Effects of two species of macroalgae—Ulva pertusa and Gracilaria lemaneiformis—on growth of Heterosigma akashiwo (Raphidophyceae)

The effects of fresh thalli, culture filtrate, water-soluble extract and dry powder of two species of macroalgae, Ulva pertusa (Chlorophyta) and Gracilaria lemaneiformis (Rhodophyta), on the growth of a bloom-forming microalga, Heterosigma akashiwo, were studied in co-culture under controlled laboratory conditions. Both fresh thalli and culture filtrate of U. pertusa and G. lemaneiformis, particularly in the form of fresh thalli, significantly inhibited microalgal growth; indeed, the microalga was completely killed during the course of the experiment. A clear concentration-dependent relationship was observed between the initial concentration of fresh thalli (either U. pertusa or G. lemaneiformis) and its inhibitory effect on H. akashiwo. Simultaneous nutrient assays showed that nitrate and phosphate were almost exhausted in G. lemaneiformis fresh thalli co-culture but remained well above nutrient limitation for microalgal growth in U. pertusa co-culture, in which the microalgal cells were completely killed. However, daily f/2 medium repletion would obviously alleviate the growth inhibition in G. lemaneiformis co-culture. Since the present study was carried out under controlled conditions, fluctuations in environmental factors (i.e., light, temperature, carbon limitation, bacterial presence and pH) were limited during the experiment. We thus concluded that allelopathy was the most likely explanation for microalgal growth inhibition in U. pertusa co-culture, while the combined roles of allelopathy and nutrient limitation were responsible for growth inhibition in G. lemaneiformis co-culture. Similarly, macroalgal water-soluble extracts and dry powders affected the co-cultured H. akashiwo greatly, with more obvious effects observed in water-soluble extract co-cultures. A dose-dependent relationship was also observed over the course of the experiment. It can be concluded that macroalgal thalli contain some bioactive compounds. The results of the present study suggest that U. pertusa and G. lemaneiformis, especially in the form of fresh thalli, effectively inhibit the growth of H. akashiwo and could thus be potential candidates for use in the control and mitigation of H. akashiwo blooms.     

DEFENSE EVOLUTION IN THE GRACILARIACEAE (RHODOPHYTA): SUBSTRATE‐REGULATED OXIDATION OF AGAR OLIGOSACCHARIDES IS MORE ANCIENT THAN THE OLIGOAGAR‐ACTIVATED OXIDATIVE BURST1

Combined phylogenetic, physiological, and biochemical approaches revealed that differences in defense‐related responses among 17 species belonging to the Gracilariaceae were consistent with their evolutionary history. An oxidative burst response resulting from activation of NADPH oxidase was always observed in two of the subgenera of Gracilaria sensu lato (Gracilaria, Hydropuntia), but not in Gracilariopsis and in species related to Gracilaria chilensis (“chilensis” clade). On the other hand, all species examined except Gracilaria tenuistipitata var. liui and Gracilariopsis longissima responded with up‐regulation of agar oligosaccharide oxidase to an challenge with agar oligosaccharides. As indicated by pharmacological experiments conducted with Gracilaria chilensis and Gracilaria sp. “dura,” the up‐regulation of agar oligosaccharide oxidase involved an NAD(P)H‐dependent signaling pathway, but not kinase activity. By contrast, the activation of NADPH oxidase requires protein phosphorylation. Both responses are therefore independent, and the agar oligosaccharide‐activated oxidative burst evolved after the capacity to oxidize agar oligosaccharide, probably providing additional defensive capacity to the most recently differentiated clades of Gracilariaceae. As demonstrated with Gracilaria gracilis, Gracilaria dura, and Gracilariopsis longissima, the different responses to agar oligosaccharides allow for a fast and nondestructive distinction among different clades of gracilarioids that are morphologically convergent. Based upon sequences of the chloroplast‐encoded rbcL gene, this study suggests that at least some of the samples from NW America recorded as Gs. lemanaeiformis are probably Gs. chorda. Moreover, previous records of Gracilaria conferta from Israel are shown to be based upon misidentification of Gracilaria sp. “dura,” a species that belongs to the Hydropuntia subgenus.     

FIRST RECORD OF GRACILARIA VERMICULOPHYLLA (GRACILARIALES,RHODOPHYTA) IN THE PO DELTA LAGOONS,MEDITERRANEAN SEA (ITALY)1

Molecular outcomes led us to report the first occurrence of the invasive alien species Gracilaria vermiculophylla (Ohmi) Papenf. in the Mediterranean Sea. This species was recorded for the first time in the Po Delta lagoons in May and October 2008, probably introduced by the importation of the Manila clam Tapes philippinarum. At present, G. vermiculophylla is spread only near some clam‐farming areas, but its diffusion is expected to increase with the colonization of other lagoons where aquaculture is spread, as already observed for other alien species such as Agardhiella subulata and Solieria filiformis. The present study supplies further information on the morphology of this species, the ecological characteristics of the colonized areas, and the most probable introduction vector, confirming that the species spreading occurs in eutrophic and turbid coastal systems.     

