Non-enzymatic isolation of somatic cells from Kappaphycus spp. and Eucheuma denticulatum (Solieriaceae,Rhodophyta)

Cell culture technology is immensely useful in somatic hybridization, induction of mutations, cloning of specific isolates and maintenance of strains of defined genotypes. However, its application in strain improvement of some tropical red macroalgae has been limited due to the difficulty of isolating viable cells from their complex intercellular matrices. A simple, non-enzymatic technique of isolating somatic cells was developed for Kappaphycus spp. and Echeuma denticulatum. Surface-sterilized tissues (0.1 g fresh weight, 2.0 mm thick discs) from subcortical and medullary layers were treated with 3% NaOH, 3% KOH, or hydrogen peroxide in phosphate buffer solution (PBH₂O₂). Tissues of K. alvarezii treated with PBH₂O₂ softened after 5 h of treatment and completely dissociated after 12 h. Viable cell counts (VCC), determined through staining with Evan’s blue, were significantly higher (2.4 × 10⁵ cells g^?1 fresh weight tissue) in K. alvarezii (‘tambalang’ strain) treated with PBH₂O₂ compared with tissues treated with carrageenase from a marine bacterium.     

Occasional pathogenic bacteria promoting ice-ice disease in the carrageenan-producing red algae Kappaphycus alvarezii and Eucheuma denticulatum (Solieriaceae,Gigartinales, Rhodophyta)

The bacterial isolates from normal and diseased branches of Kappaphycus alvarezii and Eucheuma denticulatum in the Philippines were examined for possible role in the development of the ice-ice disease. The numbers of bacteria on and in ice-iced branches were 10–100 times greater than those from normal, healthy ones. Gram-positive bacteria predominated in almost all branch sources, but with an increasing proportion of agar-lysing bacteria in branches suffering from the ice-ice disease. These agar-lysing bacteria were composed of yellow and non-pigmented, spreading colonies identified to the Cytophaga-Flavobacterium complex and the Vibrio group. Among isolates which mainly appeared on ice-iced branches, two strains, designated as P11 (Vibrio sp.) and P25 (Cytophage sp.), which showed pathogenic activity, were obtained. These strains caused early ice-ice whitening of K. alvarezii especially when subjecting branches to environmental stress, such as reduced salinity and light intensity, suggesting that these bacteria were occasionally pathogenic. This paper offers new evidence of bacterial role in the development of so-called ice-ice disease among farmed species of Kappaphycus.     

Biological activity,cDNA cloning and primary structure of lectin KSA-2 from the cultivated red alga Kappaphycus striatum (Schmitz) Doty ex Silva

The red alga (Kappaphycus striatum) has been widely cultivated as a source of carrageenophytes for industry. Previously, lectin KSA-2 from this alga has been isolated and evaluated for the strictly high-mannose N-glycan binding specificity. In this study, we have determined the antibacterial activities and cDNA cloning encoding lectin KSA-2. Complementary DNA (cDNA) cloning based on the rapid amplification of cDNA ends (RACE) elucidated the full-length sequence of KSA-2, which encoded a polypeptide of 269 amino acids including initiating methionine, with four tandemly repeated domains of about 67 amino acids, and sharing 45% sequence identity. The calculated molecular mass from the deduced sequence was consistent with that of natural KSA-2 (28,021.5 Da), which was determined by electron spray ionization–mass spectrometry. The primary structure of KSA-2 is highly similar to those of the high-mannose N-glycan specific lectins in lower organisms including Burkholderia oklahomensis EO147 (BOA), Myxococcus xanthus (MBHA) and Pseudomonas fluorescens Pf0-1 (PFL) from proteobacteria, Oscillatoria agardhii NIES-240 (OAA) from cyanobacterium, Eucheuma serra (ESA-2) and Eucheuma denticulatum (EDA-2) from macro red algae, indicating that they were closely related to each other. The active fraction KSA-2 inhibited the growth of human and shrimp pathogenic bacteria Enterobacter cloacae and Vibrio alginolyticus, respectively, although it did not affect the growth of Staphylococcus aureus, Escherichia coli, Vibrio parahaemolyticus and Vibrio harveyi, suggesting that KSA-2 caused the activity through binding to the target receptor(s) on the cell surface of E. cloacae and V. alginolyticus. These results indicate that the cultivated K. striatum is a good source of a lectin that may be useful as an antibacterial agents.     

