144 Use of Fourier Transform Infrared (FT IR) Diffuse Reflectance Spectroscopy and Hamming Distances to Study the Phycocolloid Chemosystematics of the Red Algae (Rhodophta)

Coalescence in seaweeds is known to occur in the laboratory among young and older sporelings and in the field between neighboring conspecific clumps. However, because spores and germlings are difficult to study in the field, it is as yet unknown at which stage of population development coalescence is most important. Since many seaweeds disperse aggregated propagules, often with a sticky mucilagous envelope around the spores, aggregated recruitment and coalescence might be more important at early stages of population establishment than among fully grown, well established clumps. Using recruitment plates maintained during several experimental times in the field, we are evaluating the above idea with mid-intertidal populations of Mazzaella laminarioides. During high fertility seasons, close to 45% of the spores settling within or at close (<1 m) distances of the bed exhibited aggregated recruitment, forming groups of 2 to 150+spores. The probability of aggregated recruitments is a function of dispersal distance and spore density. The number of sporelings produced is a function of spore density and coalescence. Highest after-recruitment mortality (first 15 days) occurs among solitary recruits, followed by sporelings conformed by small number of spores (2–4). Approximately 50% of the spores recruited, isolated or in group, coalesce within these 15 days, gradually forming massive sporelings with increasingly larger basal areas. Thus, after recruitment, sporelings may disappear (die), survive or coalesce. These three alternatives are integrated in a new demographic model for coalescing seaweeds (supported by grant FONDECYT 1020855).     

143 Coalescense During Early Recruitment of Mazzaella Laminarioides

Coalescence in seaweeds is known to occur in the laboratory among young and older sporelings and in the field between neighboring conspecific clumps. However, because spores and germlings are difficult to study in the field, it is as yet unknown at which stage of population development coalescence is most important. Since many seaweeds disperse aggregated propagules, often with a sticky mucilagous envelope around the spores, aggregated recruitment and coalescence might be more important at early stages of population establishment than among fully grown, well established clumps. Using recruitment plates maintained during several experimental times in the field, we are evaluating the above idea with mid‐intertidal populations of Mazzaella laminarioides. During high fertility seasons, close to 45% of the spores settling within or at close (<1 m) distances of the bed exhibited aggregated recruitment, forming groups of 2 to 150+spores. The probability of aggregated recruitments is a function of dispersal distance and spore density. The number of sporelings produced is a function of spore density and coalescence. Highest after‐recruitment mortality (first 15 days) occurs among solitary recruits, followed by sporelings conformed by small number of spores (2–4). Approximately 50% of the spores recruited, isolated or in group, coalesce within these 15 days, gradually forming massive sporelings with increasingly larger basal areas. Thus, after recruitment, sporelings may disappear (die), survive or coalesce. These three alternatives are integrated in a new demographic model for coalescing seaweeds (supported by grant FONDECYT 1020855).     

SIZE INCREMENTS DUE TO INTERINDIVIDUAL FUSIONS: HOW MUCH AND FOR HOW LONG?1

Size increments following interindividual fusions appear as a general benefit for organisms, such as coalescing seaweeds and modular invertebrates, with the capacity to fuse with conspecifics. Using sporelings of the red algae Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira and Mazzaella laminarioides (Bory) Fredericq, we measured the growth patterns of sporelings built with different numbers of spores, and the magnitude and persistence of the size increments gained by fusions. Then we studied three morphological processes that could help explain the observed growth patterns. Results indicate that in these algae, coalescence is followed by immediate increase in total size of the coalesced individual and that the increment is proportional to the number of individuals fusing. However, the size increments in sporelings of both species do not last >60 d. Increasing reductions of marginal meristematic cells and increasing abundance of necrotic cells in sporelings built with increasing numbers of initial spores are partial explanations for the above growth patterns. Since sporelings formed by many spores differentiate erect axes earlier and in larger quantities than sporelings formed by one or only a few spores, differentiation, emergence, and growth of erect axes appear as a more likely explanation for the slow radial growth of the multisporic sporelings. Erect axis differentiation involves significant morphological and physiological changes and a shift from radial to axial growth. It is concluded that the growth pattern exhibited by these macroalgae after fusion differs from equivalent processes described for other organisms with the capacity to fuse, such as modular invertebrates.     

