Histochemistry of Spore Mucilage and Inhibition of Spore Adhesion in Champia parvula , A Marine Alga

Spores of the marine red alga, Champja parvu!a, attached initially to plastic or glass cover slips by extracellular mucilage. Adhesive rhizoids emerged from germinating spores, provided a further basis of attachment and rhizoidal division formed the holdfast. Mucilage of holdfasts and attached spores stained for sulfated and carboxylated polysaccharides. Rhizoids and holdfast cells but not mucilage stained for protein. Removal of holdfasts with HCI revealed protein anchors in holdfast cell remnants. Spores detached when incubated in the following enzymes: 13-galactosidase, protease, ce!!u!ase, a-amylase, hya!uronidase, sulfatase, and mannosidase. The FITC. lectins Con A, LCA, PNA, SBA, and the lectin from ~ yi!!osa, were used to probe the mucilage of attached spores to detect the sugar haptens a-Dmannose, a-0-glucose, ~D-galactose, and N-acetylgalactosamine, whereas probing with WGA, Phyto!acca amerjcana mitogen (PWM), and UEA did not detect N-acetylglucosamine or a-L-fucose Adhesion of newly released, floating tetraspores was inhibited by cycloheximide, tunicamycin, sodium molybdate, and Con A. These results indicate that proteins, glycoproteins, sulfated polysaccharides, and a-0mannose or a-0-g!ucose, respectively, are all necessary for adhesion. Tetraspores remained attached in the presence of the inhibitors, suggesting that they do not maintain adhesion via synthesis of proteins, glycoproteins, or sulfated polysaccharides. Tetraspores killed with H2S04 or sodium azide did not attach; therefore tetraspores must be alive to attach. Tetraspores did not detach when killed with sodium azide or DIH20. Death did not result in detachment unless the mucilage was damaged by H2S04. Glycoproteins with a-0-mannose and or a-0-glucose sugar moieties may detect the substrate upon contact and convey messages to the cytoplasm which translates spatial information about adhesion into germination and rhizoid production. The sugar moieties may recognize cellular surfaces of hosts on which .Q.. parvula is epiphytic. These glycoproteins are probably embedded in a matrix of adhesive, sulfated polysaccharides which may be cross-linked with proteins, suggesting that several classes of molecules may interact to facilitate adhesion.


INTRODUCTION
Spores are dispersal and attachment units. They are the bridges between macroalgal life cycle phases. Establishment of attached adult plants depends on spore adhesion . Spores are highly vulnerable immediately after attachment; yet little is known about their survivorship during establishment Flavier and Zingmark 1993). Spore attachment must be strong enough to withstand wave action and spores must attach to substrata that receive enough light for photosynthesis . The ability to be epiphytic as well as attached to other substrata confers the advantage of an increased range of possible habitats (Hay 1986). Even though spores are the primary reproductive unit of the macroalgae, little is known about their biology in general, and settlement in particular (Amsler and Neushul 1991 ). The mechanisms and histochemistry of red algal spore attachment are relatively unknown when compared to attachment of brown or green algae or of marine invertebrates. Knowledge g·ained from investigations of red algal spore attachment can be used to increase our understanding of algal spore biology ), host-epiphyte relationships, and biofouling. Such knowledge will also make a necessary contribution to the intriguing, significant, but as yet understudied field of red algal biology ).

Red Algal Mucilage and lnltlal Spore Attachment
Red algal spores have what has been called "a ubiquitous mucilage" . There is little argument that this encompassing mucilage functions in spore adhesion, especially because red algal spores are nonmotile (Fletcher and Callow 1992). They sink very slowly ) but do not have far to fall from their relatively small parent plants ) and the thicker the mucilage layer, the faster the spore settlement . The mucilage sticks to the substratum upon contact to provide initial, albeit insecure adhesion .
Attachment of almost all red algal spores becomes progressively firmer with time . Initial attachment of Ceramjum spores is probably by the viscous mucilage envelope ; attached spores are easily washed off their substrate at this stage. Adhesive secreted from golgi vesicles after spore release forms a resistant attachment pad and further anchorage is provided by rhizoid development.
Mucilage and vesicles which probably contain adhesives are formed during sporogenesis by a variety of red algae. Mucilage is produced by the golgi apparatus and sometimes by the endoplasmic reticulum. Starch accumulates and adhesive vesicles form during sporogenesis of Haliptilon cuyjed (Vesk and Borowitska 1984). Golgi vesicles change appearance from striated to fibrous and are involved in protein turnover during carposporogenesis in Polysjphonja noyae-angliae (Wetherbee and West 1977). Proteinaceous crystalloids accumulate in vegetative cells but disappear upon sporogenesis in Wrangelia plumosa (Wetherbee et al. 1984).  found that attached spores of Porphyra yarjegata did not have adhesive vesicles, but were coated with a substance that filled an indentation of the spore and resembled the vesicle contents; a true cell wall formed after initial attachment.
Attachment is mediated by sheaths of extracellular mucilage that develop after release to surround spores of Chondrus crtspus .
Within the sheath, attached carpospores and tetraspores divide and form disclike sporelings that expand radially and form erect fronds. Without a sheath, sporelings form loosely attached semifilaments. Sheaths may provide physical conditions that are conducive to disc formation. Coalescence can occur between sporelings and between sheaths of adjacent sporelings , and may increase the chances for establishment of perenniating .Q.. crjspus holdfasts, which can regenerate up to 25% of their area if necessary (Taylor et al. 1981 ). Germinating spores also coalesce in lrjdaea laminarioides, forming common basal discs for frond and stipe emergence, with vigorous growth in the center of the disc (Martinez and Santelices 1992).
Encrusting coralline red algae have a number of ways to ensure their tenacious attachment. When Phymatolithon lenormandjj, ~ laeyjgatum, ~ polymorphum, Lithothamnion glaciale, Lithophyllum jncrustans, and Lithophyllum ~. were cultured on glass slides, spore mucilage provided initial adhesion but was invisible after the algae became calcified . As the algal crust developed, hypothallial filaments provided further attachment by flattening onto the smooth glass slides. On rough substrate, hypothallial filaments moulded to and penetrated surface irregularities. Any spaces that remained or developed between the algal crust and the substrate were filled by a layer of aragonite crystals bound with an organic component, which was probably secreted by the cells. If the corallines were deliberately removed, material left behind stained positively for polysaccharides.
Spore mucilage and rhizoids are important in host-epiphyte and hostparasite interactions. Initial attachment of the epiphyte Polysjphonja lanosa onto Ascophyllum nodosum or Fucus yesjculosjs is an interaction between spore and host mucilage . The epiphyte releases adhesive vesicles and penetrates the host with unicellular rhizoids. More spores survive on & nodosum than on .E.. yesjculosjs because lateral pit and axil sites provide shelter. Rhizoids deeply penetrate the host and remain attached after A. nodosum sheds its outer surface (Filion-Myklebust and Norton 1981 ). Choreocolax spores have enlarged vacuoles which may push the spore contents into the rhizoid when it . penetrates the host . Some parasitic spores of Harveyella mjrabilis attach with extensions of their outer coverings to walls of wounded cells of Odonthalia floccosa: others germinate in ruptured cytoplasm and attach with rhizoids that grow into the sulfated polysaccharide rich cortical cell walls . Ultrastructural studies of parasitic red algal rhizoids revealed abundant rough and smooth endoplasmic reticulum that connects with the plasmalemma, numerous ribosomes, protein bodies, mitochondria, and protein staining microbody-like organelles .
In addition to adhesion, a myriad of other functions are attributed to red algal spore mucilage. These include metal chelation (Tanaka et al. 1971 ), nutrient absorption , light absorption, resistance to abrasion, protection from physical and chemical changes , protection from toxic compounds , protection against mechanical injury Characklis and Cooksey 1983), antipredation , antibiotic West 1973), reproduction, andintercellular communication (Characklis andCooksey 1983); Sulfated mucilage serves as an ionic regulator (Kloereg and Quatrano 1988). Deformation of the mucilage could dissipate the forces of waves, thereby maintaining adhesion (Boney 1975). The mucilage also functions in dispersal. For example, mucilage of lrjdaea lamjnadojdes carpospores sticks to legs of the amphipod Hyale SQ..., or the carpospores travel unharmed through the digestive tract .

Polysaccharides In Red Algal Cell Adhesion
Carpospores and tetraspores of-Ceramjum are thought to attach with a polysacchadde that becomes hydrated and forms a viscous, macro-molecular hydrocolloid that disperses upon contact with seawater . Spores detached when polysaccharides were degraded with periodic acid and sodium tetraborate. Although stains for proteins were positive in the mucilage of unreleased spores of Ceramjum but negative following spore release,  concluded that the adhesive was not proteinaceous because agents that disrupt proteins did not cause spore detachment. The mucilage of Hypnea muscjformjs, Polysjphonja geusta, and Halarachnjon ligulatum contains a highly sulfated acidic polymer  which may be adhesive.
In addition to their role in adhesion, polysaccharides are important structural elements of cell walls of red algae ). The viscous mucilage of Rhodella reticulata is thought to serve as protection; its production increased as growth stopped . The polysaccharide component of this mucilage is synthesized and ·sulfated in the golgi apparatus, while its protein component is thought to be secreted through ducts in the endoplasmic reticulum that are fused to the plasma membrane . Dessication is prevented by sulfated capsular polysaccharides of Porphyridjum aerugineum ; in which the golgi complex incorporates approximately half of the cellular sulfate into polysaccharides . Protein rich extracellular secretions of Njzymenja australjs that contain sulfated and carboxylated polysaccharides are thought to facilitate the release of spermatia and provide protection from epiphytes and pathogens .

Glycoproteins In Red Algal Cell Adhesion
Glycoproteins have been shown to be involved in adhesion of red algal cells: in spore adhesion , in reproduction (Kim and Fritz 1993a,b;Kaska et al.1988), and in wound healing (Kim and Fritz 1993c;Watson and Waaland 1983).
Little is known about the role of glycoproteins in red algal spore attachment. Red algae may have proteins or glycoproteins that are unique to and involved in spore adhesion. Spores may carry only those proteins or glycoproteins necessary to insure their attachment, germination, and viability.
Glycoproteins, along with sulfated and acidic polysaccharides are probably adhesive molecules in tetraspores of Palmaria palmata .
Glycoproteins are active in red algal reproduction. A glycoprotein with a-D-methyl mannose residues is found on the outer surfaces of spermatia of Antjthamnjon njpponjcum, and binds with a receptor in the trichogyne to mediate gamete recognition (Kim and Fritz 1993b). Glycoproteins differ between male and female reproductive tissue in Porphyra perforata and in morphologically and functionally distinct regions of the thallus ).
Wound healing involves cell adhesion. A glycoprotein is thought to mediate wound healing in Antjthamnjon sparsum (Kim and Fritz 1993c). The hormone rhodomorphin is a · glycoprotein with a-D-mannose and/or a-Dglucose sugar haptens that is necessary for wound healing in Grjtfithsja pacjfjca (Watson and Waaland 1983).

Interactions Between Adhesive Molecules
Adhesion may involve interactions between classes of molecules.
Although red algal cell walls contain a proportionately smaller amount of proteins than carbohydrates , most algal adhesives studied thus far are variations of polysaccharide-protein complexes that are at least partially synthesized in the golgi apparatus (Willey and Giancarlo 1986).
There may be more than one adhesive or one adhesive may become functional when it is cross-linked (Wigglesworth- . Increases in the strength of red algal adhesion over time are thought to result from a "curing" process which may be polymerization or cross-linking , Heaney-Kieras et al. 1977, possibly between sulfated polysaccharides or glycoproteins and · the divalent cations Ca2+ or Mg2+ (Jones et al. 1982;).
It appears that red algal adhesion is an interaction between sulfated and acidic polysaccharides, proteins, and glycoproteins. Golgi vesicles secrete mucilage rich in sulfated and acidic polysaccharides during tetraspore development of Palmarja palmata . After mucilage deposition ceases, the golgi apparatus makes vesicles with glycoprotein rich contents.
These vesicles are abundant in released tetraspores and probably have adhesive material for spore attachment. . Both proteins and sulfated polysaccharides are found in vesicles produced by the Golgi apparatus in differentiating carposporangia of Chondda tenujssjma (Tsekos 1985).

