Ascidian News*


Charles and Gretchen Lambert
Department of Biological Science
California State University Fullerton
Fullerton, California 92634
tel. (714)773-3481 Fax (714)773-3426
e-mail: clambert@fullerton.edu or glambert@fullerton.edu

Number 36
November 1994

Having finally obtained our own email account this year, we are using and enjoying it so much that we want to help all AN subscribers to better and faster correspond with each other via email. To accomplish that, we are requesting that all of you please send us your email address (if you have one), and also your Fax # and phone #, and we will publish these in the next issue of AN (next spring). This should be a very helpful addition to the new mailing list that we sent out in AN35 last April.
We have greatly enjoyed visits from several ascidiologists this year: Danielle Georges in Feb., Teruaki Nishikawa and his family in March, and Hiromichi Koyama in November. We had a productive summer at the Friday Harbor Labs and will be returning there for the month of January 1995 as well as all of next summer.
We extend a a very big thank-you to Dr. Paolo Burighel for duplicating and mailing all of the Italian issues of AN. Dr. Hoshi has been doing the same for the Japanese issues. These two countries are our biggest foreign subscribers and account for about one fourth of our total membership. This saves us a great deal in postage costs. Please keep sending us a copy of your latest publications. We always need Work in Progress articles, abstracts from meetings attended, and thesis abstracts.

NEWS AND VIEWS

Retirements: Werner Peters, Univ. of Dusseldorf; Pierre Guerrier, Ecole Normale Superieure de Lyon, France; Jim Swinehart, Univ. of Calif. at Davis; Todd Newberry and John Pearse, Univ. of Calif. at Santa Cruz.

New journal: Invertebrate Biology (a continuation and name change of Transactions of the Amer. Microscopical Society). A call for papers by Editor-in Chief Dr. Vicki Pearse. 7th Intl. Congress on Invert. Reproduction to be held at the Univ. of Calif. Santa Cruz 5-11 August, 1995. Contact Dr. John Pearse, Inst. of Mar. Sci., UCSC, Santa Cruz, CA 95064 Fax 408-459-4882, email pearse@biology.uscs.edu, for more information. Symposia will include: Mechanisms of oocyte maturation and fertilization, Dichotomous spermatogenesis, Defense mechanisms of embryos, Gene action in early development, Insect and Crustacean endocrinology and physiology, Reproductive physiology of males, Reproductive biology in the deep sea, Phylogenetic systematics and life history, role of juveniles in life histories, Propagation of introduced species for aquaculture. Symposia and contributed papers will be published in the journal Invert. Reproduction & Development. Deadline for registration and receipt of abstracts is 31 March 1995.

WORK IN PROGRESS

We recently surveyed all the marinas of southern California, from San Diego to Santa Barbara, and find that non-native ascidians continue to invade these areas. Tentative identifications: Microcosmus squamiger, first spotted 10 years ago, is now extremely abundant. Newer arrivals include Polyandrocarpa zorritensis, Symplegma oceania, Bostrichobranchus pilularis, Pyura vittata, and Ascidia interrupta. Ciona savignyi continues to expand its distribution. We would appreciate knowing of any sightings of these or other exotics along the American Pacific coast, as well as new records of introductions in other parts of the world. For example, this past summer we found a dense population of large Styela clava at a marina on Vancouver Island; this species has not previously been recorded north of San Francisco.

Brief notes: Ralph Lewin and Lanna Cheng recently spent 17 days in Palau studying Prochloron in didemnids. They continue to make new discoveries about this fascinating organism and its relationship with ascidians.
Roberto Di Lauro has begun working at the Stazione Zoologica of Naples on isolation of homeobox genes from Ciona intestinalis.
Andy Davis (Univ. of Wollongong, Australia) has been in southern Chile on sabbatical working on Pyura chilensis.
John Ryland and Quentin Bone both visited Patricia Mather (separately) in Brisbane; John admits he was not working on ascidians (shame on him!), while Quentin is working on a book on pelagic tunicates. Motonori Hoshi is now the Editor-in-Chief of the journal Development, Growth & Differentiation. Rolf Bak is studying changes in populations of Trididemnum solidum over time.
Alan Butler is working on antifouling agents from sponges that inhibit settlement by Clavelina larvae.
Hiromichi Koyama has been at the Scripps Inst. of Oceanography in La Jolla, CA with Nick and Linda Holland for the past 2 months working on sensory corpuscles in Amphioxus.