Group Recruitment and Early Survival of Mazzaella Laminarioides

Several phycocolloid-producing Rhodophyta of significant economic importance are coalescing species, able to fuse with conspecifics during recruitment, reach larger sizes and increase their survival. In these species spores are needed to start cultivation (e.g. Gigartina, Mazzaella) or to increase the seed stocks, to renew senescent clones or to enlarge the base of genetic variation of vegetatively propagated species (e.g. Chondrus, Gracilaria, Eucheuma). This study uses Mazzaella laminarioides to evaluate some key features that influence recruitment success. Field measurements indicate that in any recruitment event a variable amount of the spores reaching a given place may form groups of 2 to over 100 coalescing spores, while field experiments support the idea that early recruitment success is a function of the number of coalescing spores forming the individual, as multisporic, coalescing recruits have higher survival rates than sporelings formed by one or a few spores. Therefore, group recruitment (spores settling and recruiting in close spatial proximity) appears as a prerequisite for sporeling coalescence and early recruitment success. In turn, laboratory experiments suggest that the frequency of group recruitment and coalescence increases with increasing spore abundance and with slight Ca⁺⁺ additions to the culture medium. These last two factors could be handled by farmers to improve the success of spore inoculations of coalescing species.     

The effects of coalescence on survival and development of <Emphasis Type="Italic">Mazzaella laminarioides</Emphasis> (Rhodophyta,Gigartinales)

The potential benefits associated with coalescence in red algae have been best documented with early stages of development. In this study, we report on the effects of the original number of spores on branching and fertility during later stages of development. Measurements for 20 months of survival rates, field growth, and fertility of a set of 96 sporelings produced in the laboratory with different number of spores, and outplanted at mid levels of the Mazzaella laminarioides belt at a locality in central Chile, indicate significant differences between multisporic holdfasts (50 and 100 coalescing spores) and uni- or oligosporic (ten spore) holdfasts in the number of branches and erect axes produced 10 and 15 months after germination. The difference is related to the persistence of clusters of axes and branch initials in deep portions of holdfasts of multisporic plantlets and their absence from equivalent portions of uni- or oligosporic holdfasts. This is an example in macroalgae of an event occurring during germination and expressing its effects in the next growing season (some 10 months after).     

INTERTIDAL AMPHIPODS AS POTENTIAL DISPERSAL AGENTS OF CARPOSPORES OF IRIDAEA LAMINARIOIDES (GIGARTINALES,RHODOPHYTA)1

Mid and late successional benthic algae have poor dispersal capacities. Mobile herbivores may increase dispersal of some algae because spores can survive digestion by grazers and stick to external body appendages. We show that carpospores are the only type of reproductive unit of the rhodophyte Iridaea laminarioides Bory that survive passage through the digestive tract of the amphipod Hyale sp. The spores also stick to the amphipods' legs and are thus carried by the amphipods in the field. Amphipods significantly increased (P < 0.01) the number of spores settling on artificial substrata in places where barriers prevented the normal ingress of algal propagules. The presence of artificial substrata that act as a refuge for the grazers also caused an increase (P < 0.01) in the number of settled spores. Amphipods also significantly increased (P < 0.01) the number of spores under an Ulva sp. canopy in laboratory experiments.     