Adhesion of Brown Algae
The adhesion of brown algal zygotes has been investigated extensively Evans et al. 1979;Ouatrano et al. 1979;Brawley and Quatrano 1979a;Kropf et al. 1989;Vreeland et al. 1992Vreeland et al. , 1993. Initial attachment of zygotes generally occurs with adhesive mucilage supplemented by adhesive zygotic walls, followed by secondary attachment with rhizoids Vreeland et al.1993). pelyetja canaliculata provides an example of such attachment. Zygotes are anchored with an enclosing mesochiton that can be said to correspond with the mucilage of red algal spores (Boney 1975) by countering water movements and assisting settlement. The mesochiton stains with alcian blue to suggest sulfated polysaccharides as initial adhesives; secondary adhesion occurs when the rhizoids provide further anchorage .
Initially, Halidrys sjliguosa zygotes are attached by a rigid, alcian blue staining, adhesive wall that is secreted by the golgi apparatus and surrounds the zygote. Because the adhesive is also part of the wall, failure is likely to occur only at the adhesive-substrate interface, and not at the zygote-adhesive interface. The adhesive wall is shed once the rhizoids appear, which is not until several days after initial attachment. The four primary rhizoids and their derivatives have adhesive mucilage, and while the thallus grows their continued division eventually forms the holdfast .
Adhesion is similar in Bifurcaria bifurcata .
Zygotes of Eucus and Ascophyllum follow the pattern of attaching initially by exuding mucilage and secondarily by the primary rhizoid which produces alcian blue staining mucilage at its tip. If the substrate is smooth, this mucilage will spread to form an adhesive, suction-like "foot". New adhesive feet are produced in pulses as the rhizoid grows. On non-smooth surfaces the rhizoid grows down into the substratum through any available crevices. The basal embryo cells produce secondary rhizoids which fan out to provide further anchorage . Glass coverslips provide an artificial substrate that differs greatly from that supplied by intertidal rocks to which Eucus adheres in nature. Rhizoids of Eucus plants grown on glass were long, thin, and tapered while those grown on rocks were more stout for a greater contact area and firmer attachment . Eucus zygotes attach more firmly when rhizoids can penetrate interstices in substrates such as porous rock and wood, and less firmly on smooth substrates such as glass where rhizoids cannot penetrate. Eucus rhizoids are anchored by a highly sulfated fucan glycoprotein, (E2), (Brawley and Quatrano 1979a) which must be sulfated before adhesion will occur; Eucus embryos grown without sulfate formed rhizoids but did not adhere. Those grown with sulfate did adhere and sulfated polysaccharides were detected at the rhizoid tips with toluidine blue and the 0-galactose specific lectin EITC-RCAI ). If embryos are grown without sulfate, E2 will not be sulfated or localized in the rhizoid (Quatrano et al. 1979). E2 is sulfated in the golgi apparatus, through which it is secreted (Evans et al. 1979) and then transported via an actin network to the rhizoid tip Kropf et al. 1989) with a vitronectin like glycoprotein, (Vn-E) ). Vn-E may begin its association with F2 during travel through the golgi apparatus. Vn-E is localized in the extracellular matrix of the elongating rhizoid tip, which anchors the zygote to the substrate. When two-celled Fucus embryos were cultured without sulfate, Vn-f was not localized in the rhizoid tip and the embryos did not adhere in the presence of the Vn antibody ).
algae. Vitronectin and fibronectin are found in humans and other mammals , and a homolog of vitronectin has been found in angiosperms . A vitronectin-like protein on the surface of carrot cells is used by the pathogenic bacterium Agrobacterjum tumefacjens as a receptor when it attaches to carrot cells (Wagner and Matthysse 1992). Vitronectin and fibronectin are adhesive proteins found on the external side of the · plasma membrane in focal adhesions, which are membranous connections between the extracellular matrix and the cytoskeleton .
Adhesive formation increased with the addition of 1 µM vanadate to Fucus zygotes cultured in artificial seawater (Vreeland et al. 1992). A peroxidase that requires vanadate may catalyze cross linkages between cell wall carbohydrates and phenolics to place the now cross linked phenolics at adhesive sites. Peroxidase activity and extracellular phenolics were found in the cell wall during initial adhesion and in the rhizoid tip after germination.
Microspheres bound to the mucilage of Fucus gardnerj zygotes to show localized patches of adhesive 3-6 h after fertilization (Vreeland et al. 1993); this corresponds with the localization of cross-linked phenolics at adhesive sites in the presence of vanadate. More microspheres bound as zygote development progressed, showing that more adhesive was being produced by the zygote. The patches of adhesive eventually grew to cover the hemisphere of the zygote oriented towards the substrate and the rhizoid upon its emergence. Because the adhesive appeared in patches, it was probably produced by cytoplasmic vesicles. The cell wall was anchored by strands of adhesive polymers. Microspheres detached from rhizoids in the presence of calcium chelators, implying the necessity of divalent cations in adhesion. It appears that adhesion of Eucus rhizoids is an interaction between many factors: mucilage, phenolics, the sulfated fucan E2, the adhesive glycoprotein vn-E, divalent cations, and positioning of these adhesive molecules through transport, cross-linking and enzyme catalysis.

Adhesion of Green 'Algae
Acidic polysaccharides , glycoproteins (Musgrave 1987;, mucopolysaccharides  and protein (Tosteson and Corpe 1975) have all been implicated as adhesives in the green algae. In addition, Ca2+ has been associated with adhesive events such as mucilage secretion  and mating reactions ).
Zoospores of Enteromorpha intestinalis from different parent sources have different adhesive abilities but detach similarly when exposed to trypsin, pronase, and ex-amylase . Because the effects of trypsin mirrored those of ex-amylase, the adhesive is thought to be a mucopolysaccharide. Within minutes of substrate contact, the adhesive is secreted. Newly attached spores were most susceptible to enzyme induced detachment but became more resistant over time.
The initial adhesive of the zygote of ~ mutabilis differs from that of the rhizoid, which provides secondary adhesion (Brc\ten 1975). Just before gametes are released, their cytoplasm contains small, electron dense vesicles. Once the zygotes form, their adhesive is probably secreted through these vesicles onto the substrate. This adhesive stains with ruthenium red for acidic polysaccharides and can be removed with pronase and a-amylase, but not with hyaluronidase or ruthenium red, which binds with acidic polysaccharides The rhizoid cells differentiate several days after the initial adhesion of the zygote and produce adhesive continuously as they grow. The resistant adhesive of the rhizoid does not stain with ruthenium red or detach with enzymes and is therefore thought to have a different chemical composition than that of the initial, zygotic adhesive .
.u.JY.a ruru:ta and Enteromorpha compressa are often epiphytic on Gracilarja chjlensjs.  found that exudates from the culture medium of ~ chilensis stimulated settlement of spores of both green algal species. Upon analysis, the exudate was found to contain sulfated galactans.
The mechanism of response by the· epiphytes to the host exudate is not yet known. A high epiphyte load can be detrimental to the farming of a, chilensjs , as the added weight can cause the host to detach. These epiphytes can compete for nutrients, light, and dissolved gases.
They can also exude allelopathic substances that are either harmful or beneficial to the host .
Exudate containing protein and carbohydrate enhances adhesion of unicellular Chlorella vulgaris to glass (Tosteson and Corpe 1975). This non-diffusible material originates from the following sources: 1) ~ yulgarjs, 2) fouled marine surfaces, 3) marine bacterial cultures and 4) natural seawater, with the latter two being the most effective. The exudate may induce adhesive polymer synthesis, stimulate secretion of the adhesive, stabilize the adhesive, or act as an adhesive substitute. ~ yulgarjs cells adhered less but began to agglutinate in a lectin induced manner when C..... yulgarjs exudate concentrations were above 0.2 ng/cell. More washed cells than unwashed cells adhered when exudate was added; washing may expose more adhesive sites or change the physiological state of the cell. Maximal adhesion of ~ yulgarjs occurs in the G2 period of interphase , possibly because the cell surface may be altered by biochemical changes between cell cycle phases. Increased adhesion may be related to possible changes in the cell proteins within the cell cycle, which in turn may influence the composition of the proteinaceous exudate, which may be adhesive or have its synthesis catalyzed by a protein (Tosteson and Corpe 1975).
Agglutinating glycoproteins on the flagellar surfaces of Chlamydomonas eugametos gametes are intrinsic ·to the membrane and have differently shaped ends; one is knoblike and the other is hooklike (Musgrave 1987), which may facilitate their mediation of flagellar adhesion between + andgametes. Adhesion of the flagellae initiates travel of a signal from the membrane to the cytoplasm of the paired cells, which fuse upon receipt of the signal. Ca2+ and cAMP interact as the messengers for this signal . Upon adhesion, intracellur cAMP levels increase sharply. Gametes will mate if given di-butyryl-cAMP. This effect was blocked by inhibitors of Ca2+ transport or utilization.
Elongated cells of the desmid Closterjum glide on substrates by mucilage that is secreted from the pole opposite the direction of motion . The mucilage is an acidic polysaccharide that stains with Ruthenium red. Dense cored vesicles from the golgi apparatus associate with microtubules and are secreted out of the cell through flask-shaped pores in the cell wall . Labeling with chlorotetracycline showed that these pores are associated with calcium rich areas on the cell surface . Pores in the poles of Closterjum ehrenbergjj cell walls secrete receptors for the ~-D-galactose specific lectin RCA120 as part of the mucilage (v. Sengbusch et al. 1982); therefore carbohydrate recognition by lectins may be part of the adhesive process for these desmids, as lectins have been found in the Chlorophyta ).

Adhesion of Diatoms
The pennate marine diatom, Amphora coffeaformis, glides on adhesive mucilage containing acidic polysaccharides that is secreted from golgi vesicles through the raphe fissure (Drum and Hopkins 1966;Webster et al. 1985). Binding of 0-glucose to chemosensory receptors on the plasma membrane may signal release of inositol triphosphate as a transducer, which then binds to a further receptor to signal release of bound Ca 2 +, opening of Ca2+ channels in the membrane, and an increase in cytoplasmic Ca2+ concentrations ; Wigglesworth-cytoplasmic transducers to translate the Ca2+ message into release of mucilage, hence adhesion. The mucilage probably remains attached to the cytoplasm through plasmalemma at the raphe; in this way it maintains contact with bundles of actin filaments that may translate adhesion into motility by moving the site of mucilage attachment along the raphe (Edgar and Pickett-Heaps 1984).
These diatoms detached and left adhesive "footpads" behind in the presence of the Ca2+ chelator, ethylene glycol-bis( N,N',N'tetraacetic acid (EGTA), to suggest that external Ca2+ may maintain adhesion by cross-linking negative charges in the extracellular matrix ). Glycoproteins and proteins may be part of the adhesive or may synthesize the adhesive, as adhesion was reduced when glycoprotein and protein synthesis were inhibited with tunicamycin and cycloheximide, respectively. Because polysaccharides have been found repeatedly in the extracellular polymers of diatoms and proteins have not often been detected, it is likely that the adhesive of diatoms is chiefly polysaccharide (Hoaglund et al. 1993).
The pennate marine diatom, Ardjssonea crystallina, has a different means of motility (Pickett-Heaps et al. 1991 ). It can change direction as it glides on mucilage that adheres to the substrate, stains with alcian blue and is secreted through terminal grooves in the trailing end of the cell. The elastic mucilage may swell after release and push the cell in the direction of travel, which is similar to the effect of mucilage on desmids. A. crystallina can also attach with thick stalks that stain with alcian blue to indicate sulfated polysaccharides as adhesives in this diatom. Diatoms with stalks were found to be more abundant in the upper, well lit but more turbulent reaches of a reservoir than were diatoms without stalks (Hoaglund and Peterson 1990). Stalked diatoms were able to survive in both the upper and the lower reaches of the reservoir while unstalked diatoms did not fare well in the upper reaches.

Adhesion of Euglenoids
Colacium libellae migrates and attaches to freshwater arthropods. After travelling through the golgi apparatus, a polymer is extruded from pores in the anterior pellicle to form an adhesive disc which stains for sulfated polysaccharides with alcian blue but not for neutral polysaccharides with periodic acid-Schiff (PAS) (Willey and Giancarlo 1986). Next, a flexible stalk forms between the adhesive disc and the euglena cell. The core of the stalk stains with PAS and its periphery stains with alcian blue. Differential staining signifies different adhesive polymers. Both the adhesive disc and stalk resist pronase digestion and remain attached to indicate polysaccharide in adhesion rather than the protein-polysaccharide complex generally found in algal cells ).

