The yield,physical and chemical properties of agar gel from Gracilaria species (Gracilariales,Rhodophyta) of the Kenya coast

Six species of Gracilaria, G. corticata J. Agardh, G. crassa Harvey, G. millardetii J. Agardh, G. salicornia (J. Ag.) Dawson, G. verrucosa (Huds.) Papenfuss and Gracilaria sp, collected from different stations along the Kenya coast were studied. The yield of hot water native agar extract ranges from 8.1–30% of dry weight, with G. verrucosa and G. salicornia having the greatest and the least yield, respectively. The gel-strength of 1.5% agar solution was highest in G. verrucosa (220 g cm^–2) and lowest in G. corticata (< 60 g cm – 2) whereas the highest gelling temperature was recorded for Gracilaria sp. (40.4 °C) and the lowest in G. verrucosa (28.9 °C). 3,6 anhydrogalactose content was the highest in G. verrucosa and the lowest in G. corticata while sulphate content was higher in the latter.     

GRACILARIA HUMMII SP. NOV. (GRACILARIALES,RHODOPHYTA), A NEW NAME FOR THE AGAROPHYTE “GRACILARIA CONFERVOIDES” HARVESTED IN NORTH CAROLINA DURING WORLD WAR II1

Gracilaria hummii Hommers. et Freshwater is proposed as a new name for the inshore cylindrical species found in North Carolina that was treated as Gracilaria confervoides (L.) Grev. during World War II, and more recently as G. verrucosa (Huds.) Papenf. Molecular evidence places G. hummii in the Gracilis‐group in Gracilaria together with G. gracilis (Stackh.) Steentoft, L. M. Irvine et Farnham, the name currently applied to specimens formerly identified as G. confervoides and G. verrucosa. G. hummii differs from G. gracilis in possessing shallower male conceptacles in which the spermatangial filament originates from a surface cortical cell rather than from a subcortical cell. The cystocarps are similar, except that the gonimoblasts of G. hummii are attached to the base of the pericarp by numerous, prominent thickened terminal tubular cells and because terminal tubular cells are absent above the midregion of the cystocarp or in the vicinity of the ostiole. The gonimoblasts are subtended by a bundle of longitudinally oriented, thick‐walled secondary filaments of a type that has not been described before in Gracilaria but that may be characteristic of some other species. G. hummii occupies a basal position in the Gracilis‐group and is distinct from all other cylindrical North Carolina Gracilaria species, according to the molecular and morphological evidence.     

Seasonality of some species ofGracilaria (Gracilariales,Rhodophyta) from Kenya

Studies on the seasonality of 5Gracilaria species (G. corticata, G. crassa, G. millardetii, G. salicornia, G. verrucosa) were carried out. The seasonal abundance of the various species varied with time and geographical location. Nevertheless, a general single peak was evident between the months of September and December.G. verrucosa on the other hand had its peak biomass in July/August.G. millardetti is the only species which apparently preferred lower salinities; the rest of the species had no special trends as far as salinity is concerned. The occurrence ofG. verrucosa for a very short period during the cool part of the year is attributed to the high temperature that prevails in the coastal waters.     

Gracilaria phuquocensis sp. nov., a new flattened Gracilaria species (Gracilariales,Rhodophyta), previously recognized as G. mammillaris,from the southern coast of Vietnam

Flattened Gracilaria species are widely distributed along the coasts of the South China Sea with more than 20 species recorded. Within the South China Sea, Gracilaria mammillaris has only been reported from Vietnam, but this species is likely restricted to the western Atlantic. This study aimed to reevaluate the taxonomic status of Vietnamese specimens of ‘G. mammillaris’ using combined morphological and molecular data. Our data clearly indicated that Vietnamese specimens were morphologically and genetically distinct from authentic G. mammillaris from the western Atlantic, and also other described flat Gracilaria species. We, therefore, propose that specimens from Vietnam originally identified as G. mammillaris be designated as a new species, Gracilaria phuquocensis sp. nov. Morphologically, G. phuquocensis can be distinguished from other flat Gracilaria species by its small thallus size, narrower blades, many medullary layers, abundant basal nutritive filaments within mature cystocarps, and tetrasporangial nemathecium. Our rbcL sequence analyses showed that the new species was sister to Gracilaria rhodymenioides from Thailand, and these two species formed a clade with cylindrical Gracilaria species. Our study contributes to clarification of the taxonomic status of misidentified specimens attributed to the flattened Gracilaria species in the South China Sea.     