THESIS ABSTRACTS:

A new member, Dr. Sjaak Lemmens, has sent the following thesis astracts summarizing some of the recent work from his lab:

1. Denise Kirkpatrick, Curtin University, Honours 1994:
The filtering capacities of four ascidians from Marmion Lagoon, Western Australia.
Experimental studies were conducted on the filtration rate of the ascidians Pyura australis (Quoy and Gaimard), Polycarpa clavata Hartmeyer, Polycarpa nigricans Heller, and Polycarpa viridis Herdman. The effects of algal concentration, temperature, and body size on filtration rate were analysed. The phytoplankton cultured for experimental use was the marine diatom Thallasiosira pseudonana. All four ascidians demonstrated temperature optima in clearance rates; those with a subtropical distribution (P. clavata; ~0.8 l/háindividual, P. nigricans ~0.23 l/hácolony.) around 22¡C, those with a temperate distribution (P. australis ~0.65 l/háind. and P. viridis ~0.25 l/háind.) around 17¡C. The filtration rate was species dependent and proved to show trends of a decrease as concentration increased, although all results were statistically non-significant. No clear correlation was found between AFDW and filtration rate, possibly due to excessive disturbances during experimentation.

2. Geordie Clapin, Edith Cowan University, Research Project 1994.
The spatial distribution and biomass of macro filter feeders in seagrass communities of Cockburn Sound. The spatial distribution and biomass of macro filter feeders in shallow waters (<10m) of Cockburn Sound Western Australia was studied, and correlated with spatial patterns in Chlorophyll a levels. Chl a levels are relatively high in Cockburn Sound (0.94 - 2.66 mgál-1), and are generally highest at the south-eastern boundary. Filter feeder distribution in Posidonia meadows follows a similar pattern: 28.6 - 41.3 g AFDWám2 at the south-eastern boundary, 9.6-15.4 g AFDWám2 at other sites. Ascidians, sponges and bivalves were dominating in Posidonia meadows, while at the Southern flats the introduced polychaete S. spallanzanii reached high densities (458.9 g AFDWám2). Other seagrasses (Heterozostera, Amphibolis) were not found at all sites. Species evenness (J') in Posidonia meadows was highest at SE sites (J': ~0.8) and lowest at Woodmans point (J': 0.5), while the number of species was highest at eastern sites (S: 11). Filter feeder biomass was considerably lower in bare sediment (3.0 g AFDWám2), Heterozostera (1.2 g AFDWám2) and Amphibolis (2.3 g AFDWám2) meadows. The spatial distribution of filter feeders correlated well with spatial patterns in chl a levels, thus showing some potential as an indicator of recent phytoplankton levels.

For complete abstracts of ascidian papers presented at the following recent meetings request copy of AN36.
Amer. Soc. for Cell Biol. meeting, San Francisco, CA, Dec. 10-14, 1994.

NEW PUBLICATIONS

Acuna, J.L. 1994. Summer vertical distribution of appendicularians in the central Cantabrian Sea (Bay of Biscay). J. Mar. Biol. Ass. U. K. 74:585-601.

Acuna, J.L. & R. Anadon 1992. Appendicularian assemblages in a shelf area and their relationship with temperature. J. Plankton Res. 14:1233-1250.

Araki, I., H. Saiga, K.W. Makabe & N. Satoh 1994. Expression of AMD1, a gene for a MyoD1-related factor in the ascidian Halocynthia roretzi. Roux's Arch. Dev. Biol. 203:320-327.

Arizza, V., N. Parrinello, M. Cammarata & A. Picciurro 1993. Immunocytochemical localization of cellular lectins in Phallusia mamillata hemocytes. Anim. Biol. 2:83-90.

Arnoult, C., D. Georges & M. Villaz 1994. Cell cycle-related fl u ctuations in oocyte surface area of the ascidian Ciona intestinalis atrmeiosis resumption. Dev. Biol. 166:1-10.

Badre, A., A. Boulanger, E. AbouMansour, B. Banaigs, G. Combaut & C. Francisco 1994. Eudistomin U and isoeudistomin U, new alkaloids from the Caribbean ascidian Lissoclinum fragile. J. Nat. Prod. 57:528-533.