Adhesion of Fungi
Ascospores of marine ascomycetes have a variety of attachment mechanisms . These include: 1) release of a mucilage drop from a polar end chamber to form an adhesive pad, 2) long, viscous threads which form when cap-like appendages uncoil, 3) sticky mucilagenous sheaths which may expand upon contact with water, 4) sticky vermiculate appendages, 5) tufts of fibrillar appendages which trap the spores on jagged substrate edges such as wood, 6) amorphous appendages which rely on contact with a large surface area of the substrate for adhesion, 7) adhesive spore walls with a sticky, fibrillar layer, and 8) combinations of the above.
Marine fungi attach to substrata by chemical or physical forces and the greater the area of contact with the substrate, the greater the attachment. The appendages of marine fungi increase the area of substrate contact.
Spores of the plant pathogen, Nectrja haematococca attach by their apices with "macroconidial tip mucilage" that labels with FITC-Con A only when the fungus is cultured in a medium that promotes adhesion (Kwon and Epstein 1988). Further investigation of the mucilage of adherent macroconidia with SDS-polyacrylamide gel electrophoresis revealed a 90 kDa glycoprotein that binds with Con A. Spores detached when exposed to protease and did not attach in the presence of Con A.

Adhesion of Marine Bacteria
Adhesive polysaccharides in bacterial mucilage attach to glycoproteins of the marine conditioning layer that becomes adsorbed to underwater marine surfaces (Baier 1980;. More bacteria attached to biofilms of previously attached bacteria of the same species than to biofilms of other species or to glass (Banks and Bryars 1992). Depending on the shape of the bacterium, adhesion will occur via mucilage to fill in the space between the substrate and the cell wall. Rods and cocci attach with strands and sheets, or less frequently with pads and capsules. Stalked bacteria attach with basal mucilage pads. Continuous colonial growth results in a film of bacteria with accompanying mucilage .

Adhesion of Animals
Marine mussels, Mytjlus ~-. attach to .surfaces by forming a byssus composed of a bundle of threads ·connecting the mussel to an adhesive plaque with a water resistant polyphenolic protein ) that attaches the plaque to the substrate . The core of the byssal threads forms from collagenous secretions of the collagen gland (Vitellaro-Zuccarello 1980); the accessory gland then secretes a protective coating composed of a proteinaceous resin and a curing enzyme, catecholoxidase , which converts 3,4dihydroxyphenylalanine (DOPA) in the resin to peptidyl DOPa-quinone, which probably acts as a cross-linker to harden the adhesive  turbulent places and has a less porous byssus, stronger attachment strength and an adhesive with a different protein composition than that of M.. edulis, which inhabits calm, sheltered environs ). phragmatopoma caljfornjca. a marine polychaete, has a stable and insoluble proteinaceous adhesive . The adhesive connects hard particles so that a tube can be made for the polychaete to live in. Three percent of the adhesive's amino acid residues are 3,4dihydroxyphenylalanine (DOPA). DOPA extracted from the adhesive signaled planktonic larvae off... caljfornjca to induce attachment and metamorphosis, thereby expanding concretions of marine polychaetes.
Barnacles, ~ (Balanus) balanojdes, disperse and explore the substrate during the cyprid stage of metamorphosis and use a temporary proteinaceous adhesive for this initial phase of attachment . Settlement occurred with greater frequency on slate. coated with proteins called arthropodins which were extracted ·from .fL. balanojdes than on noncoated slate or slate coated with bovine serum albumin. Within 24 h of selection of a final settlement site they complete metamorphosis and a more permanent proteinaceous adhesive with a higher bonding strength is formed . Barnacle adhesive is notable because of its tenacity (greater on slate than that of limpets or mussels on slate), ability to spread along fissures and attach to a variety of surfaces, and resistance to biodegradation. Although adhesion of barnacles is tenacious it is not instantaneous and enzymes probably catalyze reactions that convert water-soluble adhesive proteins to an insoluble state ).
Chemical inducers of settlement and metamorphosis have been found in a variety of marine animals. It is of interest that the red algal pigment phycoerythrobilin contains structural analogs of the neurotransmitter gammaaminobutyric acid (GABA), and that GABA induces metamorphosis in the red abalone Haliotis rubescens (Morse et al. 1979). Three crustose coralline red algal genera, Lithothamnjum, Lithophyllum, and Hildenbrandja are specific substrata for the red abalone. Another crustose coralline red alga, Hydrolithon boergesenji. produces a specific morphogen that induces attachment and metamorphosis in the coral, Agarica humilis (Morse and Morse 1991 ). Lectinlike receptors on the coral probably bind with multiple N-acetylglucosamine and galactose residues on the morphogen, which is associated with a sulfated glycosaminoglycan. Adults of the oyster, Crassostrea yjrgjnjca. and bacterial biofilms on oyster shells produce ammonium as a metabolite that induces settlement of oyster larvae (Tamburri et al. 1992).
Coelomocytes of the sea cucumber, Holothurja QQ.lli, require cations for adhesion to glass coverslips . Mg2+ ions were more effective than Ca2+ ions. Adhesion was not inhibited by the metabolic inhibitors potassium cyanide or sodium azide, but was inhibited by the inhibitor of microtubules, vinblastine. Adhesion was enhanced by coating glass coverslips with coelomic fluid or with purified 220 kDa coelomocyte aggregating factor, which is produced by coelomocytes and may e similar to fibronectin.
An elevation of intracellular pH by 0.2 -0.3 units acts as a signal to the cytoplasm of mammalian cells that a substrate has been encountered and adhesion has taken place ). If the Na+/H+ antiport that facilitates the substrate dependent pH change in mouse fibroblasts or neutrophils (Margolis et al. 1988) is blocked by amiloride, mammalian cells will adhere but will not follow the sequence of events leading to proliferation . In this sequence, cellular extensions known as lamellapodia develop and provide further anchorage, the round cells flatten and spread onto the substrate; spreading is then used as a signal for the cells to enter the S phase of mitosis and proliferate . Collisions between fibroblasts on the substrate will raise the pH, possibly because adhesive sites on the cell surface are occupied. The pH elevation was reversed when cells were detached with trypsin or EDTA. Adhesion of mammalian cells is mediated by integrins, which are transmembrane proteins that bind to the amino acid sequence Arg-Gly-Asp (RGD) on the adhesive glycoproteins vitronectin and fibronectin in the extracellular matrix . Binding of the RGD p·eptide ·to plasma membrane integrins will raise the pH in the same manner as will adhesion to a substrate ). lntegrins in turn require the divalent cations Mg2+ or Ca2+ to bind to the RGD peptide  and chlorotetracycline, a fluorescent calcium chelator, was used to localize Ca2+ at attachment points of cell membranes in neural crest cells from the salamander, Ambystoma maculatum (Moran 1984).
Glycoproteins from mammalian cell membranes and extracellular matrices characterize and influence the development of different tissue types and are involved in cellular recognition (Moscona 1974). Glycoproteins of rat cells are supplied with mannose in the endoplasmic reticulum and with galactose and fucose in the golgi apparatus before being transported to the cell surface in vesicles that release their glycoproteins to fuse with the plasma membrane. In this way, glycoproteins reach the cell surface.
Changes in adhesives on the cell surface can be important factors in diseases of animals (Travis 1993). The adhesive molecules of metastatic cancer cells differ from those of nonmetastasizing cells and these adhesive changes may be what allows metastasizing cells to break away from primary tumors, travel through the bloodstream, and start new tumors. On a surface groove of HIV viral particles there is a glycoprotein which binds with CD4, a receptor protein of human cells (Moffat 1993). If the viral particles will bind to a mimic of CD4, infection could be prevented. Initial events in phagocytosis by disease fighting macrophages involve adhesion (Aggeler and Werb 1992) and adhesive glycoproteins have been found in macrophages of mice (Tomita and Ishikawa 1992).

Influence of the Substrate on Algal Spore Adhesion
Because adhesives must bond with the substrate to attach firmly, compatibility of the substrate and adhesive is crucial (Fletcher and Callow 1992). One characteristic of the substrate that probably influences spore adhesion is surface free energy. If a substrate is hydrophobic and has high surface tension, it has low wettability and low surface free energy. On a hydrophobic Teflon surface, Fucus rhizoids were elongated and did not attach securely . Spores of Enteromorpha settled on hydrophobic surfaces more readily when they were pressed with coverslips . Surface tension varied between algal species and on different parts of a thallus; it may determine which epiphytes attach (Linskens 1963).
Scjrpus yalidus supported more algal epiphytes in the fall than earlier in the growing season, possibly because the necrotic culms had higher surface free energy due to the breakdown of the hydrophobic cuticle .
Surface texture may also influence spore attachment Fletcher and Callow 1992). Spores may detect small differences in surface profile and attachment to one of the many planes of rough surfaces increases the chances of survival by providing shelter from waves and currents. Larger spores may require large pits on rough surfaces for attachment, while smaller spores may attach to small pits on smooth surfaces  to avoid dislodgement by turbulent water beyond the boundary layer . Pelyetia fastigata embryo survival rates were greater in small depressions of red algal turf than on ridges or flat areas where the embryos would not be constantly immersed .  found that initial settlement of seaweeds did not differ on acrylic discs bolted to intertidal rocks and divided into quadrants; one was left smooth, while three were coated with 0.1-0.5 mm, 0.5-1.0 mm, or 1.0-2.0 mm particles of hard silicon dioxide. Over time, patterns of species distribution developed on the quadrants. Chondrus crispus and ~ Lactuca were abundant on the larger grades but scarce on the smooth surface. Polysjphonja harveyj was found equally on all three particle grades but was also scarce on the smooth quadrant. Corallina officjnalis thrived on the smallest particles; when it appeared on the smooth surface it did not form upright, articulated branches.

Cell-substrate Contact Influences Differentiation
The act of adhering to a substrate has profound effects on the structure and behavior of cells ). Cell-substrate interactions trigger molecular interactions which determine and regulate cell morphology, motility, growth, and metabolism . Increases in knowledge are necessary on the perception of surfaces by cells . The cytoskeleton and proteins in the membrane may interact to detect the surface free energy and other characteristics of a substrate upon contact (Van Kooten 1992) to determine if adhesion and subsequent developmental changes take place (Hanein et al.1993). Amphora coffeaformjs, a pennate diatom, synthesizes small amounts of an extracellular, water-soluble, possibly acidic polysaccharide which diffuses into the water column and may act as a substrate detector by becoming more concentrated when diffusion is blocked as the cell approaches a substrate (Wigglesworth- . Swimmer cells of the bacterium Vibrio parahaemolytjcus transform to swarmer cells if they are grown on or embedded in solid media, suggesting that contact with a surface or confinement are developmental cues which may induce bacteria to switch genes on or off to produce different phenotypes .

Lectins Can Influence Adhesion
Lectins are proteins or glycoproteins that function in cell adhesion, cell-cell recognition, and cell agglutination by binding to complementary carbohydrates (Liener et al. 1986). The word lectin is from the latin legere, meaning to pick out or choose. Lectins have a greater affinity for oligosaccharides or glycoproteins than for their corresponding sugar haptens (von Sengbusch et al. 1982). All lectins that have been found in marine red algae will bind with glycoproteins . Those with molecular weights above 60 kDa tend to have a requirement for divalent cations and will bind to monosaccharides such as Dgalactose, N-acetylgalactosamine, N-acetylglucosamine, or D-fucose.
Haemagglutination varied between lectins from tetrasporangial and cystocarpic plants of Chylocladja vertjcillata (Rogers et al. 1980). Because ~ yertjcillata is in the same family as Charopja parvula, lectins may be present in the mucilage of Q... parvula spores. Most algal. lectin literature does not focus on possible ecological or physiological functions of the lectins. Questions that arise are: 1) What is the role of marine red algal lectins?, 2) Might it be in substrate recognition during host-epiphyte interactions?, and 3) Might they be involved in spore adhesion in the intertidal realm?
An unknown chemical cue for parasitic spore attachment of Harveyella mirabiljs originates with its host, Odonthalia floccosa  and may involve enzymatic degration of sulfated polysaccharides in cell walls of the host. A substance from the host may stimulate parasitic spores to release the adhesive contents of their vesicles . Spore attachment may be initiated by carbohydrates on host cells that bind with receptors on spores: this suggests that lectins are involved in attachment of this parasitic alga. Baier (1980) suggested that there is a glycoproteinaceous conditioning layer that is adsorbed to all marine surfaces. If so, lectins or other plasma membrane receptors of adhesive organisms 'may interact with appropriate sugar haptens of glycoproteins in the conditioning layer to detect surfaces and then adhere (Wigglesworth- . Lectins may mediate in the settlement of larvae of Janua (Dexiospira) brasiliensjs, a marine polychaete, by binding with bacterial polysaccharides or glycoproteins of the surface film (Kirchman et al. 1982). Enhanced tetraspore adhesion occurred in Champja parvula spores in seawater pre-conditioned by ~ paryula tetrasporophytes (Dworetsky 1983), although a compound that enhances adhesion was not found. Perhaps the parent plants produce glycoproteins to form a marine conditioning layer that is recognized by lectins on ~ parvula spore surfaces. · Fluorescein isothiocyanate (FITC) lectin binding reflected species-specific biochemical diversity expressed at cell surfaces in a variety of algal species (von Sengbusch and Muller 1983) and differentiated between clones of unicellular algae . Therefore, differences exist in cell surfaces that may contribute to lectin/receptor interactions.  found that egg-sperm recognition in Fucus serratus is mediated by fucosyl and mannosyl containing ligands on the egg surface which bind with protein on the sperm surface to suggest a lectin and sugar interaction. There is also evidence of intraspecific variation in lectin binding. In Fucus spjralis, FITClectins were used to demonstrate that the glycan moieties of the cell surface differ between spermatozoa, oocytes, zygotes, and embryos ).
Lectins mediate adhesion in angiosperms, fungi, and bacteria. Lectin receptors in plants have been found to be glycoproteins, and glycolipids, phenolic glycosides, or glycosides of secondary metabolites (Etzler 1986).
Gladiolus gandayensjs stigmas contain (3-glycosyl specific lectins which adhere to pollen grains and probably recognize the appropriate pollen so that self pollination can be avoided ). The cx-0-mannose/cx-Dglucose specific lectin concanavalin A, (Con A), from Canayalia ensiformis reacted with receptors on the stigma surface of .G... gandayensjs to inhibit penetration of the stigma by the pollen tube, possibly by blocking pollenstigma interaction. Phaseolus yulgarjs lectin and Con A stimulated in vitro germination of Lilium longiflorum pollen . Lectin from the potato plant inhibited hyphal extension and spore germination in Botrytjs cjnerea  and wheat germ lectin bound to spores of chitinous fungi . Lectins are involved in attachment of the symbiotic bacteria, Bhizobjum leguminosarum, to root hair tips of peas (Kijine et al. 1988).