Influence of ultraviolet radiation on spore liberation in marine macroalgae Ulva fasciata (Ulvales,Chlorophyceae) and Gracilaria corticata (Gracilariales,Rhodophyceae)

The effect of ultraviolet‐B (UV‐B) and UV‐A radiation on spore liberation in the intertidal marine macroalgae Ulva fasciata Delile (Chlorophyceae) and Gracilaria corticata J.Agardh (Rhodophyceae) was investigated. The two algae were exposed to UV‐A and UV‐B radiation separately for 10, 20, 30, 45 and 60 min and percentage inhibition of spore liberation was determined in controlled laboratory conditions. The spore liberation period in UV treated algae was extended for 4 days in U. fasciata and 9 days in G. corticata. UV‐B radiation inhibited spore liberation as much as 76.6% in U. fasciata and 55.5% in G. corticata at 60 min exposure. A significant positive correlation was observed between percentage inhibition of spore liberation and length of UV‐B exposure in both U. fasciata and in G. corticata. Similarly, UV‐A radiation also inhibited spore liberation as much as 75% in the former and 50% in the latter. There was a significant correlation between inhibition of spore liberation and length of UV‐A exposure in U. fasciata and in G. corticata. Analysis of variance results showed inhibition of spore liberation at 60 min of UV exposure differed significantly with that of other exposure lengths. The present findings reveal that UV‐A radiation also had an impact on spore liberation but to a lesser extent than UV‐B radiation. Thallus thickness and plant location on the shore determines their exposure to UV radiation. High UV impact was seen for U. fasciata growing in the upper parts of the intertidal region with a thin sheet like thallus and high surface area resulting in higher inhibition of spore liberation than in G. corticata.     

Nutritional Composition and Assessment of Gracilaria lemaneiformis Bory

The chemical composition, mineral elements, vitamins, free fatty acids and amino acid content of the edible red alga Gracilaria lemaneiformis Bory, grown in the sea near Nan＇ao island, Guangdong Province, were analyzed in the present study. Gracilaria lemaneiformis Bory showed a total sugar content of 14.65%. The protein content was 21%, of which approximately 41% was determined to be essential amino acids （EAA）. The major amino acid components were glutamic acid, leucine, arginine, and alanine. Of the EAA assayed, methionine and cysteine appeared to be the most limiting amino acids compared with the EAA pattern provided by Food and Agricultural Organization of the United Nations. The total lipids content was 0.87% and comprised a high composition of unsaturated fatty acids （61%）, mainly as linoleic acid and oleic acid, and a little amount of polyunsaturated fatty acid; palmitic acid was the main component （39%） of saturated acids. Relatively high levels of vitamin C, iodine, phosphorus, and zinc were also present in G. lemaneiformis. The nutritional composition between G. lemaneiformis and Nostoc flagelliforme, a rare alga that is widely eaten in Chinese society, was compared. The results suggest that N. flagelliforme can be substituted for by G. lemaneiformis, not only because of their similar shape, but also because of their approximate nutritional composition. Gracilaria lemaneiformis may possibly serve as a potential healthy food in human diets in the future.     

SPORE RELEASE IN ACROCHAETIUM SP. (RHODOPHYTA) IS BACTERIALLY CONTROLLED1

The facultative red algal epiphyte Acrochaetium sp. liberated spores preferentially and recruited more successfully in laboratory cultures when its host Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira was present. The same effect was also induced by cell‐free medium from G. chilensis, suggesting it contained a molecular signal. Antibiotics prevented spore release in Acrochaetium sp., even when G. chilensis was present, suggesting a prokaryotic origin of the signal. Simultaneous application of N‐butyl‐homoserine‐lactone (BHL) restored the spore‐release capacity, which demonstrated that spore release was not directly inhibited by the antibiotics and indicated that bacterially generated N‐acyl‐homoserine‐lactones (AHLs) regulate spore release. An involvement of AHL was further indicated by the fact that two different halofuranone inhibitors of AHL receptors also inhibited spore release when they were applied at relatively low concentrations. Of seven different AHLs tested, only BHL induced the effect. However, BHL was only active at relatively high concentrations (100 μM), and it was not detected in spore‐release‐inducing medium of G. chilensis. Another water‐soluble AHL or an AHL structure analog is therefore probably the active compound in G. chilensis cultures. The data presented demonstrate that life cycle completion in Acrochaetium sp. strongly depends on bacteria, which are not always present in sufficient numbers on the alga itself. Exogenous bacteria that are associated with G. chilensis or with other potential substrates may therefore trigger timely spore liberation in Acrochaetium sp., provided that the necessary concentration of AHL is reached. This first finding of AHL perception in a red alga confirms that AHL signalling is more widespread among eukaryotes than was thought until recently. However, spore release of a second red alga, Sahlingia subintegra (Rosenv.) Kornmann, was unaffected by AHL, and the reaction observed is therefore not universal.     