CONVERGENT BIOLOGICAL PROCESSES IN COALESCING RHODOPHYTA

Sporeling coalescence in Gracilaria chilensis Bird, McLachlan et Oliveira produces genetically polymorphic, chimeric individuals. If this is common in red algae, it may have significant biological consequences. In this study, we evaluate the hypotheses that coalescence is widespread among the Rhodophyta and that specific and convergent morphological and ecological responses characterize this as a unique growth style among marine algae. A literature survey on coalescence was undertaken to assess the distribution of this condition in the Florideophycidae. Sixty-two (54.9%) of 113 species considered germinated to form a disk. Subsequent development in 37 of these species showed crust formation and coalescence during development with other crusts in 31 species (84%). Coalescing red algae were members of the orders Ahnfeltiales, Corallinales, Gigartinales, Gracilariales, Halymeniales, Palmariales, and Rhodymeniales. Ultrastructural studies in species of Ahnfeltiopsis, Chondrus, Gracilaria, Mazzaella, and Sarcothalia suggested a common pattern of early development. Newly released, naked spores may fuse into a single cell, as they do in Chondrus canaliculatus, or they may develop individual cell walls that later are surrounded by a thickened common wall. Ultrastructural studies demonstrated two kinds of immediate development after the first mitotic division: direct development by symmetric divisions resulting in discoid sporelings or an indirect asymmetric arrangement of divisions before a diskoid sporeling was formed. Germination in coalescing species is a linear function of the initial spore density, whereas in noncoalescing species maximum germination occurs at intermediate densities. In the field, coalescing species may recruit either from solitary or aggregated spores. However, survival is significantly higher for plantlets grown from a larger number of coalescing spores. Total number of erect axes formed by the coalesced mass is a logarithmic function of the initial number of spores. Thus, germlings grown from a larger number of coalescing spores exhibited a larger photosynthetic canopy than do plantlets grown from a few spores. Juveniles and mature clumps grown from a coalescing mass may exhibit size inequalities among erect axes, with the larger axes located toward the center of the clump. These larger axes mature first or, in some cases, are the only to produce spores. The widespread occurrence of coalescence inroughly half the number of orders of the Florideophycidae, the similarity of the coalescence process, and the finding of various adaptive traits associated with coalescence characterizes this as a unique growth style, splitting the diversity of species now included in the Florideophycidae into two major groups: coalescing and noncoalescing Rhodophyta.     

ATP and related purines stimulate motility,spatial congregation,and coalescence in red algal spores

Adenosine 5′‐triphosphate (ATP) is a versatile extracellular signal along the tree of life, whereas cAMP plays a major role in vertebrates as an intracellular messenger for hormones, transmitters, tastants, and odorants. Since red algal spore coalescence may be considered analogous to the congregation process of social amoeba, which is stimulated by cAMP, we ascertained whether exogenous applications of ATP, cAMP, adenine, or adenosine modified spore survival and motility, spore settlement and coalescence. Concentration‐response studies were performed with carpospores of Mazzaella laminarioides (Gigartinales), incubated with and without added purines. Stirring of algal blades released ADP/ATP to the cell media in a time‐dependent manner. 10–300 μM ATP significantly increased spore survival; however, 1,500 μM ATP, cAMP or adenine induced 100% mortality within less than 24 h; the exception was adenosine, which up to 3,000 μM, did not alter spore survival. ATP exposure elicited spore movement with speeds of 2.2–2.5 μm · s^?1. 14 d after 1,000 μM ATP addition, spore abundance in the central zone of the plaques was increased 2.7‐fold as compared with parallel controls. Likewise, 1–10 μM cAMP or 30–100 μM adenine also increased central zone spore abundance, albeit these purines were less efficacious than ATP; adenosine up to 3,000 μM did not influence settlement. Moreover, 1,000 μM ATP markedly accelerated coalescence, the other purines caused a variable effect. We conclude that exogenous cAMP, adenine, but particularly ATP, markedly influence red algal spore physiology; effects are compatible with the expression of one or more membrane purinoceptor(s), discarding adenosine receptor participation.     