MOLECULAR DELINEATION OF SPECIES AND SPECIES RELATIONSHIPS IN THE RED ALGAL AGAROPHYTES GRACILARIOPSIS AND GRACILARIA (GRACILARIALES)1

Delineation of species in the economically important agarophyte genera Gracilaria and Gracilariopsis has proven extremely difficult using available morphological characteristics. In this study, we examine the usefulness of two transcribed spacers for molecular systematic studies of these genera. The polymerase chain reaction was used to amplify the internal transcribed spacers (ITSs) and the intervening 5.8S ribosomal DNA of the nuclear ribosomal repeat region. In addition, a plastid spacer region and flanking regions of coding genes were amplified from the RUBISCO operon. Both regions were sequenced for individuals and populations of Gracilariopsis lemaneiformis (Bory) Dawson, Acleto, et Foldvik to determine the usefulness of these spacers in delimiting populations. These studies reveal that there is as much variation among individuals of a population as there is between individuals of geographically separate populations. In addition, the ITS spacer regions were compared between different species of Gracilariopsis and Gracilaria. The nuclear ITS spacer region is conserved at a species level in both genera and provides phylogenetically informative characters that can be used to examine species interrelationships among relatively closely related taxa. However, because of the difficulties of aligning this entire region among species from the two genera, the ITS region is not useful for examining intergenera relationships. ITS interspecies sequence comparisons indicate that Gracilariopsis lemaneiformis from California is significantly different from G. lemaneiformis from China and that a species of Gracilariopsis from Peru is more closely related to G. lemaneiformis from North Carolina than it is to the other Gracilariopsis species examined. In addition, these studies indicate that Gracilaria chilensis Bird, McLachlan, et Oliveira from New Zealand and Gracilaria tenuistipitata Chang et Xia from southeast Asia are as closely related as are Gracilaria verrucosa (Hudson) Papenfuss, G. pacifica Abbott, and Gracilaria robusta Kylin. Phylogenetic analysis of aligned plastid spacer sequences from Gracilaria and Gracilariopsis taxa provide similar conclusions about species relationships.     

Karyology and cytophotometric estimation of nuclear DNA content variation in Gracilaria,Gracilariopsis and Hydropuntia (Gracilariales,Rhodophyta)

Chromosome numbers of 1 N=24 were determined for three species of Gracilaria (G. flabelliforme P. Crouan et H. Grouan ex Schramm et Maze, G. mammillaris Montagne and G. tikvahiae McLachlan) and 1 N=32 for two species of Gracilariopsis (G. lemaneiformis (Bory) Dawson, Acleto et Folvik and G. tenuifrons (Bird et Oliveira) Fredericq et Hommersand). Karyotypes for these species exhibit a characteristic size difference between largest and smallest chromosomes. Polyvalents were a common feature of meiotic nuclei. Microspectrophotometry with the DNA-localising fluorochrome DAPI was used to quantify nuclear genome sizes. A 2 C genome size of 0·37–0·40 pg was determined for five species of Gracilaria (G. chilensis Bird, McLachlan et Oliveira, G. flabelliforme, G. mammillaris, G. pacifica Abbott, G. tikvahiae) and 0·33 pg for an isolate of G. verrucosa (Hudson) Papenfuss from Pas de Calais, France. Species of Hydropuntia (H. cornea (J. Agardh) Wynne and H. dentata (J. Agardh) Wynne) and Gracilariopsis (G. lemaneiformis and G. tenuifrons) were found to have slightly larger 2 C genome contents of 0·42–0·47 pg. No intraspecific variation in 2 C genome sizes was found in regional populations of Gracilaria tikvahiae and Gracilariopsis tenuifrons.     

