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.     

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.     

Effects of low temperature stress on the antioxidant system and photosynthetic apparatus of Kappaphycus alvarezii (Rhodophyta,Solieriaceae)

To understand the effects of low temperature stress on Kappaphycus alvarezii and the responses of antioxidant systems and photosystem II (PSII), behaviour in K. alvarezii thalli exposed to low temperatures (20°C, 17°C and 14°C) for 2 hours was evaluated. Compared with the control at 26°C, activities of some antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX) and the level of antioxidant substance (reduced glutathione) increased in K. alvarezii thalli when exposed to lowered temperatures (20°C, 17°C). Hydroxyl free radical (·OH) scavenging activity of K. alvarezii thalli also increased at 20°C and 17°C compared with the control. This indicated that the resistance to low temperature stress in the antioxidant system of K. alvarezii increased at lowered temperatures of 20°C and 17°C. However, at the lowest temperature (14°C), no significant increases of this algal antioxidant were observed. Under low temperature stress, the maximum quantum yield of PSII photochemistry (F_V/F_M) and PSII actual photochemical efficiency (Φ_PSII) decreased in K. alvarezii thalli, suggesting that the photosynthetic capacity declined. Components of the photosynthetic apparatus (such as the oxygen-evolving complex, light absorption antennas, reaction centres, electron acceptor sides and electron donor sides of PSII) were damaged by low temperature stress to varying degrees. In addition, it was found that low temperature stress led to decreases of both D1 protein and Rubisco LSU (Rubisco large subunit) protein levels. This work is a significant contribution towards understanding the basic mechanism involved in the resistance and the adaptation of K. alvarezii to low temperature stress conditions.     

Evaluation of growth rate and semi‐refined carrageenan properties of tissue‐cultured Kappaphycus alvarezii (Rhodophyta,Gigartinales)

This study aimed to evaluate and compare the quality of κ‐carrageenan obtained from tissue‐cultured and field‐cultured Kappaphycus alvarezii. Carrageenan properties including yield, viscosity, gel strength and sulfate content were studied. After 60 days of cultivation, tissue‐cultured K. alvarezii showed a higher growth rate (6.3 ± 0.01% day^?1) than field‐cultured seedlings (3.4 ± 0.3% day^?1). The obtained carrageenan yield from tissue‐cultured (67.3 ± 16.4%) was higher than field‐cultured K. alvarezii (51.5 ± 21.0%). Gel viscosity of carrageenans from tissue‐cultured K. alvarezii (1280.0 ± 25.0 cP) was found significantly higher than field‐cultured samples (87.8 ± 20.9 cP). The 1.5% gel solution of tissue‐cultured and field‐cultured K. alvarezii exhibited gel strengths of 703.5 ± 14.1 and 288.3 ± 19.3 g cm^?2, respectively. The average sulfate content of carrageenans was found to be significantly different between tissue‐cultured and field‐cultured K. alvarezii with 34.2 ± 10.9 and 7.5 ± 6.7%, respectively. Tissue culture is recommended to produce high quality seedlings by providing optimized culture conditions to the seaweed. This approach can serve as an alternative way to solve the seedling shortage problems currently faced by the seaweed industry.     

Strain selection in Kappaphycus alvarezii var. alvarezii (Solieriaceae, Rhodophyta) using tetraspore progeny

A brown strain of Kappaphycus alvarezii from the Philippines produced tetraspores in the summer and autumn (December 1995 to May 1996) in cultivation experiments in the sea at Ubatuba, São Paulo State, Brazil. In vitro tetraspore release and germination experiments showed a mass mortality two to four days after release. Only 20 plants derived from tetraspores were grown successfully for over a year in the laboratory. Large differences in morphology, colour, size and growth rates were observed amongst these plants. The individual plants differed from one another in one or more characteristic. Differences appeared in the early developmental stages and persisted through time. After ten months, the plants that grew best in laboratory culture were transferred into the sea, but the others remained very small (3 to 5 mm), even after two years. In the sea, the plants also showed individual differences in their ability to survive and grow. These results emphasise the potential of the tetraspore progeny for strain selection in K. alvarezii. The results also suggest that the tetrasporophyte used in these studies is of hybrid origin.     

Cage culture ofKappaphycus alvarezii var.tambalang (Gigartinales,Rhodophyceae)

Kappaphycus alvarezii var.tambalang was cultured in a 3 × 3 m bamboo raft installed inside a 4 × 4 m floating net cage ofLates calcarifer (sea bass) broodstock at SEAFDEC Sub-station Igang, Guimaras, Philippines, from December 1989 to May 1990. Growth and production ofK. alvarezii var.tambalang was influenced by the culture months. The highest growth rate and production were recorded in January and May, respectively, while the lowest growth rate and production were observed in March. Average growth rate (wet weight) ranged from 3.72 to 7.17% day^-1, while production ranged from 575.5 to 2377 g m^-1 line^-1. A total production of approximately 123 t (fresh) or 37 t (dried) ha^-1 in the 5-month harvest season was produced from this culture system. Cultivation ofK. alvarezii var.tambalang in cages is possible, which indicates that seaweeds can be grown with carnivorous finfish, a practice which is still untapped.     