COALESCENCE VERSUS COMPETITION: FIELD AND LABORATORY STUDIES OF INTRA‐ AND INTERSPECIFIC ENCOUNTERS AMONG COALESCING SEAWEEDS

Classical ecological theory predicts that whenever growing individuals share a common and limiting resource, such as substratum in mid‐intertidal and shallow subtidal habitats, preemptive competition will occur determining species abundance and distribution patterns. However, conspecificity of several ecologically dominant Rhodophyta may coalesce when grown in laboratory cultures. The extent at which intraspecific coalescence occurs in the field and whether the process may also happens during interspecific encounters remain to be determined. If intra‐ and interspecific coalescence effectively occurs, then coexistence through coalescence rises as an alternative to competition among red‐algal dominated intertidal and shallow subtidal communities. Populations of Mazzaella laminarioides and Nothogenia fastigiata living in mid‐intertidal, semi‐exposed rocky habitats in Central Chile are being used to test the above ideas. Intra‐ and interspecific encounters occur in the field throughout the year. Coalescence does occur among conspecific partners but it has not been detected in interspecific encounters. Rather, a thick interface of compressed cells, necrotic tissues and cyanobacterial nodules is formed between the two contacting partners. In addition, observations of laboratory cultures indicate that spore germination, germling survival and differentiation of erect axes in bispecific cultures may be reduced when compared to single‐species controls. Interspecific differences in growth and differentiation rates appear as the mechanisms explaining a lack of coalescence and negative effects during interspecific contacts. On the other hand, the existence of conspecific coalescence in the field suggests this process should be considered as a real alternative to intraspecific competition among coalescing Rhodophyta.     

LIGHT BOUNDARIES AND THE COUPLED EFFECTS OF SURFACE HYDROPHOBICITY AND LIGHT ON SPORE SETTLEMENT IN THE BROWN ALGA HINCKSIA IRREGULARIS (PHAEOPHYCEAE) 1

We examined the effects of light and surface hydrophobicity individually and in tandem on Hincksia irregularis (Kützing) Amsler spore settlement. Hincksia irregularis spores were determined to be negatively phototactic by the use of computer‐assisted motion analysis. Spore settlement was significantly influenced by surface hydrophobicity and by light, individually and in tandem. Experiments conducted using culture plates modified to reduce well edge artifacts revealed significantly higher settlement on hydrophobic surfaces and in dark environments when compared with negatively charged surfaces and lighted environments. Experimental light/dark boundaries elicited distinct spore settlement responses, with spores displaying dissimilar settlement patterns on plates with different surface hydrophobicities. The results of this study indicate H. irregularis spores possess the capacity for complex responses to their environment. These complex responses may influence dispersal and aid spores in the detection of suitable settlement locations in marine microenvironments.     

Effect of bacterial biofilms formed on fouling-release coatings from natural seawater and Cobetia marina,on the adhesion of two marine algae

Previous studies have shown that bacterial biofilms formed from natural seawater (NSW) enhance the settlement of spores of the green alga Ulva linza, while single-species biofilms may enhance or reduce settlement, or have no effect at all. However, the effect of biofilms on the adhesion strength of algae, and how that may be influenced by coating/surface properties, is not known. In this study, the effect of biofilms formed from natural seawater and the marine bacterium Cobetia marina, on the settlement and the adhesion strength of spores and sporelings of the macroalga U. linza and the diatom Navicula incerta, was evaluated on Intersleek® 700, Intersleek® 900, poly(dimethylsiloxane) and glass. The settlement and adhesion strength of these algae were strongly influenced by biofilms and their nature. Biofilms formed from NSW enhanced the settlement (attachment) of both algae on all the surfaces while the effect of biofilms formed from C. marina varied with the coating type. The adhesion strength of spores and sporelings of U. linza and diatoms was reduced on all the surfaces biofilmed with C. marina, while adhesion strength on biofilms formed from NSW was dependent on the alga (and on its stage of development in the case of U. linza), and coating type. The results illustrate the complexity of the relationships between fouling algae and bacterial biofilms and suggest the need for caution to avoid over-generalisation.     

SPORELING COALESCENCE IN CHONDRUS CRISPUS (RHODOPHYCEAE)1

Coalescence of developing sporelings of Chondrus crispus Stackhouse was observed. Juvenile tetra-sporophytes showed a higher proportion of coalescence than developing gametophytes. Stages of complete coalescence between different sporelings are illustrated. Coalesced sporelings exhibit vertical and horizontal alignment of cells, as well as “cuticular” continuity and secondary pit connections between adjacent, coalesced sporelings. Ultimately the cells in the center of the coalesced sporelings produce upright, multiaxial fronds that grow more rapidly than fronds of non-coalesced sporelings. Other red algae, such as Gracilaria verrucosa (Hudson) Papenfuss and Gigartina stellata (Stackhouse) Batters also show a similar sequence of sporeling coalescence and enhanced growth. The ecological significance of sporeling coalescence is discussed.     