Stress tolerance of the invasive macroalgae <Emphasis Type="Italic">Codium fragile</Emphasis> and <Emphasis Type="Italic">Gracilaria vermiculophylla</Emphasis> in a soft-bottom turbid lagoon

Invasive species are often hypothesized to have superior performance traits. We compared stress tolerance (as change in biomass) of the invasive macroalgae Codium fragile ssp. tomentosoides and Gracilaria vermiculophylla to the native macroalgae Fucus vesiculosus, Agardhiella subulata, Hypnea musciformis and Ulva curvata in Hog Island Bay, a shallow lagoon in Virginia, USA. We hypothesized that the success of the two aliens is due to their high tolerances of turbidity, sedimentation, desiccation, grazing and nutrient enrichment. Like many lagoons, Hog Island Bay is characterized by extensive intertidal mudflats, high turbidity and sedimentation, and high densities of omnivorous mud snails. Nutrient enrichment may also become a problem as land use practices in adjacent watersheds change. Contrary to our hypothesis, C. fragile was less resistant to sedimentation, desiccation and grazing than other algae and had low growth at all light and nutrient levels. This suggests that any superior performance of this invasive species compared to native algae is probably limited to microhabitats where stress is minimal and where bivalve shells facilitate recruitment and long-term persistence. In contrast, G. vermiculophylla was resistant to desiccation, burial and grazing, and was not negatively influenced by either high or low light or nutrient levels. These traits reflect the current success of G. vermiculophylla in already invaded lagoons and estuaries, and indicates that it will likely continue its spread in European and North American turbid and tidal soft-sediment systems.     

PHYLOGENY AND SYSTEMATICS OF THE MARINE ALGAL FAMILY GRACILARIACEAE (GRACILARIALES,RHODOPHYTA) BASED ON SMALL SUBUNIT rDNA AND ITS SEQUENCES OF ATLANTIC AND PACIFIC SPECIES1

We sequenced the small subunit rDNA and internal transcribed spacer region of Gracilariaceae from the tropical Atlantic and Pacific, with emphasis on flattened or compressed species. Sequence comparisons confirmed three main lineages of Gracilariaceae: Curdiea/Melanthalia, Gracilariopsis/Gracilariophila, and Gracilaria. The Curdiea/Melanthalia diverged early in the family. Gracilariopsis was paraphyletic, because at least one Gracilariophila species evolved from it. The Atlantic Gracilariopsis were monophyletic and separated from the Pacific lineages. The Gracilaria included all species referable to its own species and to Hydropuntia, which was paraphyletic, formed by distantly related lineages. The new combination Gracilaria pauciramosa (N. Rodríguez Ríos) Bellorin, M. C. Oliveira et E. C. Oliveira is proposed for Polycavernosa pauciramosa N. Rodríguez Ríos. Recognition of subgenera within Gracilaria, based on spermatangial arrangement, was not supported. Instead, infrageneric groups were delineated by geographic origins and combinations of reproductive characters. Most Pacific species with either “textorii” or “verrucosa” type spermatangia were deeply separated from Atlantic species. Within the Atlantic Gracilaria, a lineage encompassing mostly tropical cylindrical species with “henriquesiana” type spermatangia and distinctive cystocarp anatomy was recognized. A lineage was also retrieved for cold water stringy species with verrucosa type spermatangia. Several species from the western Atlantic are closely related to Gracilaria tikvahiae McLachlan with nearly identical morphology. On the other hand, most flattened species from the tropical Atlantic were closely related despite their diverse morphologies. The interpretation of our data in addition to the literature indicates that more populations from the Indo‐Pacific must be studied before a general picture of Gracilariaceae evolution can be framed.     

UPTAKE AND RELEASE OF NITROGEN BY THE MACROALGAE GRACILARIA VERMICULOPHYLLA (RHODOPHYTA)1

Macroalgae, often the dominant primary producers in shallow estuaries, can be important regulators of nitrogen (N) cycling. Like phytoplankton, actively growing macroalgae release N to the water column; yet little is known about the quantity or nature of this release. Using ¹⁵N labeling in laboratory and field experiments, we estimated the quantity of N released relative to assimilation and gross uptake by Gracilaria vermiculophylla (Ohmi) Papenfuss (Rhodophyta, Gracilariales), a non‐native macroalgae. Field experiments were carried out in Hog Island Bay, a shallow back‐barrier lagoon on the Virginia coast where G. vermiculophylla makes up 85%–90% of the biomass. There was good agreement between laboratory and field measurements of N uptake and release. Daily N assimilation in field experiments (32.3±7.2 μ mol N·g dw^?1·d^?1) was correlated with seasonal and local N availability. The average rate of N release across all sites and dates (65.8±11.6 μ mol N·g dw^?1·d^?1) was 67% of gross daily uptake, and also varied among sites and seasons (range=33%–99%). Release was highest when growth rates and nutrient availability were low, possibly due to senescence during these periods. During summer biomass peaks, estimated N release from macroalgal mats was as high as 17 mmol N·m^?2·d^?1. Our results suggest that most estimates of macroalgal N uptake severely underestimate gross N uptake and that N is taken up, transformed, and released to the water column on short time scales (minutes–hours).     