Genetic diversity of Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae,Rhodophyta) in Southeast Asia

The commercial importance of carrageenophytes Kappaphycus and Eucheuma is well known, with much interest in terms of cultivation, marketing, and research. Considering the many lucrative prospects, these red seaweeds were introduced into various parts of the world for farming, where merely a few were comprehensively documented. Despite being extensively cultivated throughout Southeast Asia, the genetic diversity of Kappaphycus and Eucheuma is poorly studied, where heavy reliance is placed on the use of local or commercial names for identifications. This study used the mitochondrial-encoded cox1 and cox2–3 spacer genetic markers to investigate the Kappaphycus and Eucheuma haplotypes, cultivated and wild, available throughout Southeast Asia. Concatenated cox1–cox2–3 spacer datasets were also analyzed. The near full-length cox1 gene is preferred at revealing the genetic diversity of Kappaphycus and Eucheuma, provided a larger reference database is available. Both molecular markers were capable of delineating common members of the genus Kappaphycus (i.e., Kappaphycus alvarezii, Kappaphycus striatus, and Kappaphycus cottonii) and Eucheuma denticulatum, and revealed interesting genotypes and new species which may be potential alternatives to the common cultivars as well as materials for research. The relative scarcity of Eucheuma species is discussed and future sites for sampling are recommended.     

Growth rate,carrageenan yield and gel properties of culturedkappa-carrageenan producing red algaKappaphycus alvarezzi (Doty) Doty in the subtropical waters of Shikoku,Japan

Kappaphycus alvarezii (Doty) Doty from the Philippines was grown in the subtropical waters of Shikoku Is., Japan (33°2635 N, 133°2500 E). The growth rate, carrageenan yield, viscosity and gel strength were compared to values for plants cultured in the Philippines and between spring-summer, autumn-winter and winter periods in Japan. Maximum daily growth rates up to 8% occurred in Japan in October to November (autumn) while die-back occurred in winter due to low seawater temperature (15 °C). The average growth rate (ca 5%) in spring-summer to autumn was comparable to that in the Philippines. During the winter (December), the daily growth rate was approximately 1%. Gel strength, determined in a 1.5% solution in both water and 0.2% KCl, was 850 g cm^-2 and 2000 g cm^-2, respectively. Sol viscosity was 890 cP at 75 ° C. These values are quite similar to values obtained in the Philippines.     

A study of the phycocolloids from Gelidium madagascariense and Eucheuma denticulatum (Rhodophyta) collected on the south coasts of Madagascar

Several species of red algae known to contain agar or carrageenan are abundant on the southeast and southwest coasts of Madagascar. The agarophyte Gelidium madagascariense, collected in the Fort Dauphin area, has been exported on a small scale to Japan for several years. FT-IR and GLC analysis show that it contains an agar polymer, of which the methylated fraction contains predominantly 6-O-methyl galactose and traces of 4-O-methyl galactose. Eucheuma denticulatum grows profusely on the coral reefs in the Tulear area. FT-IR and GLC analysis show that it contains iota-carrageenan, with no methyl-derivative contaminants. The 3,6-anhydro-galactose content was found to be rather low, suggesting a possible contamination with deviant iota. The economic potential of other sources of phycocolloids, as well as the possibility of farming different species of Eucheuma in the Tulear area, is discussed.     