Inhibitors of Adhesion
Within an organism, disruption of many biosynthetic and metabolic processes can interfere with adhesion, as in Amphora coffeaformjs Cooksey 1986,1988) where adhesion was inhibited by tunicamycin, a glycoprotein synthesis inhibitor; cycloheximide, a protein synthesis inhibitor; D-600, a Ca2+ transport inhibitor; podophyllotoxin, a depolymerizer of microtubules, and CCCP, an energy uncoupler. This implicated glycoproteins, proteins, calcium transport, the cytoskeleton, and energy as necessities for adhesion.

Biosynthesis inhibitors restricted adhesion and germination in other
organisms. Chlorella yulgaris did not adhere when treated with actinomycin D, which inhibits DNA dependent RNA synthesis, and adhesion was reduced by 36% with puromycin, a protein synthesis inhibitor .
This suggests that protein synthesis is less important than production of messenger RNA in .C.. yulgaris . Spore adhesion but not spore germination of the plant pathogenic fungus, Nectria haematococca was inhibited by Con A . Spore germination of the freshwater fungus Aphanomyces astacj was inhibited by actinomycin D and by cycloheximide, which inhibits translation on cytoplasmic ribosomes .
Cell-cell recognition leading to gamete pairing can also be inhibited .
.C.hlamydomonas reinhardti gametes did not form pairs when synthesis of glycpproteins on their flagellar surfaces was inhibited with tunicamycin£.
although mutants that are resistant to tunicamycin will form pairs when exposed to tunicamycin (Dutcher and Gibbons 1988). In the dinophyte, A1exandrjum Qatenella, gamete pairing was inhibited by tunicamycin and by con A, although Con A inhibition was reversible with the addition of a-Dmannose or a-D-glucose  Adhesion of animals can also be inhibited. The red abalone, Haliotis r_ubescens, did not undergo metamorphosi'S in the presence of Con A (Kirchman et al. 1982). This suggests that glucose or mannose haptens are involved in substrate identification, which must take place prior to adhesion and subsequent metamorphosis. Coelomocytes of the sea cucumber Holothuria Willi attached in the presence of cytochalasin 8, which disrupts microfilaments, but not in the presence of the anti-tubulin cytoskeletal inhibitor, vinblastine . Therefore, microtubules but not microfilaments may be involved in attachment of coelomocytes. Metabolic energy may not be required for coelomocyte attachment, as it was not inhibited by the metabolic inhibitors potassium cyanide and sodium azide. It is possible that the microtubules were in place before metabolism was blocked.
Molybdate is a competitive inhibitor of the ATP sulfurylase reaction, the first step in sulfate activation . Inside the cell, molybdate is transported by the sulfate carrier system to inhibit the formation of adenosine 3'-phosphate 5'-phosphosulfate, the activated donor for sulfate transfer reactions. In this way, molybdate blocks sulfate transfer to polysaccharides en route to the cell surface . In Porphyrjdjum aerugineum, sulfation of polysaccharides was inhibited by molybdate, and inhibition was reversed by the removal of molybdate . Adhesion was reduced when molybdate was added to mechanically separated cells of erasjola stipitata. and was reversed by the addition of sulfate before or during molybdate addition . Because molybdate competes with ATP sulfurylase, enzymes can be important catalysts of the synthesis of adhesives.
Sulfation is not the only process necessary for adhesion in Prasjola stipjtata. Protein synthesis and photosynthesis are also required . Cycloheximide inhibits translation on cytoplasmic ribosomes and chloramphenicol inhibits translation on plastid ribosomes. Addition of these inhibitors to Prasiola stjpjtata resulted in 90% and 40% inhibition of adhesion, respectively. Inhibition of photosynthesis can also reduce adhesion.

Biofouling
Biofouling can be defined as the attachment of marine organisms to anthropogenic structures placed in oceans . Marine algae can attach to these structures to become biofouling organisms (Terry and Pickens 1986). Biofouling communities may be established successionally: first, a layer of non-living material becomes attached to submerged surfaces. Bacteria attach to this layer, followed by diatoms and other microorganisms (Floodgate 1971 ). The attachment and development of algal spores and animal larvae forms the mature biofouling community, but the presence of seaweed and animal larvae can by delayed until a surface coated with an antifoulant loses toxicity  ).
Biofouling increases the frictional resistance of ships, which reduces speed and decreases engine and fuel efficiency. Supertankers and other vessels must be drydocked every 1-2.5 years to remove biofouling organisms and to apply new antifouling paint . In 1981 the U. S. Navy spent $360 million on biofouling related drydocking and an additional $1 00 million a year is spent by the U. S. Navy on hull cleaning, paint removal, repainting, and toxic water and grit disposal (Alberta et al. 1992). Time spent on biofouling control is also critical.
To control biofouling organisms, an agent must interfere with adhesion or other cellular processes of potential biofoulers. Unfortunately, such interference is often toxic to biofouling and other organisms. Traditionally, such compounds as tributyl tin have been used, but these are not environmentally acceptable . By reducing surface free energy and weakening attachment strength, hydrophobic coatings have potential as antifoulants ) The synergistic combination of silicone and fatty acid mixtures is now in wide use as an antifoulant ). One course that may lead to safe, efficient biofouling control is that of using molecules similar to those that are peceived by cells as extracellular signals to adhere, but that would instead signal cells not to adhere (Wigglesworth- . Another potential type of antifoulant is the non-leaching biocide that adsorbs to surfaces by ion exchange and kills potential foulers upon contact . By definition, fouling organisms would be most likely to contact the coated surface and encounter the biocide. Therefore, untargeted organisms would not be effected by the biocide, provided that it did not leach into the water. One example of such a biocide is 3-(trimethoxysilyl)propyloctadecyl-dimethyl ammonium chloride (Dow Corning 5700) which was found to be toxic to biofoulants. This "non-leaching" biocide had the disadvantage of leaching into solutions and reducing growth of test populations of Amphora and Dunaljella by 10% and 30%, respectively. An improved method of bonding the biocide to surfaces would prevent leaching.
More barnacles, Balanus balanoides. and blue mussels, Mytilus edulis, grew in tidepools dominated by Hildenbrandja prototypus than in those dominated by Balfsia yerrucosa . Tannins isolated from Eucus yesjculosjs and Ralfsja yerrucosa were toxic to plankton.
The branch tips of Sargassum natans and · .S... flujtans have antibacterial activity, are essentially free of epibiota, and contain tannin . Epifauna, and especially hydroids, died when exposed to homogenates of the branch tips with 0.1 -0.8% tannic acid. Paint and varnish with 4-8% tannic acid on panels inhibited barnacles and algae. Seawater is an excellent medium for tannin toxicity because the alkaline pH favors algal tannin extraction and salt acts with the tannins to precipitate proteins . Antifoulants with kelp or tannins were patented from 1880 to 1900 . Algal tannins appear to be quite viable as marine antifoulants.
Extracts from marine animals may also become important controls of biofouling. The gorgonian coral, Pseudopterogorgja acerosa, is almost never host to biofouling organisms (Stochaj and Targett 1993). When polar organic metabolites were extracted from the coral, dissolved in methanol, and administered to the fouling diatom species, Njtzchia, for 48 h at 48 µg/ml, chlorophyll was lost, photosystems I and II were possibly disrupted, there was a decrease in cell carbohydrate, and no photosynthesis or growth took place.
Fatty acids isolated from the marine sponge, Phyllospongja papyracea. have been shown to have antifouling activity against Mytilus edulis ). This sponge also has few epibionts. Limpets may prove useful in biofouling control as they are grazers of attached algae and reduced biofouling cover by 80-90% when transplanted to submerged panels of shipping steel ).
Hopes for a biological and environmentally safe biofouling control agent have long been voiced (Sieburth 1965;Fletcher and Callow 1992;Stochaj and Targett 1993) and an understanding of the biology of red algal spore adhesion will be useful in attaining this goal.

Champja paryula
The organism used in this study of spore adhesion was Champja parvula (C. Ag.) Harvey, a temperate and tropical marine red macroalga (Taylor 1957) that can be free floating, epiphytic, or attached. In the seagrass beds of Biscayne Bay, Florida, its most common substrate is Thalassja testudjnum (Humm 1964). It can attach to rocks or to anthropogenic marine surfaces Mathieson 1983, Lethbridge et al. 1988) and can therefore be used as a means by which to determine the mechanisms of biofouling. It lives as an annual in Narragansett Bay, Rhode Island (Villalard-Bohnsack et al. 1 ga8), where its habitat varies from open ocean to estuary to tidepool, although it appears to prefer oceanic water (Harlin and Rines1993).
Tetrasporophytes of .C... parvula are found in summer , Harlin et al. 1992a). This alga has been widely used in toxicity studies because its reproduction is as sensitive to the heavy metals Ag, Cd, Cu, and Pb as are the most sensitive marine animals, and it is also sensitive to cyanide, arsenite and arsenate , Thursby and Steele 1984, Thursby et al. 1985. Finally, .Q.. paryula has potential for use in genetic studies ) because of spontaneous mutations that affect morphology and pigmentation and follow Mendelian patterns of transmission.
As early as 1892, Davis published an illustration of a spore with its mucilage . Champja· paryula produces two sets of spores; carpospores and tetraspores. Both sets of spores must attach before they can germinate (Dworetsky 1983) and establish adult plants; therefore spore adhesion is vital for completion of the life cycle. Attachment may act as a signal for germination and its accompanying physiological and morphological changes.
Because spore adhesion is a crucial stage in the life cycle of .C... parvula, and because spores can be easily obtained in culture, this alga was selected to examine mechanisms of spore adhesion. Light and scanning electron microscopy, histochemical studies, and interference with adhesion via inhibitors of biochemical pathways were used to investigate: 1) anatomical changes occurring during spore adhesion and germination, 2) the composition of the extracellular mucilage, and 3) the classes of molecules involved in initiation and maintainence of spore adhesion.

Culture
Qhampja paryula was obtained from the Environmental Protection Agency and Science Applications International Corporation in Narragansett, Rhode Island. Champja paryula was cultured in Percival Growth Chambers at 20° C, + or -2° C with cool white light at 60 -80 microeinsteins illuminated from above and below on a 16:8 light-dark regime. The culture vessels were aerated 500 or1000 ml flasks of filtered seawater from Narragansett Bay. The seawater was autoclaved (20 min, 121° C) to insure a unialgal culture. One ml modified 'f' medium per 100 ml seawater was added after autoclaving.
Modified 'f medium is also known as GP2 and consists of 6.35 g sodium nitrate, 0.64 g sodium phosphate, 133 mg EDTA, 51 mg sodium citrate, 9. 75 mg iron, and 1 O ml vitamin solution (2.0 g thiamine-HCI, 1.0 mg biotin, 1.0 mg 812 in 100 ml DIH20) in 1 liter DIH20 (Guillard and Ryther 1962;Thursby and Steele 1986). Nutrients and seawater were changed weekly and excess algae discarded. Flasks were acid washed with 1 O -15% HCI or washed with RBS 35 (Pierce Co.), rinsed 5 min with DIH20 to eliminate any lethal traces of detergent and autoclaved 20 min at 121° C.