Gracilaria vermiculophylla: A western Pacific species of Gracilariaceae (Rhodophyta) first recorded from the eastern Pacific

We report Gracilaria vermiculophylla (Ohmi) Papenfuss from the Pacific coast of North America based on a morphoanatomical revision and comparison of sequences of the nuclear gene coding for the small subunit of ribosomal RNA and the internal transcribed spacers of populations from Baja California, Mexico and Hiroshima, Japan. It is the first convincing report of this species out of its center of distribution in western north Pacific, where it has been considered as a synonym of former ‘G. verrucosa’ records. G. vermiculophylla also occurs in central California as indicated by internal transcribed spacer sequences of a previously unknown Gracilaria Greville material. In the northeastern Pacific G. vermiculophylla is characterized by a robust somewhat vermiform, well‐branched cylindrical thallus, with gradual cell size transition from cortex to medulla, deep spermatangial conceptacles, regular chains of carposporangia and carposporangial initials, downwardly oriented tubular nutritive cells, but rarely with upwardly oriented tubular nutritive cells. In some features of its cystocarp anatomy G. vermiculophylla is related to Gracilariopsis E. Y. Dawson or Hydropuntia Montagne and it is a relevant species for discussions about Gracilariaceae genera.     

Ricerche sui pigmenti fotosintetici di Gracilaria verrucosa (Huds.) Papenfuss. Dosaggio quantitativo della ficoeritrina

Abstract

Research on photosynthetic pigments from Gracilaria verrucosa (Huds.) Papenfuss. Quantitative analysis of phycoerythrin. – Among biliproteins of Rhodophycean algae, phycoerythrin is very interesting for constant distribution, in large amounts, in the different species. Phycoerythrin's extinction coefficients have been already calculated for some Bangiophycean and only two species of Rhodophycean algae. In this work we have analyzed all photosynthetic pigments and isolated pure phycoerythrin from Gracilaria verrucosa (Huds.) Papenfuss [= Gracilaria confervoides (L.) Grev.] belonging to Gigartinales. The aim of our research was to evaluate the quantitative ratio between this pigment and the others in a sistematic group for which there are only qualitative data. Phycoerythrin of Gracilaria is the R-classical type, with three absorption maxima. Average values of absorption coefficients, calculated on a large number of samples are E₁ ^%cm (565 nm) = 53.8, E₁ ^%cm (540 nm) = 48.4, E₁ ^1%cm (498 nm) = 49.1. These values are different from those obtained for R-phycoerythrin of Ceramium rubrum and B-phycoerythrin of Rhodochorton floridulum, both species belonging to Floridean algae. Our several experiments by chromatography and gel-electrophoresis process have demonstrated that the R-phycoerythrin we isolated is substantially pure. Thus we suggest that different absorption coefficient values are due either to a more complicated protein in the pigment molecule or to a different chromophore/protein ratio. This could result very interesting for the chemotaxonomic and phylogenetic aspect, but the small number of algae till now analyzed does not allow to draw any general conclusion.     

Effects of temperature and salinity on the growth of Gracilaria verrucosa and G. chorda, with the Potential for mariculture in Korea

Effects of temperature and salinity on the growth of the two agarophytes, Gracilaria verrucosa (Hudson) Papenfuss and Gracilaria chorda Holmes were examined in Korea. Both species grew over a wide range of temperatures (10–30 ^∘C) and salinities (5–35‰), and grew well at 17–30 ^∘C and a salinity of 15–30‰. In culture, G. verrucosa grew faster than G. chorda and their maximum growth rates were 4.95% day⁻¹ (30 ^∘C, 25‰) and 4.47% day⁻¹ (at 25 ^∘C, 25‰), respectively. In the field population the maximum growth and fertility of G. chorda were observed in summer. The growth rate of G. verrucosa was slightly higher than that of G. chorda for 2 weeks on the cultivation rope and in culture but it was much lower after being contaminated with epiphytes. The biomass of the epiphytes was 0.82 g dry wt. per host plant in G. verrucosa and 0.001 g in G. chorda. G. chorda exhibited resistance to epiphytism and grew 7 times in length and the dry weight increased 15 times after 55 days. In conclusion, G. chorda appears to be a good agarophyte with a fast growth rate and resistance to epiphytesm, and compared with G. verrucosa, has good potential for commercial cultivation.     