QUANTIFICATION OF THE EFFECTS OF SPORELING COALESCENCE ON THE EARLY DEVELOPMENT OF GRACILARIA CHILENSIS (RHODOPHYTA)1

Sporeling coalescence in species of Gracilariales and Gigartinales is predicted to result in larger basal areas of growing disks as well as earlier initiation, increased abundance, and faster growth rates of erect shoots as compared to noncoalescent sporelings. These responses have been interpreted as providing mutual benefits for organisms living in aggregation, counterbalancing disadvantages associated with crowding. Quantitative evaluations of sporelings of Gracilaria chilensis failed to support several of these predictions. Sporelings were grown in the laboratory from a range of single sporelings to coalescent masses of 20 sporelings. Coalescent sporeling masses of G. chilensis exhibited larger basal areas than noncoalescent ones, but because the specific growth rates were inversely related to the original number of carpospores, no significant differences in actual area increments, during most of the experiment, were found among sporelings derived from one, two, or three to five coalescing sporelings. Initiation of erect shoots occurred at a similar time, regardless of their origin, i.e. coalescent or noncoalescent. Abundance of erect shoots was only loosely related to the number of coalescing sporelings. Even though by the end of the experiment (week 18), the total length of the longer erect shoots arising from coalescent sporeling masses was significantly greater than that of shoots arising from noncoalescent sporelings, total length was independent of the original number of coalescing sporelings. Furthermore, specific elongation rates between week 12 and week 18 were significantly greater for noncoalescent sporelings than for coalescent sporeling masses. Quantitative screening of other species seems necessary before generalizations on the ecological advantages of sporeling coalescence in seaweeds can be made.     

Individual and Coupled Effects of Echinoderm Extracts and Surface Hydrophobicity on Spore Settlement and Germination in the Brown Alga Hincksia irregularis

Marine substrata possess cues that influence the behavior of fouling organisms. Initial adhesion of fouling algal zoospores to surfaces is also theorized to depend primarily upon interactions between substrata and spore cell bodies and flagellar membranes. In an effort to identify cues and surface characteristics that influence spore settlement and early development, the effects of bioactive echinoderm extracts, surface charge, and surface hydrophobicity were examined individually and in tandem on zoospore settlement and germination in Hincksia irregularis. Experiments utilizing 96-well plastic culture plates confirmed that spore settlement and germination were significantly affected by surface charge and hydrophobicity as well as by echinoderm metabolites, both individually and in tandem. Spore settlement rates in the dark over 30?min were >?400% higher on hydrophobic surfaces than on positively and negatively charged surfaces. Spore germling numbers were >?300% higher on hydrophobic surfaces than on positively and negatively charged surfaces when spores were allowed to settle in the light for 30?min and the settled spores allowed to subsequently germinate for 24?h. Spore germling numbers were consistently >?25% higher on hydrophobic surfaces than on positively and negatively charged surfaces when equal numbers of spores were allowed to completely settle in the light and subsequently germinate for 24?h. H. irregularis germ tube lengths were also significantly longer on positively charged plates than on negatively charged plates. All echinoderm extracts tested had significant effects on germination and settlement at levels below those of estimated ecological concentrations. Short-term (30?min) exposure and subsequent germination experiments indicated that higher concentrations of extracts had rapid toxic effects on algal spores. Synchronous effects of echinoderm extracts and plate charge upon spore settlement varied considerably and did not show a strong dose response relationship. Long-term (24?h) exposure of spores to echinoderm extracts had dosage dependent effects on germination and spore survival. The results of this study indicate that H. irregularis spores possess the capacity for complex responses to their environment, utilizing combined cues of surface charge, surface energy and biochemistry to determine where to settle and germinate. These responses may aid spores in the detection of suitable substrata and conditions for settlement in the marine environment.     