Identification of random amplified polymorphic DNA (RAPD) markers highly linked to sex determination in the red alga Gracilaria gracilis

The random amplified polymorphic DNA (RAPD) technique was employed in the haplo-diploid dioecious species Gracilaria gracilis to identify sex-linked PCR markers. Sixty-nine decamer oligonucleotide primers were tested on two bulks of DNA, one from five haploid males and the other from five haploid females. One of these primers (OPD13) generated a 430-bp fragment specific to males and a 620-bp fragment specific to females. The diploid individuals (tetrasporophytes) showed the co-occurrence of these two fragments. In order to verify the linkage between the sexual phenotypes and these markers, a progeny array of 59 haploid individuals (male and female) born on a diploid individual was analysed, in all of which the two markers produced by the OPD13 primer segregated perfectly with sex.     

GENETICS OF GRACILARIA TIKVAHIAE (RHODOPHYCEAE). X. STUDIES ON A BISEXUAL CLONE12

A male done of the red alga Gracilaria tikvahiae McLachlan spontaneously produced a bisexual frond which remained bisexual in subsequent subcultures. Both male and female components of bisexual fronds were functional; however, some unusual results were obtained in crosses. When bisexual fronds were crossed with a normal haploid male, the resulting carpospores all developed into diploid male gametophytes. When bisexual plants were self fertilized, all the carpospores yielded diploid bisexual gametophytes. Only when bisexual plants were crossed to normal haploid females, did carpospores develop into diploid tetrasporophytes as they normally do. The F₁ gametophyte generation obtained from these tetrasporophytes, however, included not only females and males but also bisexual plants, in a 2:1:1 ratio. These results are consistent with the interpretation that bisexual plants have a recessive mutation of a gene other than the primary sex determining locus, and that this mutation is expressed only in male plants. It is suggested that the altered gene may ordinarily have a regulatory function in the maintenance of the dioecious condition.     

SYSTEMATICS OF THE GRACILARIACEAE (GRACILARIALES,RHODOPHYTA): A CRITICAL ASSESSMENT BASED ON RBCL SEQUENCE ANALYSES1

Generic concepts in the economically important agarophyte red algal family Gracilariaceae were evaluated based on maximum parsimony, Bayesian likelihood, and minimum evolution analyses of the chloroplast‐encoded rbc L gene from 67 specimens worldwide. The results confirm the monophyly of the family and identify three large clades, one of which corresponds to the ancestral antiboreal genera Curdiea and Melanthalia, one to Gracilariopsis, and one to Gracilaria sensu lato, which contains nine distinct independent evolutionary lineages, including Hydropuntia. The species currently attributed to Hydropuntia comprise a single well‐supported clade composed of two distinct lineages. The two most basal clades within Gracilaria sensu lato deserve generic rank: a new genus centered around G. chilensis Bird, McLachlan et Oliveira and G. aff. tenuistipitata Chang et Xia and a resurrected Hydropuntia encompassing primarily Indo‐Pacific (G. urvillei [Montagne] Abbott, G. edulis [S. Gmelin] P. Silva, G. eucheumatoides Harvey, G. preissiana [Sonder] Womersley, and G. rangiferina [Kützing] Piccone) and western Atlantic species (G. cornea J. Agardh, G. crassissima P. et H. Crouan in Mazé et Schramm, G. usneoides [C. Agardh] J. Agardh, G. caudata J. Agardh, and G. secunda P. et H. Crouan in Mazé et Schramm). Cystocarpic features within the Gracilaria sensu lato clades appear to be more phylogenetically informative than male characters. The textorii‐type spermatangial configuration is represented in two distinct clusters of Gracilaria. The rbc L genetic divergence among the Gracilariaceae genera ranged between 8.46% and 16.41%, providing at least 2.5 times more genetic variation than does the 18S nuclear rDNA. rbc L also resolves intrageneric relationships, especially within Gracilaria sensu lato. The current number of gracilariacean species is underestimated in the western Atlantic because of convergence in habit and apparent homoplasy in vegetative and reproductive anatomy.