A seasonal study of the red seaweeds Solieria tenera and three species of Gracilaria from Jamaica

Three species of Gracilaria viz G. cervicornis, G. domingensis, G. verrucosa and Solieria tenera (Gigartinales, Rhodophyta) have been studied for their monthly variation in dry wt yields, ash, soluble carbohydrate, protein, lipid and insoluble carbohydrate contents for one year. The dry wt yields were higher in G. domingensis. Soluble carbohydrate and ash contents showed an inverse relationship in all the species. Protein content was lower, below 5% of the dry wt for all the species. A comparison of protein:carbohydrate ratios showed a similarity between morphologically similar G. domingensis and G. cervicornis in that there was less variation in the ratio. From this, it is assumed that flattened morphology probably is more efficient in maintaining nutrient balance to keep up the growth rates.     

Differentiation of Male Gametes in Gracilaria verrucosa (Rhodophyta, Gracilariales)

The development of male gametes (spermacia) in the red alga Gracilaria verrucosa has been studied using methods of transmission electron microscopy. Early spermatangia located along the wall of the conceptacle show an elongated shape in the thin sections. In the central part of the electron-dense cytoplasm of these cells there is a nucleus; numerous fibrous vesicles are arranged in the periphery. During the process of differentiation, the spermatangia become more rounded in shape and a large spermatangial vesicle is developed. The subsequent development of spermatium is accompanied by polarization of the spermatangium and the subsequent excretion of the spermatangial vesicle. The spermatia are oval cells containing a nucleus and fibrous vesicles. The process of differentiation of male gametes in G. verrucosa does not differ from that in five species of the genus Gracilaria, where it has already been studied. However, any conclusions about the degree of similarity between the spermatia in all the studied species can be made only after a detailed comparative analysis of the ultrastructural characteristics of these gametes.     

Bioremediation efficiencies of <Emphasis Type="Italic">Gracilaria verrucosa</Emphasis> cultivated in an enclosed sea area of Hangzhou Bay,China

Due to the discharge of nutrients into the East China Sea, severe eutrophication has appeared in Hangzhou Bay. Therefore, we cultivated Gracilaria verrucosa on a large scale in the Jinshan enclosed sea with an area of 1.72 km² in the northern part of Hangzhou Bay to perform bioremediation. The Fengxian enclosed sea with an area of 2.3 km² and 50 km far from Jinshan was used as the control. The results showed that the Hangzhou Bay was severely eutrophicated before G. verrucosa cultivation. During the period of cultivation between August 2006 and July 2007, the annual growth rate of G. verrucosa was 9.42% day⁻¹, and the sea water quality was improved from worse than grade IV to grades II–III, with the concentration of dissolved inorganic nitrogen (DIN) and PO₄-P significantly lower than that in the Fengxian enclosed sea (p < 0.01). The concentration of NH₄-N, NO₃-N, NO₂-N, and PO₄-P after G. verrucosa cultivation was decreased by 54.12%, 75.54%, 49.81%, and 49.00%, respectively. The density of phytoplankton in the Jinshan enclosed sea with cultivation of G. verrucosa was 6.90 –126.53 × 10⁴ cells m⁻³, which was significantly lower than that in the Fengxian enclosed sea. In addition, species diversity, richness, and evenness was significantly increased after cultivation of G. verrucosa in the Jinshan enclosed sea compared with that in the Fengxian enclosed sea. The density of Skeletonema costatum, Prorocentrum micans, and Prorocentrum donghaiense, which were the usual species of red tides at the coastal sea of China, in the Jinshan enclosed sea with cultivation of G. verrucosa was significantly lower. Based on these results, if the water quality in the Jinshan enclosed sea were to be maintained at grade I (DIN ≤0.20 mg  L⁻¹) or II (DIN ≤0.30 mg  L⁻¹), 21.8 t or 18.0 t fresh weight of G. verrucosa need to be cultivated, respectively. These results indicated that large-scale cultivation of G. verrucosa could play a significant role in the bioremediation of Hangzhou Bay.     

