) and its binding proteins in the larval settlement of the barnacle, Balanus ( = Amphibalanus) amphitrite. The full length of CaM gene was cloned from stage II nauplii of B. amphitrite (referred to as Ba-CaM), encoding 149 amino acid residues that share a high similarity with published CaMs in other organisms. Quantitative real-time PCR showed that Ba-CaM was highly expressed in cyprids, the stage at which swimming larvae are competent to attach and undergo metamorphosis. In situ hybridization revealed that the expressed Ba-CaM gene was localized in compound eyes, posterior ganglion and cement glands, all of which may have essential functions during larval settlement. Larval settlement assays showed that both the CaM inhibitor compound 48/80 and the CaM-dependent myosin light chain kinase (MLCK) inhibitor ML-7 effectively blocked barnacle larval settlement, whereas Ca(2+)/CaM-dependent kinase II (CaMKII) inhibitors did not show any clear effects. The subsequent real-time PCR assay showed a higher expression level of Ba-MLCK gene in larval stages than in adults, suggesting an important role of Ba-MLCK gene in larval development and competency. Overall, the results suggest that CaM and CaM-dependent MLCK function during larval settlement of B. amphitrite.