Phases of the Life Cycle
Carpogonia of female gametophytes were fertilized by spermatia of male gametophytes to obtain carposporophytes, which grew within the female gametophyte and produced cystocarps, inside of which were formed carpospores. These carpospores were released and attached to cover slips and germinated to form tetrasporophytes which underwent meiosis to produce tetraspores. After release, the tetraspores attached to cover slips and germinated to produce male and female gametophytes, thereby completing the life cycle. STAINS. Attached spores, germinated spores, and plants on plastic cover slips were stained with the following: 0.5% alcian blue HCI pH 0.5, 30 min, DIH20 rinse, for sulfated polysaccharides; 0.5% alcian yellow HCI pH 2.5, 30 min, DIH20 rinse, for carboxylated polysaccharides McCully et al. 1980); 0.3% alcian blue pH 1.0 in 0.9 M MgCl2, 30 min, DIH20 rinse, for sulfated polysaccharides; 0.3% alcian blue pH 2.5 in 3% acetic acid, 30 min, DIH20 rinse, for sulfated and carboxylated polysaccharides ); 0.5% toluidine blue 0 pH 1.0, 1 min, DIH20 rinse, for sulfated polysaccharides; and 0.1 % Heath's neutral red, Heath's methylene blue, and Heath's toluidine blue, 5 min, for sulfated polysaccharides (Heath 1961 ). PAS (periodic acid-Schiff) stained for neutral polysaccharides .

Collection of Spores
Coomassie blue (0.1 %) G-250, 5 min, DIH20 rinse, ) and 0.1 % fast green, 1 min, DIH20 rinse, stained for proteins (Klein and Klein 1970), and potassium iodine, IKI, 5-1 o min stained for starch (Klein and Klein 1970). Blue fluorescence in algae cultured 24 h in 0.0025% Biofluor indicated cellulose, as did a blue stain resulting from placing algae in IKI 30 min, followed by 1 drop 65% H2S04 between the cover slip and microscope slide (Klein and Klein 1970). from developing holdfasts. This concentration was used because it was high enough to detach branches but low enough to leave holdfast remnants behind. Holdfast remnants resulting from this treatment were stained with fast green and alcian blue as described above.
ENZYMES. Spores and germinated spores attached to plastic cover slips were incubated in a 1 .0 mg/ml enzyme and DIH20 or autoclaved seawater solution at 37° C for 1 h, following the methods of . Enzymes tested were protease (0.7 -1.0 units/mg), trypsin (10,000 units/mg), pepsin (3,200 -4, INHIBITORS. To determine their effects on spore adhesion and detachment, cycloheximide (a protein synthesis inhibitor), tunicamycin (a glycoprotein synthesis inhibitor), sodium molybdate (a polysaccharide sulfation inhibitor), the a-D-mannose/a-D-glucose specific lectin Con A, sulfuric acid, deionized water, and 0.01 % sodium azide (the latter three of which are lethal to~ parvula) were added to spores. Because cycloheximide and tunicamycin are insoluble in seawater, they were first dissolved in 100 µI DMSO. Molybdate forms a precipitate in seawater, so molybdate assays were conducted in 1 ml GP2/100 ml DIH20. Inhibitors were added to either 1) free floating spores to assess inhibition of attachment, or to 2) spores attached to plastic cover slips to assess detachment of spores. Spores were placed in 1 O µg/ml to 20 µg/ml to minimize spore death. Spore viability after exposure to the above agents and in a control group was determined by assessing spores for color change from red to green or white, and using a dye exclusion assay by staining dead tissue with 0.5% trypan blue ) dissolved in seawater so that the trypan blue solution would not be lethal to the spores.

Anatomy of Spore Adhesion
Tetrads of tetraspores were released as a unit and did not separate upon

FITC-LECTINS
To test the hypothesis that the mucilage of Q.. parvula spores and holdfasts contains a variety of sugar haptens, FITC-lectins were used as probes.
Following incubation in FITC-lectins, the mucilage of .Q... parvula spores and holdfasts fluoresced with 5 of the 8 lectins tested (Table 1 ). Because fluorescence occurred with lectins of differing specificities, I conclude that there is a variety of sugar haptens in the mucilage of the spores and holdfasts.
Three of the lectins tested resulted in little to no fluorescence; indicating that their specific sugars are not present, are present in minimal quantities, or were inaccessible in the spore mucilage. The lectins that did not cause fluorescence are a-L-fucose specific UEA 1, (Fig. 7), and the (N-acetyl-~(1-4)-D-glucosamine)2 or N-acetylglucosamine specific lectins WGA, (Fig. 8) and Phytolacca amerjcana mitogen (PWM). (Fig. 9).
In the mucilage of .Q.. paryula a-D-mannose and a-D-glucose were abundant The a-D-mannose and a-D-glucose specific lectin Con A produced green fluorescence in the mucilage around spores, sporelings, around the perimeters of holdfasts, and on branches. The mucilage of tetrads did not fluoresce before release ( Fig. 1 O), but began to fluoresce just after release (Fig. 11 ). Mucilage of attached spores fluoresces (Fig. 12), but not in the control, (Fig. 13). In Fig. 14, also a control, the rhizoids have appeared. A coverslip was inverted to reveal the bases of two developing holdfasts; fluorescent, adhesive mucilage of the rhizoids is visible in the holdfast on the right (Fig. 15). Fluorescent mucilage of a sporeling is illustrated in Fig. 16, and in Fig. 17 Therefore, sugars in this group appear to be well represented in the mucilage of .C... parvula spores.
Scale bars = 50 µm. Fig. 10 -tetrads (T) before release.from tetrasporophytic branch (8)  The results of histochemical staining of extracellular mucilage, spores, and rhizoids of .C... parvula.are summarized in Table 2. Staining of the rhizoids was consistent with that of the spores but different than that of the mucilage. One major difference was that the rhizoids and spores stained for protein with fast green (Fig. 23) and coomassie blue (Fig. 24) whereas the mucilage did not. In addition, the polysaccharide composition of the mucilage differed from that of the rhizoids and spores. Rhizoids and spores stained for neutral polysaccharides ( Fig. 25) with PAS but did not stain for carboxylated polysaccharides.with alcian yellow. Mucilage did not stain for neutral polysaccharides but did stain for carboxylated polysaccharides (Fig. 26). sporelings soaked in 7% HCI for 24 h detached intact from the substrate or broke at the branch-holdfast juncture. Holdfast remnants had fast green staining proteinaceous attachment points which appear to anchor the rhizoid and holdfast cells to the substrate. Protein anchors can be seen at the sites of rhizoid attachment (Fig. 37). The mucilaginous portion of the holdfast remnants stained for sulfated polysaccharides with toluidine blue (Fig. 38), or with alcian blue HCI pH 1.0.  •

Maintenance of Spore Adhesion
Enzymes were used to determine which classes of molecules play a role in maintaining adhesion. While exposure to all of the enzymes tested resulted in at least some spore detachment, the quantity and pattern of detachment varied among enzymes (Table 3). Spores detached at the mucilage/substrate interface or at the spore/mucilage interface to· teave behind a spore socket, which appeared to be an adhesive disc of mucilage. There were no differences in detachment observed between tetraspores and carpospores.
Control groups of spores in seawater or DIH20 at pH 4.5, 5.0, 5.5, 7.3 or 8.0 had only 0-10% detachment following 24 h of 37° C incubation or shaking, or with incubation followed by shaking. Control groups of spores did not detach when touched with a dissecting needle.
The cell walls and mucilage of spores and sporelings were disrupted and destroyed after exposure to cellulase. Spores incubated in cellulase detached completely without being shaken and left no remnants of holdfasts on the cover slips. Cells of attached sporelings separated from each other.
Spores detached at the spore-mucilage interface following exposure to protease, trypsin, pepsin, a-amylase and polygalacturonase. The detachment pattern varied among these enzymes. With the proteolytic enzymes, adhesive discs which stained for sulfated polysaccharides with alcian or toluidine blue were left behind (Fig. 39). These discs and any remaining attached spores or sporelings detached after 24 h of shaking.
With a-amylase, adhesive discs were left on the cover slips when spores detached (Fig. 40). Sporelings often detached at the holdfast-branch juncture, leaving behind the developing holdfast. After shaking, only faint traces of spore mucilage or sporeling holdfasts remained and stained for sulfated polysaccharides with alcian blue. Branches of sporelings broke off of holdfasts and spores detached to leave behind their adhesive discs, or spore sockets, after polygalacturonase exposure. However, fewer than 25% of the spores detached and the spore sockets remained attached after shaking.
Of the enzymes which resulted in detachment at the mucilage-coverslip interface, J}-galactosidase was the most effective. All spores and sporelings detached after incubation and shaking. Spores exposed to hyaluronidase at pH 8.0 had only limited detachment. Branches broke off sporelings and left behind the holdfasts, and spores detached at the spore-mucilage interface.
The remaining spore sockets stained with alcian blue. At pH 5.5, they detached upon touch at the mucilage -substrate interface after exposure but before shaking. As with hyaluronidase, a decrease in the pH of mannosidase from 8.0 to 4.5 resulted in increased detachment of spores and sporelings.
The algae detached at the mucilage-cover slip interface and did not detach unless touched.
Only 10-25% of the spores detached when exposed to 1.0 or 8.5 mg/ml sulfatase at pH 8.0. At pH 5.0, detachment did not increase to 50-75% until the concentration was increased to 8.5 mg/ml. Even so, the spores did not detach until after being shaken for 24 h and not unless they were touched. Spores exposed to sulfatase at either pH detached at the mucilage-cover slip interface and not at the spore-mucilage interface (Fig. 41 ).

Detachment of Tetraspores
Attached tetraspores did not detach in the presence of the biosynthesis inhibitors cycloheximide, tunicamycin, or sodium molybdate (N = 15), data not shown. Nor did tetraspores detach after exposure to Con A. The ranges of concentrations for tetraspore detachment assays were the same as those used in the tetraspore adhesion inhibition assays in the next unit.. Therefore, holdfasts were more resistant to detachment than tetraspores were.

Tetraspores soaked for 24 h in H2S04 or
Tetraspores remained attached in seawater from pH 4.5 to pH 7.5-8.0.
Tetraspores killed by soaking in DIH20 for 24 h did not detach, (N = 30), data not shown. Tetraspores turned white but remained attached even after submersion in DIH20 for 2 months. Tetraspores soaked in 0.01 % sodium azide for 24 h were dead but did not detach. Therefore, tetraspores did not need to be living to maintain adhesion, although only viable tetraspores could initiate adhesion. Tetraspores did not attach after 24 h exposures to 0.01 % sodium azide in seawater. This is evidence that tetraspores must be viable to attach. The acids H2S04 or HCI (pH 0.8, 1.4, and1 .6) are lethal to tetraspores are corrosive to the mucilage so treatment with acid did not differentiate between mucilage destruction and cell death. What little attachment that did occur probably took place before the acid could contact and damage the tetraspores, which were added to the seawater before the acid was so that parent plants would not be destroyed by the acid.

Inhibition of Tetraspore Adhesion
Tetraspore adhesion was inhibited following exposure to cycloheximide ( Fig. 42), tunicamycin (Fig. 43), Con A (Fig. 44), and sodium molybdate ( precipitates in seawater, the assays were conducted in 1 ml GP2/100 ml DIH20, in which the tetraspores remained viable for 24 h, the length of the assay. Although tetraspore adhesion was reduced from an average of 50 -60% in seawater to 45 -50% in 1 ml GP2/100 ml DIH20. spore adhesion averaged 26.0% less in 0.05 M sodium molybdate, the lowest concentration tested. Because cycloheximide and tunicamycin are insoluble in seawater, they were first dissolved in dimethyl sulfoxide (DMSO) to make a stock solution of 1 mg inhibitor/ml DMSO. As a control, 100 µI DMSO per ml seawater was administered to free floating tetraspores. It did not interfere with attachment interfere with attachment solely by their physical presence.
To determine if the doses of cycloheximide, tunicamycin, Con A, and sodium molybdate that were administered in the adhesion assays were lethal to tetraspores, 0.5% trypan blue stain was used. Tetraspores killed with 0.01 % sodium azide or DIH20 stained with 0.5% trypan blue, whereas tetraspores that were exposed to the inhibitors did not stain with 0.5% trypan blue at the concentrations used in the adhesion assay. Nor did exposed tetraspores change color from red to green, an indication of viability. These compounds were therefore not lethal to tetraspores at the tested concentrations.