Ballarin, L., F. Cima & A. Sabbadin 1993. Phagocytes and phagocytosis in the colonial ascidian Botryllus schlosseri. Anim. Biol. 2:136.

Bollner, T., P.W. Beesley & M.C. Thorndyke 1992. Pattern of substance P- and cholecystokinin-like immunoreactivity during regeneration of the neural complex in the ascidian Ciona intestinalis. J. Comp. Neurobiol. 325:572-580.

Bontemps, N., I. Bonnard, B. Banaigs, G. Combaut & C. Francisco 1994. Cystodamine, a new cytotoxic fused polyaromatic alkaloid from the Mediterranean ascidian Cystodytes dellechiajei. Tetrahedron Lett. 35:7023-7026.

Boulanger, A., E. AbouMansour, A. Badre, B. Banaigs, G. Combaut & C. Francisco 1994. The complete spectral assignment of didemnin H, new constituent of the tunicate Trididemnum cyanophorum. Tetrahedron Lett. 35:4345-4348.

Brokaw, C.J. 1994. Control of flagellar bending: a new agenda based on dynein diversity. Cell Motil. Cytoskel. 28:199-204.

Brokaw, C.J. 1994. Microtubule sliding in reduced-amplitude bending waves of Ciona sperm flagella: bending waves attenuated by lithium. Cell Motil. Cytoskel. 27:150-160.

Da Rocha, R.M. & F. Monniot 1993. Didemnum rodriguesi sp. nov., a new didemnid tunicate common to southern Brazil and New Caledonia. Annales de l'Institut oceanographique 69:261-265.

Davis, A.R. & G.A. White 1994. Epibiosis in a guild of sessile subtidal invertebrates in south-eastern Australia: a quantitative survey. J. Exp. Mar. Biol. Ecol. 177:1-14.

Dayton, P.K., B.J. Mordida & F. Bacon 1994. Polar marine communities. Amer. Zool. 34:90-99.

De Bernardi, F., U. Fascio & C. Sotgia 1993. Some new data on the endodermal cells of the ascidians. Anim. Biol. 2:137.

P>Doi, Y., M. Ishibashi & J. Kobayashi 1994. Isolation and structure of smofuridins B-G from the Okinawan marine tunicate Aplidium multiplicatum. Tetrahedron 50:8651-8656.

Downey, J.C. & C.C. Lambert 1994. Attachment of the ascidian sperm surface egg receptor N-acetylgucosaminidase to the cell membrane. Molec. Repro. & Develop. 38:453-458.

Durante, K.M. & K.P. Sebens 1994. Reproductive ecology of the ascidians Molgula citrina Alder & Hancock, 1848 and Aplidium glabrum (Verrill, 1871) from the Gulf of Maine, U.S.A. Ophelia 39:1-21.

Elola, M.T. & G.R. Vasta 1994. Lectins from the colonial tunicate Clavelina picta are structurally related to acute-phase reactants from vertebrates. Ann. N. Y. Acad. Sci. 712, Conference on Primordial Immunity: Foundations for the Vertebrate Immune System:321-323.

Ge, T., H.M. Lee & C.R. Tomlinson 1994. Identification of an antennapedia-like homeobox gene in the ascidians Styela clava and S. plicata. Gene 147:219-222.

Godeaux, J. 1992. Analyse des travaux traitant du plancton de la Meditarranee occidentale (1989-1990). Rapports Commission Int. Mer Mediterranee 32:359-386.

Godeaux, J. 1993. The state of the art of plankton research in two hypersaline bodies of water, the Levantine Basin and the Arabian Gulf. Progress in Belgian Oceanographic Research Royal Acad. Belgium:263-275.

Hirose, E., T. Ishii, Y. Saito & Y. Taneda 1994. Phagocytic activity of tunic cells in the colonial ascidian Aplidium yamazii (Polyclinidae, Aplousobranchia). Zool. Sci. 11:203-208.

Holland, L.Z. & R.L. Miller 1994. Mechanism of internal fertilization in Pegea socia (Tunicata, Thaliacea), a salp with a solid oviduct. J. Morph. 219:257-267.