Adhesion Strength of Settled Spores of the Green Alga Enteromorpha

Strengths of attachment of spores of the green fouling alga Enteromorpha to glass have been measured using a modified water jet apparatus. Surface pressures of ～250 kPa were required to quantitatively remove attached spores after 4 h contact with a surface. The development of adhesive and cohesive strength is highly time-dependent; after 8 h in contact with a surface spores did not detach, even at pressures in excess of 250 kPa. Spores settled in groups are more resistant to detachment than single spores, which suggests that the adaptive value of gregarious settlement behaviour may lie in the greater resistance of groups to detachment forces in a naturally turbulent environment. The interfacial forces exerted as water impinges on the surface and the derivation of adhesion strength values in terms of wall shear stress are discussed and compared with those obtained by other methods. A surface pressure of 250 kPa approximates to 325 Pa wall shear stress. Calculation using the power-law formula predicts that detachment forces of this magnitude are unlikely to be realized at operating speeds for most vessels and that most Enteromorpha spores would not detach from untreated hulls.     

SETTLEMENT AND SURVIVAL OF POLYSIPHONIA LANOSA (CERAMIALES) SPORES ON ASCOPHYLLUM NODOSUM AND FUCUS VESICULOSUS (FUCALES)1

The settlement patterns of spores of Polysiphonia lanosa (L.) Tandy on Ascophyllum nodosum (L.) Le Jolis and Fucus vesiculosus L. were studied using a flow tank. Settlement sites were defined as ‘sheltered’ or ‘exposed.’ Surface area calculations revealed non-random settlement on A. nodosum, with higher than expected spore frequencies on the thallus and lateral pits and lower than expected frequencies on the vesicles. Settlement of F. vesiculosus was random and significantly lower than on A. nodosum. On the shore, survival of sporelings from September (post-sporulation) to May (pre-sporulation) was highly non-random on both basiphytes. On A. nodosum, lateral pits ('sheltered') showed the highest survival frequency. Here the proportion of surviving sporelings increased over the study period, whereas the proportion on open thallus area ('exposed') decreased. On F. vesiculosus also preferential survival occurred on ‘sheltered’ sites such as vesicle/thallus interfaces and wounds. Between September and May, all P. lanosa sporelings were lost from ‘exposed’ areas (thallus surface and vesicles). Overall, frequencies of surviving sporelings were much greater on A. nodosum than on F. vesiculosus. These results are discussed with reference to basiphyte morphology, epiphyte removal mechanisms and the survival stratagy of P. lanosa.     

EFFECT OF ROCKY INTERTIDAL AMPHIPODS ON ALGAL RECRUITMENT: A FIELD STUDY1

Evidence about the potential of mobile marine invertebrates to act as algal spore dispersal agents is presently circumstantial. Using a field correlational and experimental protocol, our study tested the hypothesis that amphipods can increase the spore recruitment of the red alga Iridaea laminarioides Bory. Iridaea laminarioides spore recruitment onto glass slides was measured at a site with high amphipod abundance and a site with low density of amphipods. To evaluate the effect of an Ulva canopy on recruitment, replicated glass slides with and without a surrounding Ulva canopy were installed at both sites. The number of I. laminarioides spores recruited on the glass slides was four to eight times higher at the high amphipod abundance site than at the low density site. However, the presence of an Ulva canopy covering the glass slides did not significantly increase the recruitment of I. laminarioides. Because the abundance of I. laminarioides, the proportion of cystocarpic plants, and the percentage of open cystocarps only differed slightly between the low and high abundance amphipod sites, we suggest that the variation in recruitment between the sites is due to the differences in amphipod abundance (and their movements) and not to differences in spore production. Moreover, the presence of I. laminarioides cystocarps showing amphipod grazing scars was significantly higher at the high amphipod density site than at the low density site.     