DISCUSSION
Spore attachment in C. parvula is mediated in a number of ways. First, the spores are surrounded by extracellular mucilage. Upon attachment, this mucilage forms a circular attachment pad with sides that slope down from the spore to the perimeter of the pad. Second, cellular rhizoids with their own mucilage are formed upon germination and provide secondary anchorage.
These rhizoids will undergo cell division, forming the holdfast. Third, although the mucilage is smooth and uniform in appearance, it actually has a complex composition. Adhesion involves several classes of molecules which may interact and facilitate adhesion. These are sulfated polysaccharides, proteins, and glycoproteins. The sugar moieties cx-D-mannose or cx-D-glucose appear to be necessary for adhesion. Fourth, the spores must be living in order to attach. Therefore, adhesion is based on more than one characteristic of the spores.

Spore Adhesion -Anatomy
The mucilage of .Q... parvula spores is smooth and sheetlike; it forms the basis of initial attachment. The emergence of one cellular rhizoid per spore upon germination provides further anchorage. While the sporeling holdfast develops from rhizoidal cell division, upright branches form from non rhizoidal cells. This differs from the sporeling ontogeny of Chondrus crjspus, in which the spore undergoes division and forms a multilayered discoid sporeling before branches are formed .
The edge of the mucilage appears to stick tightly to the substrate upon examination with SEM or light microscopy. It may be that the edge of the mucilage and its interaction with the cover slip are important in maintaining adhesion, possibly by a mechanism similar to that of a suction cup.
Substrate differences can influence spore adhesion in red algae. Boney (1978) found that spores of Porphyra schjzophylla attached more slowly to negatively charged plastic cover slips than ·to uncharged cotton strings.
Because spores of .Q... paryula can attach to plastic that is probably neutrally charged and hydrophobic, and to glass that is probably negatively charged and hydrophilic, to rocks, and because this alga can be epiphytic, .Q... parvula can attach to surfaces with a variety of chemical characteristics.

Tetrad Adhesion and Coalescence
In culture, the four spores composing .C.... paryula tetrads were released together and did not separate before germination. This observation supports those of Steele et al. (1986); Each spore within the tetrad germinated to produce one rhizoid. The cells derived from the original four spores coalesced to form a germling gametophyte. Sporeling coalescence occurs in Gracilaria ~rrucosa, Gjgartjna stellata, and Chondrus crjspus . In parasitic marine red algae, genetically different vegetative filaments can fuse into pseudoparenchyma, resulting in different reproductive stages on the same thallus . While the coalescence of Q.. parvula sporelings originates from non-separation of the four spores of the tetrad, .C.... crjspus coalescence is a result of a high density of spores or sporelings growing together. As ~ crjspus coalesced sporelings are enclosed by a common cuticle , coalesced sporelings of .Q... parvula are surrounded by common extracellular mucilage. In ~ crjspus. juvenile tetrasporophytes coalesce more than sporeling gametophytes do . .Q... paryula carpospores are released singly and do not coalesce after germination into juvenile tetrasporophytes. Coalescence of gametophytic sporelings of .Q... g,aryula originates from non-separated tetrads and occurs frequently, if not always in culture. However, wave action in the intertidal.zone probably separates the spores of the tetrad before they attach.
If the spores of the tetrad do not separate in the intertidal zone, then coalescence in ~ parvula may confer advantages to sporelings as they establish themselves in intertidal marine habitats. One possibility is that an immediately larger sporeling size may contribute to faster growth. Adjacent, coalesced sporelings of .Q... crispus grow faster with more upright, branched fronds than non-coalesced sporelings do and also have secondary pit connections . The mucilaginous spore coating in .C.. crjspus has been thought to increase spore cohesiveness and longevity (Maggs and Cheney 1990). In the sand scoured habitat of a yerrucosa. Jones   (Table 1 ).
Because lectins have a greater affinity for sugar haptens of glycoproteins than for unincorporated sugars (von Sengbusch et al. 1982), it is likely that the FITC-lectins bound with sugar moieties of glycoproteins in the spore mucilage.
Con A and LCA are specific for a-0-mannose, and to a lesser extent, a-0glucose (von Sengbusch 1982, Goldstein and Poretz 1986. Spore mucilage and the mucilage around the periphery of developing holdfasts fluoresced with both Con A and LCA. Compared to Con A, LCA has a weaker affinity for polysaccharides and a greater affinity for glycoproteins (Goldstein and Poretz 1986), suggesting that glycoproteins with a-0mannose or a-0-glucose haptens are adhesive components of the spore mucilage in .C... parvula. In other organisms, a-0-mannose and a-0-glucose have been found in outer cell walls and are involved in cell adhesion (Herth et al. 1982;Watson and Waaland 1983;Kim and Fritz 1993b;. Con A bound to cuticle, cell walls and intercellular mucilage of Polysiphonja (Oiannelidis and Kristen 1988). In Antitharnnion nipponjcurn, Con A can inhibit gametic binding (Kim and Fritz 1993b). Con A demonstrated that a-0-mannose and/or a-0-glucose are constituents of the red algal hormone rhodomorphin, found in Grjffithsja .D.acjfic.a. (Watson and Waaland 1983). The spikes of Acanthosphaera zacharjasi_ (Chlorococcales) are bundles of cellulosic microfibrils which are extensions of the outer cell wall layer and bind with FITC-Con A at their bases (Herth et al. 1982). In Djctyostelium amoebae, Con A labelled membrane glycoproteins at the site of initial cell settling and spreading and on the edges or beneath the ultrathin lamellae  y;c;a yjllosa lectin and SBA are two lectins from the Nacetylgalactosamine/galactose group which gave positive results with ~ garyula mucilage. It is of interest that the lectin from ~ villosa is also specific for glycoproteins with one or two terminal N-acetyl-a-galactosamine groups linked to serine or threonine (Goldstein and Peretz 1986) and SBA has specificity for oligosaccharide molecules with terminal a or b linked Nacetylgalactosamine ). This is further evidence that glycoproteins are found in the mucilage of C... paryula spores, although polysaccharides are present.
FITC-lectins revealed no differences in fluorescence among the mucilage of spores, holdfasts, and branches, which can also adhere to plastic or glass This differed from Antithamnion nipponicum, in which Kim and Fritz (1993a) used FITC-lectins and lectin gold labelling to find that N-acetyl-glucosamine, P-D-galactose, and a-L-fucose in the spermatial cell walls but not in the vegetative cell walls. Herth et al. (1982) found that cell walls and spike bases of Acanthosphaera zachariasj bound with the lectins Con A, RCa-1, PNA, to a lesser extent with WGA, and not with UEA, while the distal ends of the cellulosic spikes did not bind with any of the lectins. Spatial differences in sugars which bound to FITC-lectins were minimal in Q.. paryula, although PNA Yielded scattered fluorescence near the periphery of the spore mucilage to indicate patches of a-D-galactose or galactosyl (~ -1,3) Nacetylgalactosamine. Fluorescence with all other FITC-lectins that were tested indicated that the variety of sugars present in the mucilage of .Q,_ parvula were otherwise distributed evenly within the mucilage.
Q... paryula did not bind with the n-acetyl glucosamine specific lectin WGA, the (N-acetyl-~-(1-4)-D-glucosamine)2 specific lectin from Phytolacca ,americana, or the a-L-fucose specific UEA. ' In another marine red alga, for:phyra perforata, extracted glycoproteins did not bind with biotinylated WGA but did bind with UEA (Kaska et al.1988), and glycoproteins differed between morphologically distinct regions of the thallus. While this work did not analyze spore mucilage, glycoproteins (18-68 kD) from the holdfast bound with Con A and an18 kD glycoprotein bound to n-acetyl galactosamine specific SBA. If the glycoproteins were adhesive, then a-D-mannose, a-D-glucose and nacetyl galactosamine may be adhesive sugar moieties in the holdfast of .P...

perforata.
The mucilage of .Q,_ parvula has a uniform appearance. However, it binds with lectins of different specificities, indicating a complex composition. This is in accordance with the findings of von Sengbusch and Muller (1983), who used lectin binding to demonstrate that many algal species with visually uniform sheaths or mucilage were composed of molecular mosaics and that variations were species-specific. Furthermore, lectin binding patterns varied between developmental states in some algae. von Sengbusch and Muller ( 1 983) postulate that such variation is useful in intracellular communication and as a means for a cell to recognize "self and non self". However, the cytoplasm of the spore does not contact the substrate upon initial adhesion, whereas protein anchors of rhizoid and holdfast cells do contact the substrate. Spore mucilage and rhizoids are temporally and spatially separated, as the spore mucilage is present before the rhizoids are formed and the rhizoids have their own mucilage.
It may be that small amounts of proteins and glycoproteins which were not detectable with fast green or coomassie blue acted within a matrix of polysaccharides to facilitate adhesion. The extracellular mucilage of forpbyrjdium cruentum consists largely of polysaccharide, but 1-2 % of the mucilage is protein covalently linked to the polysaccharide (Heaney-Kieras 1977). In three red algal species,  found that alcian blue stained the mucilage while coomassie blue did not. It is possible that protein-polysaccharide bonds or heavily glycosylated proteins obscured the binding sites of the protein stains Evans 1981, Oiannelidis and. If proteins are indeed present in the mucilage, then rhizoid and mucilage adhesive composition may be more similar than suggested by histochemical stains. Because rhizoids and not mucilage stained with fast green and coomassie blue, proteins may be present in greater quantity in the rhizoids than in the mucilage. Another possibility is that proteins are not present in the mucilage but appear with the rhizoids upon germination when they participate in adhesion. · · With PAS, the mucilage did not stain but the rhizoids did stain. It may be that the sulfated mucilage did not stain with PAS because sulfation blocks the relevant hydroxyl groups necessary for periodate oxidation . Positive staining of the rhizoid with Heath's stains and with some of the alcian blue stains is evidence that the rhizoids do have sulfated polysaccharides. PAS staining was not blocked in the rhizoids.
Sulfated polysaccharides of the mucilage would probably not have blocked PAS staining. Therefore, neutral polysaccharides would not be found in the mucilage. This is similar to the findings of , in which the mucilage of Hypnea muscjformjs did not stain with PAS but the cell walls did stain.
Alcian blue at pH 0.5, 1.0, or 2.5 stained spores but was also effective at detaching them. While low pH will detach spores, it may be that alcian blue detaches spores by binding with adhesive sulfated polysaccharides. Alcian blue also stained holdfast remnants and spore sockets following exposure to a-amylase or protease. Positive alcian blue stain, spore detachment with sulfatase, and inhibition of sulfation and spore attachment with molybdate all implicate sulfated polysaccharides as adhesive molecules or as molecules that form connections with adhesive molecules that are necessary for adhesion Proteins and polysaccharides appear to play a role in adhesion of ~ g,arvula, but in Ceramium polysaccharides and not proteins are thought to be adhesive . The mucilage of unreleased ,eeramjum spores stained for polysaccharides and proteins; after spore release, polysaccharides but not proteins were detected in the mucilage.  concluded that the lack of staining for proteins after spore release may be caused by 1) dilution of the proteins in seawater, 2) unavailability of the now cross-linked proteins, 3) artifacts of staining, or 4) absence of proteins. Agents that disrupt protein bonds did not detach Ceramium spores or did so to only a limited extent, whereas disruption of polysaccharides led to detachment of spores. Perhaps proteins of Ceramjum spores play a non-adhesive role or are involved in initiating adhesion but not in maintenance of adhesion. · Diffe·rential staining suggesting variation in adhesives has also been found within the stalk of the euglenoid flagellate Colacium (Willey and Giancarlo 1986) and in the green alga ~ mutabilis, where staining differed between the zygote and rhizoids .
The fibrillar component of algal cell walls is generally cellulose but can be xylan or mannan . The composition of reproductive and vegetative cell walls sometimes differs, as in Antjthamnion .Di.pponicum. in which the vegetative cells but not the spermatia were labelled with Calcofluor white (Kim and Fritz 1993a). While ~ parvula spore, rhizoid, and branch cell walls stain for cellulose with it is as yet unknown whether spermatia contain cellulose. Such information would be useful in determining the similarities between gametes, spores, and other reproductive structures in C.. paryula.