Horton, P.A., R.E. Longley, O.J. McConnell & L.M. Ballas 1994. Staurosporine aglycone (K252-c) and arcyriaflavin A from the marine ascidian, Eudistoma sp. Experientia 50:843-845. Isaacs, S., Y. Kashman & Y. Benayahu 1994. 3-hydroxy-7-phenyl-4E,6E-heptadienoic acid from an ascidian Didemnum granulatum. J. Nat. Prod. 57:648-649.

Ishii, T., Y. Saito & Y. Taneda 1993. Histological differences between the color patterns of two strains of the compound ascidian Polyandrocarpa misakiensis. Zool. Sci. 10:977-982.

Ishii, T., Y. Taneda & Y. Saito 1994. Polarity and body pattern in ascidian chimeras formed between normal (situs normale viscerum) and reversed (situs inversus viscerum) bodies. J. Exp. Zool. 269:336-348.

Jeffery, W.R. 1993. Role of cell interactions in ascidian muscle and pigment cell specification. Roux's Arch. Dev. Biol. 202:103-111.

Jeffery, W.R. & B.J. Swalla 1993. An ankryin-like protein in ascidian eggs and its role in the evolution of direct development. Zygote 1:197-208.

Kawamura, K. & S. Fujiwara 1994. Transdifferentiation of pigmented multipotent epithelium during morphallactic development of budding tunicates. Int. J. Dev. Biol. 38:369-378.

Kumazaki, T., S. Ishii & H. Yokosawa 1994. Identification of the reactive site of ascidian trypsin inhibitor. J. Biochem. 116:787-793.

Lambert, C.C., G.P. Gonzales & K.M. Miller 1994. Independent initiation of calcium dependent glycosidase release and cortical contractions during the activation of ascidian eggs. Dev., Growth & Differ. 36:133-139.

Lane, N.J., G. Martinucci, R. Dallai & P. Burighel 1994. Electron microscopic structure and evolution of epithelial junctions. pp. 23-43 in Molecular Mechanisms of Epithelial Cell Junctions: From Development to Disease, ed. vol., ed. by Citi, S.

Litaudon, M., F. Trigalo, M.T. Martin, F. Frappier & M. Guyot 1994. Lissoclinotoxins: antibiotic polysulfur derivatives from the tunicate Lissoclinum perforatum. Revised structure of lissoclinotoxin A. Tetrahedron 50:5323-5334.

Manni, L., G. Zaniolo & P. Burighel 1994. An unusual membrane system in the oocyte of the ascidian Botryllus schlosseri. Tiss. & Cell 26:403-412.

Marikawa, Y., S. Yoshida & N. Satoh 1994. Development of egg fragments of the ascidian Ciona savignyi: the cytoplasmic factors responsible for muscle differentiation are separated into a specific fragment. Dev. Biol. 162:134-142.

Martinucci, G.B., F. Lafargue & P. Burighel 1993. The sperm of didemnids (Tunicata, Ascidiacea), colonial ascidians with internal fertilization. Anim. Biol. 2:132.

Meedel, T.H. & K.E.M. Hastings 1993. Striated muscle-type tropomyosin in a chordate smooth muscle, ascidian body-wall muscle. J. Biol. Chem. 268:6755-6764.

Michibata, H. 1993. The mechanism of accumulation of high levels of vanadium by ascidians from seawater: biophysical approaches to a remarkable phenomenon. Adv. Biophys. 29:105-133.

Miya, T., K.W. Makabe & N. Satoh 1994. Expression of a gene for major mitochondrial protein, ADP/ATP translocase, during embryogenesis in the ascidian Halocynthia roretzi. Dev., Growth & Differ. 36:39-48.

Monniot, C. 1994. Pyura lignosa (s.s.), a Pacific ascidian from Central America, with descriptions of species confused under this name. Zool. J. Linn. Soc. 110:41-51.

Monniot, C. & F. Monniot 1994. Additions to the inventory of eastern tropical Atlantic ascidians; arrival of cosmopolitan species. Bull. Mar. Sci. 54:71-93.

Monniot, C. & F. Monniot 1994. Ascidians collected in the Weddell Sea by the RV "Polarstern" (EPOS cruise leg 3). Bull. Mus. natl. Hist. nat., Paris 16:13-37.

Monniot, F. 1993. Ascidies de Nouvelle-Caledonie XIII. Le genre Polysyncraton (Didemnidae). Bull. Mus. natl. Hist. nat., Paris 15:3-17.