Seasonal variation in growth and carrageenan yield in cultivated Kappaphycus alvarezii (Doty) Doty on the coastal waters of Ramanathapuram district,Tamil Nadu

Kappaphycus alvarezii, a kappa carrageenan yielding red seaweed, was cultivated in Indian subtropical waters on the Palk Bay side of Bay of Bengal in Ramanathapuram district, Tamil Nadu, at three different sites: Vedalai, Munaikadu, and Thonithurai, for 1 year from April 2011 to March 2012. The maximum crop yields of 416 ± 51 and 550 ± 41 FW raft^?1 were recorded at Vedalai and Munaikadu, respectively, in December 2011, whereas a maximum of 326 ± 49 FW raft^?1 was recorded at Thonithurai in April 2011. Similarly, the maximum daily growth rates (DGRs) of 4.30 ± 0.51 % and 4.92 ± 0.40 % were obtained at Vedalai and Munaikadu, respectively, in December 2011, and 3.76 ± 0.48 % was recorded at Thonithurai in April 2011. Variations in environmental parameters like seawater temperature, salinity, nitrate, and phosphate were found to influence the growth of the seaweed. The maximum carrageenan content as a percentage of dry weight was 29.30 ± 0.61 % (Vedalai), 31.00 ± 0.71 % (Munaikadu), and 29.10 ± 1.12 % (Thonithurai) in March 2012. The data on moisture content (%), clean anhydrous weed/salt ratio, and carrageenan yield obtained in this study fulfilled those of international standards. Further, the results indicated that large-scale cultivation by either NGOs or self-help groups (SHGs) may be taken up along the coastal waters for the benefit of the poverty-stricken coastal people as their livelihood, as the growth rate obtained in this study compared well with that recommended for commercial cultivation elsewhere.     

Introduction of non-native species of Kappaphycus (Rhodophyta, Gigartinales) in subtropical waters: Comparative analysis of growth rates of Kappaphycus alvarezii and Kappaphycus striatum in vitro and in the sea in south-eastern Brazil

We compared the growth rates of Kappaphycus alvarezii (Doty) Doty ex P. Silva and Kappaphycus striatum (Schmitz) Doty, both, in vitro under different conditions of light and temperature, and in the sea. Temperature was the most important factor controlling the growth of both species, in vitro and in the field. In the sea there was a clear seasonal pattern in growth rate, attributed to temperature and salinity variation. The lower growth rates were registered in winter and spring, and the highest in summer and autumn months for both species. Based on growth rate in the field, and the production of viable tetraspores during the summer in Kappaphycus striatum, we conclude that is more profitable, and ecologically safer, to only continue with the introduction program of Kappaphycus alvarezii.     

Parasitic dinoflagellates and ellobiopsids (Ellobiopsidae) of the coastal waters of the sea of Japan

Five species of parasitic dinoflagellates of the genera Protoodinium, Paulsenella, Duboscquella, Syndinium, and Dissodinium and two species of ellobiopsids of the genus Ellobiopsis are described. Figures and distribution data are provided. All species, except for Dissodinium pseudolunula Swift ex Elbrachter et Drebes, are first records for the Russia Far-Eastern seas.     

Growth of various ecotypes ofLemna paucicostata in Japan under various temperature conditions,and their wintering forms

Lemna paucicostata in Japan is classified into 4 types, N-1, N-2, K and S types. S type strains which are distributed in southern Japan grew more rapidly than other strains at the temperatures of both 27 and 17C. With the extension of the culture period at 17 C to which the plants had been transferred from 25C, the growth rate increased greatly in S type strains, while it decreased in N-1 type strains which are distributed widely from Hokkaido to Kyushu, and particularly in those distributed in the northern area. S type strains survived winter mostly in the form of normal fronds under natural conditions at Kyoto (minimum temperature is lower than OC), while most of the fronds of N-1 type strains died after producing seed before winter. Thus S type strains are considered to have adapted to the climate of southern Japan so as to survive winter in the form of fronds, while N-1 type strains overwinter in the form of seed. N-2 type strains which are distributed in the northern part of the area adjacent to the Japan Sea produced turion-like fronds during winter under natural conditions, while none of the other strains did. The K type strain which was found only at the campus of Kyoto University overwintered only in the form of fronds which produce no seed.     