Micrograzers and spore release in Iridaea laminarioides Bory (Rhodophyta: Gigartinales)

Preferential consumption of algal reproductive tissues has been predicted to potentially reduce algal fitness by reduction of reproductive output. This study shows that the amphipod Hyale media (Dana) has a marked trophic preference for mature cystocarpic tissues of Iridaea laminarioides Bory. However, during the feeding process the amphipod tears the cystocarps releasing large numbers of spores into the water column. Germination and growth rate experiments indicate that grazer-mediated release does not affect further spore development. Field populations of I. laminarioides might have unopened cystocarps even in senescent, decaying fronds, while the total number of open cystocarps in mature and senescent fronds increases at higher amphipod density sites, suggesting a facilitation mechanism. Laboratory observations indicate spores can stick to the legs and body sides of the amphipods while a fraction of the ingested spores survives passage through the amphipod digestive tract. Thus, the amphipod seems ecologically important not only as a grazer but also as spore releaser and disperser.     

Individual and coupled effects of echinoderm extracts and surface hydrophobicity on spore settlement and germination in the brown alga Hincksia irregularis

Marine substrata possess cues that influence the behavior of fouling organisms. Initial adhesion of fouling algal zoospores to surfaces is also theorized to depend primarily upon interactions between substrata and spore cell bodies and flagellar membranes. In an effort to identify cues and surface characteristics that influence spore settlement and early development, the effects of bioactive echinoderm extracts, surface charge, and surface hydrophobicity were examined individually and in tandem on zoospore settlement and germination in Hincksia irregularis. Experiments utilizing 96-well plastic culture plates confirmed that spore settlement and germination were significantly affected by surface charge and hydrophobicity as well as by echinoderm metabolites, both individually and in tandem. Spore settlement rates in the dark over 30 min were > 400% higher on hydrophobic surfaces than on positively and negatively charged surfaces. Spore germling numbers were > 300% higher on hydrophobic surfaces than on positively and negatively charged surfaces when spores were allowed to settle in the light for 30 min and the settled spores allowed to subsequently germinate for 24 h. Spore germling numbers were consistently > 25% higher on hydrophobic surfaces than on positively and negatively charged surfaces when equal numbers of spores were allowed to completely settle in the light and subsequently germinate for 24 h. H. irregularis germ tube lengths were also significantly longer on positively charged plates than on negatively charged plates. All echinoderm extracts tested had significant effects on germination and settlement at levels below those of estimated ecological concentrations. Short-term (30 min) exposure and subsequent germination experiments indicated that higher concentrations of extracts had rapid toxic effects on algal spores. Synchronous effects of echinoderm extracts and plate charge upon spore settlement varied considerably and did not show a strong dose response relationship. Long-term (24 h) exposure of spores to echinoderm extracts had dosage dependent effects on germination and spore survival. The results of this study indicate that H. irregularis spores possess the capacity for complex responses to their environment, utilizing combined cues of surface charge, surface energy and biochemistry to determine where to settle and germinate. These responses may aid spores in the detection of suitable substrata and conditions for settlement in the marine environment.     

Activity of Commercial Enzymes on Settlement and Adhesion of Cypris Larvae of the Barnacle Balanus amphitrite,Spores of the Green Alga Ulva linza,and the Diatom Navicula perminuta

Fouling species produce adhesive polymers during the settlement, adhesion and colonization of new surfaces in the marine environment. The present paper tests the hypothesis that enzymes of the appropriate specificity may prevent biofouling by hydrolysing these adhesive polymers. Seventeen commercially available enzyme preparations designed originally for bulk use in a range of end-use applications were tested for their effects on the settlement and/or adhesion of three major fouling species, viz. the green alga Ulva linza, the diatom Navicula perminuta and the barnacle Balanus amphitrite. The serine-proteases were found to have the broadest antifouling potential reducing the adhesion strength of spores and sporelings of U. linza, cells of N. perminuta and inhibiting settlement of cypris larvae of B. amphitrite. Mode-of-action studies on the serine-protease, Alcalase, indicated that this enzyme reduced adhesion of U. linza in a concentration-dependent manner, that spores of the species could recover their adhesive strength if the enzyme was removed and that the adhesive of U. linza and juvenile cement of B. amphitrite became progressively less sensitive to hydrolysis as they cured.