Assessment of populations ofGracilaria chilensis (Graeilariales,Rhodophyta) utilizing RAPDs

Phenotypic variability and mixing of material due to massive cultivation for commercial purposes has contributed to the taxonomic confusion ofGracilaria in Chile. At least four species with cylindrical thalli and similar morphology have been recorded. However, since establishment ofG. chilensis, most of the collected thalli have been attributed to this species despite the lack of diagnostic features. In an attempt to resolve whetherGracilaria from 3 localities where it grows in natural and artificial populations belongs to the same species, gametophytic samples were compared by applying RAPD-PCR to their total DNA. This was analysed using 25 different 10-mer primers from which 21 revealed polymorphism within and between populations. Similarity matrices and cluster analyses were performed based on the presence/absence of bands representing fragments of DNA generated by random amplification. Similarity values between two of the populations were equivalent to those detected within a third, indicating the mixing of genetic material due to transplant between the two former localities. Similarities between samples of ChileanGracilaria andG. tenuistipitata from Sweden are considerably lower (0.45–0.53) than those between populations from Chile (0.74–0.88), confirming the existence of a single specific taxon,G. chilensis, in these three localities.     

A Light Microscope Study of Carposporophyte Development in Gracilaria verrucosa (Hudson) Papenfuss

Gracilaria verrucosa is a very common marine red alga of Greekcoasts. The diploid carposporophyte which develops attachedto the female gametophyte of Gracilaria is described. The immaturecystocarps are very small while the mature ones are globose,ostiolate and are borne profusely all over the surface of thethallus. The earliest observed fusion cell is small and fusesprogressively with adjacent vegetative cells to form a largemultinucleate cell. From this fusion cell gonimoblast initialsoriginate, dividing further and giving rise to a large numberof gonimoblast cells. The resultant carposporophyte consistsof a basal-central, multinucleate cell surrounded by a conicalor hemispherical mass of gonimoblast cells. Chains or clustersof successively maturing carpospores are borne from the terminalgonimoblast cells. The liberation of mature carpospores takesplace through the ostiole of the cystocarp. The liberated carposporeslack cell walls and are naked in a mucilage mass. Gracilaria verrucosa (Hudson) Papenfuss, Gigartinales, Gracilariaceae, Rhodophyta, carposporophyte, development     

Growth,photosynthesis and agar in wild-type strains of Gracilaria verrucosa and G. conferta (Gracilariales,Rhodophyta), as a strain selection experiment

The local species Gracilaria conferta and the foreign G. verrucosa were grown together under a wide range of photon flux density and temperature conditions. Gracilaria verrucosa showed a higher growth rate, especially under low temperatures, and higher photosynthetic performances as well as higher ribulose-1,5-bisphosphate carboxylase activity as compared with G. conferta. Gracilaria verrucosa also showed a better quality and yield of agar, suggesting that this species could be more suitable than G. conferta for outdoor cultivation in Israel and may improve winter growth in ponds. Growth rate and agar quality (gel strength) were rated as the most suitable characteristics influencing the preference of strains for outdoor cultivation.     

Cloning and characterization of two types of ubiquitin genes from Gracilariopsis lemaneiformis (Gracilariales, Rhodophyta)

Two types of ubiquitin genes were isolated from the marine red alga Gracilaria lemaneiformis: a ubiquitin-52 amino acid fusion protein gene, and a 6-unit polyubiquitin gene. Alignment of polyubiquitins among three red algae (Gracilaria lemaneiformis, Gracilaria verrucosa, Aglaothamnion neglectum) and other species revealed that there were six ubiquitin repeats in all three red algae polyubiquitins, and that glutamine was the final amino acid residue in the terminal repeat of the polyprotein in the two Gracilaria sequences. Southern blot analysis revealed that both genes were encoded by low-copy number genes. Semi-quantitative RT-PCR was performed to investigate the expression of these two genes in two phases of G. lemaneiformis. The result revealed that the monoubiquitin was phase-relative, and upregulated in tetrasporophytes compared with female gametophytes. The polyubiquitin gene was expressed at similar levels in both phases.