Growth characteristics of the heterotrophic bacterial population of water and bottom sediments in tanks under different trophic conditions

Treatment of single and double doses of fertilizers had a definite response on the bacterial population of water and bottom sediments in tanks after 7 days (F_2,21 3.61; P < 0.05), but did not cause any significant change after day 32 (F_2,18 1.67; P > 0.05). Although the generation time of water bacteria did not vary much in these treatments of trophic gradients (P > 0.05), there was always a distinct variation (P < 0.01) in the generation time of the bacterial population of bottom sediments. The population size of water bacteria was shown to maintain an inverse relationship with the diurnal variation of temperature (P < 0.05) and dissolved oxygen (P < 0.05) of the water. The changes in the size of the bacterial population of water were positively correlated with the variations of hardness (P < 0.05), chloride (P < 0.05) and ammonia nitrogen (P < 0.05) of water. Although the relationships between the heterotrophic bacterial population and the inorganic phosphate of water was not convincing (P > 0.05), an inverse relationship was established between the bacterial community of bottom sediments and the phosphate content of the water (P < 0.01).     

Decomposition and nitrogen release from the foliage litter of fir (Abies sachalinensis) and oak (Quercus crispula) under different forest canopies in Hokkaido, Japan

When two tree species co-occur, decomposition and nitrogen (N) release from the foliage litter depend on two factors: the forest floor conditions under each canopy type and the species composition of the litter. We conducted an experiment using fir and oak to answer several questions regarding decomposition beneath canopies of the two species and the effects of litter species composition on decomposition. We compared the rates of decomposition and N release from three different litters (fir needle, oak leaf, and a mixture of the two) in 1-mm-mesh litterbags on the forest floor under three different canopies (a 40-year-old fir plantation, large oak trees, and mixed fir and oak trees) in Hokkaido, Japan, over a 2-year period. Beneath each of these canopy types, the litter decomposition rate and percentage of N remaining in the litterbags containing a mixture of fir and oak litter were not significantly different from the expected values calculated for litterbags containing litter from a single tree species. Oak leaf litter decomposed significantly faster than fir needle litter beneath each canopy type. The litter decomposition rate was significantly higher beneath the fir canopy than under the oak canopy, and was intermediate under the mixed canopy of fir and oak. No net N release, that is, a decrease in the total N compared to the original amount, was detected from fir litter under each canopy type or from oak leaf litter beneath the oak canopy. N increased over the original amount in the fir litter beneath the oak canopy and the mixed canopy of fir and oak, but N was released from the oak litter under the fir canopy and the mixed canopy of fir and oak. These results suggest that oak leaf litter blown onto fir forest floor enhances nutrient cycling, and this might be a positive effect of a mixed stand of conifer and broad-leaved trees.     

Development of the embryo nest ofVespa affinis (Hymenoptera: Vespidae) in Southern Japan

Summary The aerial nests ofVespa affinis on Ishigaki Island, Japan, were found mainly within 1 m of the ground, attached to a twig roughly 4 mm in diameter. These are probably adaptations to survive adverse weather conditions. The favoured nesting habitat was in open farmland areas rather than in the natural forest. The entire envelope and more than half of the comb of the embryo nest is constructed during the first 10–12 days. The first workers emerge around 34 days after nest initiation. The duration of larval development was found to be linked to the number of larvae present in the nest, with the earlier progeny developing faster. The changes in the ovarian index of the mother queen during the early stages of nesting are described.     