Enzymatic Analysis of Adhesion
Enzyme, FITC-lectin, and stain data suggest that the mucilage of .C...
Qarvula is composed of a variety of molecules and that adhesion is mediated by more than one class of molecule. A variety of enzymes were effective in detaching spores (Table 3).
Proteins are instrumental in the maintenance of spore adhesion and their role in adhesion may include interactions with polysaccharides. Following exposure to protease or trypsin, spores detached from plastic cover slips only after 24 h of shaking. The adhesive was weakened but did not fail until an outside force was applied. This implies that spores have a means of attachment in addition to proteins. After shaking, spore sockets which stained for sulfated polysaccharides with alcian blue were left behind. Proteins may connect the spore with the mucilage or act as bridges between adhesive molecules contacting the substrate and the spore.
Proteins and polysaccharides play adhesive roles in other species of algae. The Ceramjum adhesive often fails at the cell-adhesive interface . When the crustose coralline red alga, f,twmatoljthon !aevjgatum, was removed from glass slides (Walker and Moss   1 g84) an inner circle which stained darkly with PAS was left behind. Loosely attached 3.5 h old Eucus zygotes detached from slides when shaken and left behind sticky rings which may contain the known mucilage components, alginate and fucan (Vreeland et al. 1993). In Colacium. cells were digested by pronase and trypsin but adhesive discs and stalks were left intact and stained with alcian blue (Willey and Giancarlo 1986 (Stephenson and Stephenson 1972), and the sulfatase used in this work was purified from the limpet Patella yulgata. Perhaps grazing limpets dissolve or digest red algae with sulfatase and red algae have evolved a resistance to sulfatase.
Hyaluronidase cleaves sulfated polysaccharide chains (Morse and Morse 1991) and binds with glycoproteins to render ruthenium red staining impossible (Trelease 1980). It was effective in detaching spores of ~ parvula, possibly by disrupting sulfated polysaccharides or by binding with adhesive 91 , ! I I proteins glycosylated with sulfated polysaccharide chains. Zygotes, but not rhizoids, of 1Jbra mutabilis detached in hyaluronidase .
Detachment of spores in mannosidase, spore mucilage fluoresced with a-Dmannose/a-0-glucose specific FITC-Con A, and Con A inhibited attachment of spores. This is evidence that a-0-mannose is present in the mucilage and necessary for adhesion and adhesion maintenance. Kim and Fritz (1993b) have found that a-D-methyl-mannose is necessary for sperm-trichogyne binding in Antithamnion nipponicum. Although spores detached at the mucilage -substrate interface in a-amylase,.rhizoids and spores but not spore mucilage stained with IKI for starch. Starch appears to play a role in maintaining spore adhesion in an as yet unknown manner. Zoospores of the green alga Enteromorpha had reduced adhesion in the presence of aamylase . In cellulase, spores detached completely without being shaken and left no remainders on the cover slips. It is likely that cellulase degraded the cellulose in the cell walls, facilitating detachment. In Gracjlarja lemanaeformjs, cellulase produced microcracks in the external thallus wall and collapsed the internal cell walls (San Martin et al. 1988). A mixture of enzymes from digestive tracts of the sea snail Littorina littorea and cellulase was used to degrade the thallus sheath and cell walls of Porphyra leucosticta .

Spore Adhesion using Cycloheximide
Tetraspore adhesion in .C.. parvula was inhibited in the presence of 0.25 µI cycloheximide per ml seawater (Fig. 42) which inhibits protein synthesis by inhibiting translation on cytoplasmic ribosomes .thereby limiting the supply of new proteins. Proteins play a role in adhesion within 24 h of tetraspore release, the length of the incubation in cycloheximde. Adhesive proteins appear to be synthesized de novo after tetraspore release, which is when the cycloheximide contacted them to inhibit protein synthesis. This differs from Djctyosteljum, where gene expression, early synthesis and storage of proteins which coat tetraspores occurs well in advance of use of the proteins .
If adhesive proteins had been present upon tetraspore release, adhesion probably would still have occurred because the cycloheximide would not have disrupted extant proteins. Another possibility is that cycloheximide would not inhibit adhesion if an adhesive protein was synthesized in advance and ubiquinated upon or prior to spore adhesion. Ubiquitin is a regulatory protein that complexes with other cellular proteins and is found in all eukaryotic cells.
One ubiquitinated protein became more abundant during gamete induction in Cblamydomonas in the presence of cycloheximide .
The vital stains neutral red and cresyl blue can enter red algal spores and leave the mucilage unaffected, which shows that the mucilage does not prevent substances from diffusing in and out of red algal spores (Boney 1975).
This suggests that cycloheximide can diffuse into the cytoplasm of ~ parvula tetraspores, as does the resultant reduced adhesion in the presence of cycloheximide.
Protein synthesis was also shown to play a role in the settlement of Haljotjs rutescens larvae. Settlement occurred in the presence of cycloheximide but did not occur in the presence of two other protein synthesis inhibitors, emetine and anisomycin (Fenteany and Morse 1993), which by virtue of their lipophilic structure may have been better able to diffuse into larval tissues.

Spore adhesion using Tunicamycin
Adhesion decreased in newly released, free floating tetraspores when glycoprotein synthesis was inhibited with 0.5 µg tunicamycin per ml seawater ( Fig. 43). Glycoproteins play a role in adhesion within 24 h of spore release, the length of the incubation in tunicamycin. Glycoproteins must be synthesized de novo after tetraspore release for adhesion to occur, which is when the tunicamycin was in contact with the tetraspores. If the glycoproteins had been synthesized before tunicamycin contact, the tetraspores probably would have adhered because tunicamycin would not have disrupted extant glycoproteins.
Tunicamycin is a competitive inhibitor of UDP-N-acetylglucosamine-1-Ptransferase mediated asparagine-linked glycosylation (Dutcher and Gibbons 1 988). Asparagine linked glycosylation appears to be necessary for adhesion in .C... garvula tetraspores, and it is perhaps this step in glycoprotein synthesis which occurs after tetraspore release. Proteins to be glycosylated must be present and able to be glycosylated after tetraspore release, but they would not necessarily need to be synthesized after tetraspore release, although cyctoheximide inhibition of protein synthesis shows that this is probably the case. The desmid Closterium has a glycoprotein which is involved in and synthesized prior to gamete release , as shown by the addition of inhibitors of metabolism before release which then blocked subsequent gamete release . .C... parvula appears to differ from Closterjum in synthesizing its adhesive glycoproteins after tetraspore release.
Although the rhizoids and not the mucilage stained for proteins, spores can attach with their mucilage before germination and appearance of the rhizoids.
Cycloheximide and tunicamycin interfered with attachment in tetraspores that had not yet formed rhizoids, therefore proteins and glycoproteins play an adhesive role in the mucilage. If rhizoidal proteins were the sole adhesive proteins, then cycloheximide and tunicamycin would have blocked adhesion only when the rhizoids appeared.

Spore Adhesion using Sodium Molybdate
Tetraspore adhesion in .Q.. paryula was inhibited by the presence of sodium molybdate (Fig. 44). This is evidence that sulfation of polysaccharides is necessary within 24 h of tetraspore release for tetraspores to adhere.
Tetraspores do not appear to have a sufficient store of sulfated Polysaccharides before release, or they would be able to adhere in the presence of molybdate. Tetraspores given molybdate in 1 ml GP2 per ml deionized water were red and did not stain with trypan blue, implying viability.
Although molybdate did not cause tetraspore color change or spore death, it appears that it did enter the cytoplasm because tetraspore adhesion was reduced in its presence.

Spore Adhesion using Con A
In the presence of Con A, attachment of tetraspores of ~ parvula was reduced (Fig. 45). As this lectin is a-0-mannose or a-0-glucose specific; it is possible that these sugars incorporated into glycoproteins are adhesive molecules. However, one must not overlook the possibility that Con A inhibited adhesion by altering a cellular function that was necessary for adhesion, as opposed to interfering with the actual adhesive. The inhibition of tetraspore adhesion by both Con A and tunicamycin points to glycoproteins as adhesive molecules with a-0-mannose and/or a-0-glucose sugar haptens.
The sugars may contact the substrate and anchor the adhesive glycoproteins.
In other organisms, a-0-mannose and/or a-0-glucose have been found to be necessary in reproduction and morphogenesis (Kirchman 1982;Kim and Fritz 1993b;. Antjthamnion nipponjcum bases sperm-trichogyne recognition on an interaction between surface carbohydrates and receptors on the gametes. This recognition was inhibited by Con A, which blocked spermatial binding to trichogynes, and by the corresponding carbohydrate, a-0-methyl mannose, which blocked trichogyne receptors (Kim and Fritz 1993b). In the dinoflagellate Alexandrjum ~enella.. Con A and tunicamycin inhibited sexual attachment in gamete pairs and the addition of a-0-mannose or a-0-glucose overcame inhibition by Con A to indicate these sugars as components of cellular agglutinins . Con A inhibited larval settlement and subsequent metamorphosis in the marine polychaete Janua (Dexjospjra) brasiliensjs (Kirchman 1982). Macroconidial adhesion of the plant pathogenic fungus, Nectrja baematococca, was blocked by Con A .
such inhibition is not limited to Con A. Fusion between male and female gametes of the brown alga, Ectocarpus siliculosus, was inhibited when WGA, a lectin from Trjticum yulgaris. was added to female gametes or when the complementary sugar, N-acetylglucosamine was added to male gametes .

Spore Adhesion -Viability
Tetraspores of .Q.. parvula must be alive in order to attach. Tetraspores killed with 0.01 % sodium azide did not attach. Sodium azide interferes with mitochondria but leaves the rest of the tetraspore relatively intact and does not disrupt the mucilage. Therefore, we know that tetraspore adhesion is an active process and does not rely solely on inherent stickiness of the mucilage.
In a multicellular organism, most cells are either attached to other cells within the organism or to a cellular or noncellular substrate ). Detection of substrata by attaching organisms is an area that deserves further exploration (Wigglesworth- . Spores may derive germination cues from the substrate. An as yet unknown molecular messenger may translate information about the substrate into adhesive synthesis and subsequent spore germination. Polarity of rhizome emergence would be influenced by such a signal, as the rhizoids always germinate downward between the spore and the substrate. .Q... parvula spores sink through water to attach to a substrate; developmental signals to germinate may be cessation of sinking or a tactile response to contact with the substrate.
Spores will attach to and germinate on tetrasporophytes and gametophytes. This implies that there is no antagonism between spore adhesives and the mucilage surrounding the branches of the adult plants.
Adjacent cells within a .Q... paryula plant do not germinate, so they do not derive germination cues from adjacent cells. When C.. parvula branches come into contact with a substrate, they will produce rhizoids to form a holdfast.
Therefore, the branch can perceive a cue from the substrate.

Spore Detachment
Tetraspores did not detach in the presence .of cycloheximide, tunicamycin, molybdate, or Con A. Therefore, continued synthesis of proteins, glycoproteins, and sulfated polysaccharides are not necessary for adhesion maintainence. Synthesis inhibitors could not disrupt previously synthesized molecules that were already in place as adhesives. Alpha-D-mannose and/or a-D-glucose would not be available for binding with Con A if they were adhering to the substrate; therefore Con A was not effective in detaching tetraspores. The adhesive disc did not detach without physical disruption by scraping or chemical disruption with the acids H2S04 and HCI or with enzyme exposure. Tetraspore viability is also not necessary for adhesion maintainence, as attached tetraspores killed with 0.01 % sodium azide or by immersion for 2 months in DIH20 did not detach.
When treated with enzymes, spores sometimes detached and left behind the adhesive disc or remnants thereof. This implies that once the spore has adhered and the adhesive is firmly attached to the substrate, the adhesive may be inert and independent of the spore. Enteromorpha jntestjnaljs zoospores which had settled for more than 5-1 O min did not contain •settlement vesicles" whereas swimming, (pre-settlement) spores did have these vesicles . When inhibitors of trypsin were added to spores which were already in the presence of trypsin, no increase in the number of settled spores was seen when the inhibitors were added later than 5-10 min.after settlement. This suggests that adhesive synthesis occurs within minutes of settlement. Synthesis of sulfated polysaccharides is necessary for adhesion maintenance  in Prasiola stjpjtata (Chlorophyta), in which the presence of molybdate led to cell detachment but was partially reduced by the addition of sulfate. It appears that in ~ parvula.
synthesis of the adhesive occurs after tetraspore release and diminishes after adhesion, as synthesis inhibitors did not cause detachment.
Spore sockets are formed when the spore detaches but leaves behind its adhesive material. Spore sockets can occur naturally when spores detach with no chemical treatment (Dworetsky 1983). Spore sockets may also result When the adhesive fails at the spore-mucilage interface. The adhesivesubstrate interface may be maintained by interatomic forces . Such forces would explain why adhesive discs, or spore sockets, can remain on the substrate after the spore is detached. Because the adhesive remains attached to the substrate, the adhesive is probably independent of the spore. This may allow the spore to remain attached to the substrate when it is no longer alive. One possibility is that remnants of the adhesive provide an attachment point for future spores of ~. parvula.