Monniot, F. 1994. Ascidies de Nouvelle-Caledonie XIV. Le genre Diplosoma (Didemnidae). Bull. Mus. natl. Hist. nat., Paris 16:3-11.

Montgomery, D.W., G.K. Shen, E.D. Ulrich & C.F. Zukoski 1994. Immunomodulation by didemnins - invertebrate marine natural products. Ann. N. Y. Acad. Sci. 712, Conf. on Primordial Immunity: Foundations for the Vertebrate Immune System b:301-314.

Mukai, H. 1994. The occurrence of glomerulocytes in the compound styelid ascidiansMetandrocarpa and Polyzoa<.U>. Sci. Rep. Fac. Educ. Gunma Univ. 42:89-96.

Nakatani, Y. & H. Nishida 1994. Induction of notochord during ascidian embryogenesis. Dev. Biol. 166:289-299.

Niwa, H., M. Watanabe, H. Inagaki & K. Yamada 1994. Didemnilactones A and B and neodidemnilactone, three new fatty acid metabolites isolated from the tunicate Didemnum moseleyi (Herdman). Tetrahedron 50:7385-7400.

Ohtsuka, Y., H. Nakae, H. Abe & T. Obinata 1994. Immunochemical studies of an actin-binding protein in ascidian body wall smooth muscle. Zool. Sci. 11:407-412.

Pancer, Z., H. Gershon & B. Rinkevich 1994. Direct typing of polymorphic microsatellites in the colonial tunicate Botryllus schlosseri (Ascidiacea). Biochem. Biophys. Res. Commun. 203:646-651.

Patricolo, E. & C. Mansueto 1994. Onset of DNA synthesis in experimentally activated ascidian eggs. J. Exp. Zool. 269: 373-377.

Peddie, C.M. & V.J. Smith 1994. Blood cell-mediated cytotoxic activity in the solitary ascidian Ciona intestinalis. Ann. N. Y. Acad. Sci. 712, Conf. on Primordial Immunity: Foundations for the Vertebrate Immune System:332-334.

Pestarino, M. 1993. Localization of peptide histidine isoleucine-like immunoreactivity in the gut of the ascidian, Styela plicata. Anim. Biol. 2:91-95.

Pestarino, M. 1994. A possible immunomodulatory role of endozepine-like peptides in a tunicate. Annals of the New York Academy of Sciences 712, Conf. on Primordial Immunity: Foundations for the Vertebrate Immune System:365-367.

Raftos, D. 1994. Allorecognition and humoral immunity in tunicates. Ann. N. Y. Acad. Sci. 712, Conf. on Primordial Immunity: Foundations for the Vertebrate Immune System:227-244.

Rinkevich, B., T. LilkerLevav & M. Goren 1994. Allorecognition xenorecognition responses in Botrylloides (Ascidiacea) subpopulations from the Mediterranean coast of Israel. J. Exp. Zool. 270:302-313.

Roberts, D.E., S.R. Fitzhenry & S.J. Kennelly 1994. Quantifying subtidal macrobenthic assemblages on hard substrata using a jump camera method. J. Exp. Mar. Biol. Ecol. 177:157-170.

Rodrigues, S.A. & R.M. da Rocha 1993. Littoral compound ascidians (Tunicata) from Sao Sebastiao, Estado de Sao Paulo, Brazil. Proc. Biol. Soc. Wash. 106:728-739.

Rudi, A., I. Goldberg, Z. Stein, F. Frolow, Y. Benayahu, M. Schleyer, et al. 1994. Polycitone a and polycitrins A and B: new alkaloids from the marine ascidian Polycitor sp. J. Org. Chem. 59:999-1003.

Saito, Y., E. Hirose & H. Watanabe 1994. Allorecognition in compound ascidians. Int. J. Dev. Biol. 38:237-248.

Scippa, S. & G. Iazzetti 1993. Vacuolated cells in the body cavity of Phallusia mammillata larvae before and after hatching. Anim. Biol. 2:135.

Sensui, N., M. Ishikawa & M. Morisawa 1994. Oocyte maturation and furrow formation in an unfertilized egg by fusion with a fertilized egg or blastomeres in the ascidian Ascidia sydneiensis divisa. Dev., Growth & Differ. 36: 373-380.