Growth and survival performance of the gametophyte of Gigartina skottsbergii (Rhodophyta, Gigartinales) under defined nutrient conditions in laboratory culture

Gigartina skottsbergii is a commercially important carrageenan producer that has been suffering severe extraction pressure in Chile’s Magellan Region and Cape Horn Archipelago since 1998. In order to create baseline information for its cultivation and repopulation, we studied the effects of agricultural fertilizers on growth of G. skottsbergii early developmental stages. The culture media utilized were: a) seawater + Bayfoland, b) seawater + Superphosphate, c) seawater + Urea, d) seawater + Provasoli and e) seawater as a control. The culture conditions were: a) 12L:12D photoperiod; b) temperature 8 ± 1°C and c) irradiance at 45 μmol photons m⁻² s⁻¹. After 60 days, higher relative growth rates between treatments were observed; the treatments that included Bayfoland and Provasoli showed greater growth (382 ± 55 and 378 ± 50 μm, respectively,) compared to Superphosphate (88 ± 16 μm), control (78 ± 10 μm) and Urea (70 ± 11 μm) treatments, after 81 days. The Urea treatment and the control had inhibitory effects on G. skottsbergii germlings growth and survival, as evidenced by progressive loss of pigmentation and death after 60 days. These results showed that Bayfoland was an excellent alternative to develop cultures.     

Growth and photosynthesis in seedlings of Hizikia fusiformis (Harvey) Okamura (Sargassaceae, Phaeophyta) cultured at two different temperatures

This study examined temperature acclimation, growth, and photosynthetic characteristics of the zygote-derived seedlings of Hizikia fusiformis (Harvey) Okamura (Sargassaceae). The seedlings were cultured at 15°C or 25°C for 4 weeks. The average relative growth rate was significantly higher in seedlings acclimated at 25°C. The photosynthetic rate measured at 15°C was much higher in seedlings grown at 15°C than those grown at 25°C, indicating photosynthetic acclimation to a lower temperature. At 35°C, the photosynthetic rate of 15°C-grown seedlings was drastically decreased, whereas that of 25°C-grown seedlings was significantly increased. The maximum relative electron transport rate (rETR_max) measured at the respective growth temperature was significantly higher in seedlings grown at 25°C than at 15°C. At a measuring temperature of 35°C, the rETR_max in both 15°C- and 25°C-grown seedlings were considerably reduced with regard to those measured at 15°C or 25°C. Our results suggested that, compared with the seedlings grown at 25°C, those acclimated at a lower temperature could be disadvantaged under adverse conditions such as increased temperatures.     

Growth rate of the carrageenophyte Kappaphycus alvarezii (Rhodophyta, Gigartinales) introduced in subtropical waters of São Paulo State, Brazil

Twenty branches of Kappaphycus alvarezii (Doty) Doty ex P. Silva produced in unialgal culture and weighing about 3 g each were transferred into the sea monthly from October 1995 to October 1996 at Ubatuba Bay, São Paulo State, Brazil (23°26.9′S, 45°0.3′W), an area with mean monthly seawater temperature from 20.3 to 28.5°C (extremes: 17.0–31.0°C). All plants were harvested after the second month and a new series of growth experiments was performed using cuttings weighing from 100 to 150 g. The cultivation experiments were carried out using a floating system. Daily growth rates were calculated based on monthly weight measurements of each individual. Small branches produced in vitro showed high survival rates when introduced into the sea during all seasons. Growth rates observed for the transplanted branches during the first and second months in the sea were higher (6.5–10.7% day‐¹) than for subsequent cuttings produced in the sea (4.5–8.2% day‐¹). The latter values are in the range reported for other regions. Seasonal variation of growth rates was clearly related to seawater temperature. These results show that the commercial cultivation of K. alvarezii is technically feasible at Ubatuba Bay using a floating raft culture method.     