Summary
Inhibition of protein synthesis with cycloheximide, of glycoprotein synthesis with tunicamycin, of polysaccharide sulfation with molybdate, and binding of con A to a-D-mannose or a-D-glucose resulted in reduced tetraspore adhesion without cell death. The biosynthesis inhibitors and Con A were added to tetraspores immediately after tetraspore release, therefore adhesion probably relies on synthesis of proteins, glycoproteins, and sulfated polysaccharides and on the availability of a-D-mannose or a-D-glucose after release but before adhesion. If the inhibitors had not blocked adhesion, then adhesive molecules could be synthesized before tetraspore release.
Adhesion of ~ parvula spores requires proteins, glycoproteins, sulfated polysaccharides, and a-0-mannose or a-0-glucose, which may be sugar

Further Investigations
Fucoidan (F2) is a highly sulfated fucan glycoprotein  which must be sulfated for the rhizoids of Eucus embryos to adhere Kropf et al. 1989). F2 sulfation may be necessary for F2 to associate and co-localize in the rhizoid tip with Vn-F, a vitronectin-like glycoprotein ) and this co-localization may be reflected in the changes in the cell surface of Fucus zygotes and rhizoids as detected with FITC-lectins by .  between isomorphic stages of the marine red alga lrjdaea lamjnarjojdes (Luxuro and Santelices 1989). Are the changes between life cycle stages accompanied by corresponding changes in the proteins or glycoproteins present in each life cycle stage of this alga? SDS PAGE electrophoresis and dual staining of the resultant gels with coomassie blue and silver were used to determine whether or not protein or glycoprotein change occurs between life cycle phases in this marine red alga.

Proteins and Glycoprotelns In Algal Life Cycles
There is evidence to suggest that protein and glycoprotein composition may change within the life cycle of an alga. Glycoproteins of Porphyra ~rtorata. a marine red alga, were found to differ in male and female reproductive tissue and in morphologically and functionally distinct regions of the thallus ). Even though Porphyra blades appear to be simple they are morphologically and physiologically complex. Isolates of cells from functionally different areas of the blade regenerated to form different thalli (Polne-Fuller and Gibor 1984). Proteins from cuticles of Chondrus crispus tetrasporophytes were not the same as those from male and female gametophytes . Synthesis of a few specific proteins was found to change during early embryogenesis in Fucus sp., a brown alga (Kropf et al. 1989b). In Fucus serratus, monoclonal antibodies were used to show that glycoproteins on the egg surface are organized by differing composition and size ).  found seasonal fluctuations in the protein levels of Fucus sp., and Pelvetia canaliculta. Seasonal increases in Euchema nudum protein quantity were more closely related to growth or reproductive stages of the alga than to environment . Amino acid composition changes during cyst formation, storage and subsequent germination in the dinoflagellate Scrjppsiella trochoidea (Lirdwitayaprasit et al. 1990). Haemagglutination, possibly caused by lectins (which are proteins or glycoproteins), differs between tetrasporophytic and cystocarpic plants of Chylocladja yertjcillata (Rogers et al. 1980). Therefore, it appears possible that .C.... parvula has temporal and stage-specific protein variation.
Algae may have proteins or glycoproteins that are unique to and involved in spore adhesion or reproductive events. Spores may carry only those proteins or glycoproteins necessary to insure their attachment, germination, and viability. In spermatial vesicles and on the outer spermatangial surface, a glycoprotein with a-D-methyl mannose residues accumulates and is involved in sperm-trichogyne recognition in Antithamnjon nipponicum (Kim and Fritz 1993b). Gametes and zoospores of~ mutabilis were found to have a 51 .4 kDa protein that may represent tubulin (Guliksen et al. 1982). Ubiquitin is a protein found in all eukaryotic cells that conjugates with other proteins to regulate cellular processes such as development and differentiation. The 28 kDa ubiquinated protein level was elevated during gamete induction in Chlamydomonas while other ubiquinated protein levels decreased or remained the same . A glycoprotein induces the release of gametic protoplasts of the Closterjum peracerosum-strjgosumlittorale complex and its activity can be inhibited by metabolism inhibitors applied before the gametic protoplast release stage .
Biochemical changes between cell cycle phases may alter cell surface characteristics, and in unicellular -chlorella yulgaris. adhesion varies at different points throughout the cell cycle, with maximum adhesion in the G2 period of interphase . A proteinaceous adhesive or synthesis of an adhesive may reflect protein change in the cell cycle (Tosteson and Corpe 1975  .

Change in Proteins of Dlctyostellum
Three major proteins compose the extracellular spore coat of Dictyostelium giscoideum. Synthesis of these proteins is temporally and cell type specific, as it occurs in prespore cells shortly after aggregation. Spore coat proteins are stored in pre-spore vesicles during the slug stage, and finally secreted during spore encapsulation .

Cell Differentiation and Gene Expression
Swimming cells differentiate into swarmer cells upon surface contact in the bacterium Vibrio parahaemolyticus. Signals from the substrate must be detected by sensors which then control expression of specific genes ). Using laf:lux reporter gene fusions,  detected luminescence when the cell differentiation genes were activated upon swarmer cell surface contact, demonstrating that differentiation involves activation of specific genes. Bacteria associated with surfaces may have different phenotypes than aqueous bacterial inhabitants because contact with the surface may be instrumental in switching certain genes on or off.
In Porphyra tenera, polysaccharides and proteins differed between the thallus and conchocelis phases. The major amino acid of the conchocelis phase is aspartic acid and in the thallus phase the major amino acids are glycine and alanine   were placed in pre-chilled mortar and pestles filled with liquid nitrogen. After the liquid nitrogen boiled away, the frozen algae was ground to a powder and kept on ice. To each sample, 1.0 ml of protein extraction buffer consisting of 0.121 g Tris, 0.5 ml Triton X-100, 0.5 g SDS and 1.0 ml J}-mercaptoethanol in 100 ml DIH20 (Kropf et al. 1989b), with protease inhibitors ( ANALYSIS. Lowry protein determination (Lowry et al. 1951) yielded protein concentration in µg/µI and was used to calculate sample volumes for electrophoresis. 10% SDS 1.5 mm polyacrylamide1-D gels (Laemmli 1970) were stained with 0.1 % coomassie brilliant blue or dual stained with 0.1 % coomassie and silver (De Moreno et al. 1985) to detect modified proteins (Dzandu et al. 1984). Molecular weights were determined using Sigma molecular weight standards (kOa range) and the gels were dried in a Hoefer were not demonstrated to change between stages of the life cycle. Fig. 1. is a gel stained with coomassie blue and Fig. 2 is a coomassie blue and silver dual stained gel of comparisons between stages.of the life cycle. In Fig. 2, there is a protein of approximately 53 kOa in the tetrasporophytic tissue that appears as a thinner and more pale band in the male and female tissue.
However, this is the only gel that showed this difference in concentration in the 53 kOa protein. SOS-PAGE electrophoresis and subsequent coomassie-silver dual staining of basal portions of free floating tetrasporophytes and male and female gametophytes did not reveal any differences between basal proteins and those from whole floating plants.
Glycoproteins were detected with silver staining in tetrasporophytes and in male and female gametophytes (Fig. 2). Bands on dual stained gels that turned brown are modified proteins, those that stained blue are not modified.

Discussion
One dimensional gel electrophoresis was more effective than isoelectric focusing in this work because t~e protein content of .Q.. parvula is not sufficient to warrant the analysis of a single sample spread over an entire gel, as is required for isoelectric focusing. One dimensional gels were also more effective because samples from different stages of the life cycle could be directly compared within a single gel.
In .Q.. parvula. 35s-methionine labeling was weak, possibly because of the high level of sulfation in the extracellular mucilage. Apparently, the 35 Smethionine was incorporated into the extracellular mucilage. 14C-L-amino acid mixture also resulted in low levels of labelling in ~ paryula. which resulted from most of the label going to the spiritoaquaeous fraction. This is similar to what occurred in the red algae Gracilaria and Ahnfeltia, in which super 14C-galactose and super 14C-sodium bicarbonate labelling resulted in the following distribution of label: 1) spiritoaquaeous fraction, 2) polysaccharides, 3) proteins, and 4) cellulose ). In Rhodella  used 14C-arginine to study synthesis of the protein component of the solubilized mucilage of this unicellular red alga because it is the only amino acid that Rhodella will use as a nitrogen source; Rhodella was grown without inorganic nitrogen. The labelled arginine was detectable in solubilized mucilage after 9-1 O h.
Methionine levels were higher in conchocelis phases of the red alga Porphyra lenera than in thallus phases . Kropf et al. (1989b)  It would be interesting to study the protein composition of the spore and spore mucilage to determine if there are proteins unique to the spore stage of the life cycle. Such proteins could be involved in development or they could be related to spore adhesion, as the spores must attach before they can germinate to establish the next life cycle phase (Dworetsky 1983). However, it is difficult to obtain spores in sufficient numbers for electrophoresis.  used polyacrylamide gel disc electrophoresis to fractionate 14C-L-arginine and 35S04 labelled Rhodella mucilage. The mucilage was recovered by freeze-drying and dissolving 0.2% mucilage in electrophoresis running buffer. The mucilage would not form a true solution so not all of the sample could enter the gel and be electrophoresced . .Q.... paryula spore mucilage may be obtainable by freeze drying if sufficient quantities of spores were available. Electrophoresis of spore mucilage would be instrumental in determining the composition of the mucilage.  stained the 2.5% acrylamide Rhodella mucilage gels with PAS or toluidine blue for polysaccharides, amide black for protein, or assessed 14C-arginine (for protein) and 3Sso4 (for polysaccharides) labelling results by scintillation counting.
Electrophoresis did not reveal any consistent protein or glycoprotein differences between male, female, and tetrasporophytic plants. There were also no protein or glycoprotein differences found between basal and branch proteins of free floating male, female, and tetrasporophytic plants. Because sos-PAGE electrophoresis reveals the 30-50 most common proteins, it could be that there are existing protein differences, but that the proteins differing between life cycle phases are not within the 30-50 most common. The most prevalent proteins in .C... parvula have a molecular weight of approximately 20 kDa, which corresponds with the molecular weight of the phycobiliproteins, phycoerythrin, phycocyanin, and allophycocyanin .
While this work did not reveal any stage-specific proteins, it did provide a Luxuro, C. and B. Santelices. 1989. Additional evidence for ecological differences among isomorphic reproductive phases of lrjdaea p.
Thursby, G. B. and R. L. . Comparison of short-and long-term sexual reproduction tests with the marine red alga Champja parvula.
3. Wash 500 ml and 1 I Erlenmeyer flasks in Alconox, acid wash in1 :5 solution of HCl:DH20, and rinse with DIH20. Alternatively, culture flasks may be washed in RBS 35 and rinsed with DIH20.
4. Add 300 ml seawater to 500 ml flask or 800 ml seawater to 1 I flask.
6. Cap flasks with foam stoppers and insert pipette between flask wall and stopper. Attach aeration tube to pipette at one end, and to aquarium pump at other end.  Immerse algae 1 min and rinse with OIH20.
a. Fast green stains protein green. Immerse algae 1 min or less and rinse with OIH20.
1 o. IKl-H2S04 stains cellulose blue. Immerse algae in IKI for 30 min, invert cover slip onto microscope slide and place a drop of 65% H2S04 under the cover slip to diffuse into the tissues in 1-2 min. Observe quickly without rinsing.
11. Cellulose emits blue fluorescence when stained with Biofluor. Algae was cultured with 0.0025% Biofluor for 24 h and examined with fluorescence microscopy.
3. Divide each solution into 4 aliquots, each in a 25 ml beaker.
4. Place the same volume of DIH20 in 4 beakers, and the same volume of autoclaved seawater in 4 beakers.

5.
Add algae attached to cover slips and cover with Parafilm. 6. Incubate in 37° C oven for 1 h.