Shen, G.Q. & B.J. Baker 1994. Biosynthetic studies of eudistomin H in the tunicate Eudistoma olivaceum. Tetrahedron Lett. 35:4923-4926.

Shen, G.Q. & B.J. Baker 1994. Biosynthetic studies of the eudistomins in the tunicate Eudistoma olivaceum. Tetrahedron Lett. 35:1141-1144.

Swalla, B.J., M.E. White, J. Zhou & W.R. Jeffery 1994. Heterochronic expression of an adult muscle actin gene during ascidian larval development. Dev. Genetics 15:51-63.

Swersey, J.C., C.M. Ireland, L.M. Cornell & R.W. Peterson 1994. Eusynstyelamide, a highly modified dimer peptide from the ascidian Eusynstyela misakiensis. J. Nat. Prod. 57:842-845.

Szczepankiewicz, B.G. & C.H. Heathcock 1994. Total synthesis of diplamine, a cytotoxic pyridoacridine alkaloid from a Pacific tunicate. J. Org. Chem. 59:3512-3513.

Takahashi, H., K. Azumi & H. Yokosawa 1994. Hemocyte aggregation in the solitary ascidian Halocynthia roretzi: plasma factors, magnesium ion, and met-lys-bradykinin induce the aggregation. Biol. Bull. 186:247-253.

Todd, C.D. & M.J. Keough 1994. Larval settlement in hard substratum epifaunal assemblages: a manipulative field study of the effects of substratum filming and the presence of incumbents. J. Exp. Mar. Biol. Ecol. 181:159-187.

Toop, T. & M.G. Wheatly 1993. Some behavioural and physiological responses of the ascidian Styela plicata (Lesueur) during acclimation to low salinity. Mar. Behav. & Physiol. 24:33-44.

Tosti, E. & B. Dale 1994. Regulation of the fertilization current in ascidian oocytes by intracellular second messengers. Molec. Repro. & Develop. 37:473-476.

Tsukamoto, S., H. Hirota, H. Kato & N. Fusetani 1994. Effect of urochordamines on larval metamorphosis of ascidians. Experientia 50:680-683.

Tsukamoto, S., H. Kato, H. Hirota & N. Fusetani 1994. Narains: N,N-dimethylguanidinium styryl sulfates, metamorphosis inducers of ascidian larvae from a marine sponge Jaspis sp. Tetrahedron Lett. 35:5873-5874.

Ueki, T. & N. Satoh 1994. An ascidian homolog of SEC61 is expressed predominantly in epidermal cells of the embryo. Dev. Biol. 165:185-192.

Ueki, T., S. Yoshida, Y. Marikawa & N. Satoh 1994. Autonomy of expression of epidermis-specific genes in the ascidian embryo. Dev. Biol. 164:207-218.

Uyama, T., Y. Moriyama, M. Futai & H. Michibata 1994. Immunological detection of a vacuolar-type H+ - ATPase in vanadocytes of the ascidian Ascidia sydneiensis samea. J. Exp. Zool. 27:148-154.

Vazquez, E. 1994. A new species of the genus Aplidium savigny, 1816 (Ascidiacea, Polyclinidae) from the Spanish Atlantic coast. Sarsia 79:53-57.

Wada, H. & N. Satoh 1994. Details of the evolutionary history from invertebrates to vertebrates, as deduced from the sequences of 18S rDNA. Proc. Natl. Acad. Sci. 91:1801-1804.

Yasuo, H. & N. Satoh 1993. Function of vertebrate T gene. Nature 364:582-583.

Yasuo, H. & N. Satoh 1994. An ascidian homolog of the mouse Brachyury (T) gene is expressed exclusively in notochord cells at the fate restricted stage. Dev., Growth & Differ. 36:9-18.

Yokobori, S.-I., T. Ueda & K. Watanabe 1993. Codons AGA and AGG are read as glycine in ascidian mitochondria. J. Molec. Evol. 36:1-8.

Zaniolo, G., L. Manni & P. Burighel 1993. Ovulation and embryo-parent relationship in Botrylloides aff. violaceus (Tunicata). Anim. Biol. 2:139.

Zaniolo, G., L. Manni & P. Burighel 1994. Ovulation and embryo-parent relationships in Botrylloides leachi (Ascidiacea, Tunicata). Invert. Repro. & Develop. 25:215-225.