Growth of larval and juvenile Diaphus theta (Pisces: Myctophidae) in the transitional waters of the western North Pacific

Diaphus theta is one of the most common myctophid fish species in the subarctic and transitional waters of the North Pacific. The growth of larval and juvenile D. theta was investigated using sagittal otolith increment analysis of specimens caught in transitional waters of the western North Pacific. Samples taken over a 24-h period demonstrated that otoliths exhibited daily growth cycles, allowing accurate determination of age. Calcification of the incremental zone of otoliths took place only at night, suggesting that the formation cycle of the increment of juvenile D. theta was different from that of shallow-water fishes and would be related to their diel vertical migration. The relationships between standard length (SL) and daily growth increment (D) were expressed as linear equations: SL = 2.65 + 0.141D (r ² = 0.942) for larvae of 5.1–9.6 mm SL and SL = 3.54 + 0.129D (r ² = 0.933) for juveniles of 13.7–27.6 mm SL. The growth rates were 0.14 mm d⁻¹ in larvae and 0.13 mm d⁻¹ in juveniles; this is slow compared with tropical or subtropical mycto-phid species, in which growth occurs at about twice these rates. The larval period, including the metamorphic stage, was long compared with species at lower latitudes and was estimated to be 71 days. The slow growth rate and long period of larval stage of D. theta would be the life history pattern of high-latitudinal species adapted to a low-temperature habitat. Received: March 23, 2001 / Revised: July 5, 2001 / Accepted: July 19, 2001     

Variable growth rates of the tropical estuarine fish barramundi Lates calcarifer (Bloch) under different freshwater flow conditions

Relationships between freshwater flows and growth rates of the opportunistic predatory finfish barramundi Lates calcarifer in a dry tropical estuary were examined using data from a long-term tag-recapture programme. Lagged effects were not investigated. After accounting for length at release, time at liberty and seasonal variation ( e.g. winter, spring, summer and autumn), growth rates were significantly and positively related to fresh water flowing to the estuary. Effects were present at relatively low levels of freshwater flow ( i.e. 2·15 m³ s⁻¹, the 5th percentile of the mean flow rate experienced by fish in the study during time at liberty). The analysis, although correlative, provides quantitative evidence to support the hypothesis that freshwater flows are important in driving the productivity of estuaries and can improve growth of species high in the trophic chain.     

Differences in growth,pigment composition and photosynthetic rates of two phenotypes Microcystis aeruginosa strains under high and low iron conditions

This paper provided insight into the influence of iron on the growth of Microcystis aeruginosa strains related to different phenotypes of this species. In this research it was intended to compare the growth, pigment composition, photosynthetic efficiency and extracellular polysaccharides production of unicellular and colonial strains of M. aeruginosa. A significantly growth inhibition under iron-limited condition on unicellular M. aeruginosa was noted, whereas the colonial strain could maintain a steady growth along with the culture time. This observation was reconfirmed by the content of chlorophyll a. Compared with unicellular strain; the colonial strain exhibited a higher PSII maximum light energy transformation, photosynthetic oxygen evolution and extracellular polysaccharides (EPS) production in iron-limited condition. Further, in order to gain more information about the accessibility of iron in the two phenotypic Microcystis, we found the two strains could produce hydroxamate-type siderophores, the content of siderophores produced by the colonial strain was more than those in unicellular strain under the iron-limited condition. It was interpreted as an adaptation to the dilute environment. Our results demonstrated that the colonial phenotypes possessed stronger ability to endure iron-limited condition than unicellular strain by higher pigment contents, higher photosynthetic activities, higher EPS production and higher siderophores secretion. It might elucidate that the colonial M. aeruginosa bloom can sustain in eutrophic reservoirs and lakes.     

Germination and survival of tetraspores of Kappaphycus alvarezii var. alvarezii (Solieriaceae, Rhodophyta) introduced in subtropical waters of Brazil

The carrageenophyte Kappaphycus alvarezii was introduced in 1995 and vegetatively propagated in Ubatuba, São Paulo State, Brazil, for the purpose of commercial cultivation. This species produces tetraspores mainly in the austral summer and fall. Tetraspore germination and survival were studied under different conditions of temperature, photon flux density, and photoperiod in the laboratory. Field experiments were also carried out. Although tetraspores of K. alvarezii germinated, they had low survival rates, most dying after 20 days. Recruitment of K. alvarezii tetraspores did not occur in experiments conducted in the field. The results indicated that the establishment of K. alvarezii via spore production in the natural environment of the south‐east coast of Brazil is rather remote.