Use of plant growth regulators in micropropagation of Kappaphycus alvarezii (Doty) in airlift bioreactors

The effects of plant growth regulators on callus induction rate and regeneration of K. alvarezii explants was evaluated. K. alvarezii calluses were induced in vitro with kinetin (K), 6-benzylaminopurine (B), 1-naphtalene acetic acid (N) and spermine (S). After 30 days, K. alvarezii explants produced filamentous calluses and isolated crystalline filaments growing from the medullar region and from cortical cells at the cut edge. The plant growth regulators 1-naphtalene acetic acid (1 mg L⁻¹) and 6-benzylaminopurine (1 mg L⁻¹) and the 1-naphtalene acetic acid + kinetin + spermine (1, 1, 0.018 mg L⁻¹ respectively) combination produced 85 to 129% more calluses, with significant differences versus the control (p<0.05). Spermine at 0.018 mg L⁻¹ produced calluses in the apical, intercalary and basal regions of explants. Spermine also reduced callus induction time to 7 days, which is faster than previously reported induction times with other plant growth regulators. An airlift bioreactor was designed and characterized to micropropagate K. alvarezii calluses. The bioreactor had mixing times ranging from 4.6–10.3 s at T ₉₀ and T ₉₅, which is shorter than those for the Fernbach (5.2–13.4 s) and balloon flasks (6.3–17.3 s). Mixing time standard deviations were smaller for the bioreactor (1.1–4.6) than for the Fernbach (9.3–13.6) and balloon flasks (5.5–15.8), suggesting an adequate flow regime within the bioreactor. The results are useful for improving callus induction in K. alvarezii and propagating microplantlets in an airlift bioreactor, and provide baseline data for macroalgal bioreactor culture.     

Cultivation and carrageenan yield and quality ofKappaphycus alvarezii in the waters of Vietnam

The carrageenan-producing red algaKappaphycus alvarezii (Doty) Doty was brought to Vietnam from Japan in 1993. Branch fragments of this species were cultivated in a pond, lagoon, inlet and offshore in Vietnam for the first time. The best daily growth rate (DGR) of plants grown in the lagoon area attained 9–11 % day^–1 in May to June (cold season). The water temperature and salinity in this area ranged from 27.2–32.4 °C and 31.4–33.7 °C, respectively. DGR of plants grown in the inlet ranged from 7 to 9% day^–1 in June. Grazing by fish has been observed to occur in this area. The DGR of plants grown in the pond ranged from 5–6% in January–July, but decreased to less than 4% day^–1 in August (hot season). K. alvarezii in Vietnam showed a carrageenan yield of 18.8–24.6% and gel strength of 1566–1712 g cm^–2. These values are similar ones obtained fromK. alvarezii cultivated in the Philippines and Indonesia.     

Temperature and salinity responses of drifting specimens of Kappaphycus alvarezii (Gigartinales,Rhodophyta) farmed on the Brazilian tropical coast

Bioinvasion events causing serious environmental damage have been a concern with the mariculture of Kappaphycus alvarezii (Doty) Doty ex P.C. Silva, suggesting the importance of studying the biological aspects of drifting specimens of K. alvarezii for monitoring programs. The present study aims to evaluate the tolerance and growth of drifting color variants of K. alvarezii under different temperatures and salinities to determine their physiological capacity for growing outside cultivation rafts. Drifting color variants were collected in Paraíba State, Brazil, in November 2011(dry month) and August 2012 (rainy month), and cultivated in the laboratory under different temperatures (20, 24, 28, and 32 °C) and salinities (15, 25, 35, 45, and 55 psu). Growth rates as well as pigment and protein contents were determined. Results showed that drifting specimens collected in the dry month showed higher tolerance to variation in temperature (20 to 28 °C) and salinity (25 to 35 psu) than drifting specimens collected in the rainy month. Higher growth rates occurred in samples cultured at 20 and 24 °C (2.8–3 % day^?1) and 25 to 35 psu (3.4–3.5 % day^?1), suggesting temperature and salinity optima. Higher phycobiliprotein levels were observed in the red and brown variants under hypersaline conditions (45 and 55 psu). Higher chlorophyll a contents were associated with samples cultivated at 20–24 °C and 24–35 psu. Based on the results of the present study, drifting specimens collected in dry month are more tolerant to temperature and salinity variations, suggesting that the drifting K. alvarezii should be monitored especially during this period to prevent its establishment outside the cultivation rafts and dispersion along the northeastern coast of Brazil.     

Relationships among Eucheuma denticulatum,Eucheuma isiforme and Kappaphycus alvarezii (Gigartinales,Rhodophyta) based on nuclear ssu-rRNA gene sequences

The nuclear genes encoding small-subunit ribosomal RNAs (ssu-rDNAs) of the carrageenophytes Eucheuma denticulatum, E. isiforme and Kappaphycus alvarezii were amplified by the polymerase chain reaction, cloned and sequenced. The sequences range from 1767 (K. alvarezii) to 1781 (E. isiforme) nucleotides in length, and have guanine+cytosine contents between 51.2% (E. isiforme) and 51.5% (E. denticulatum). Pairwise sequence identities among these sequences ranged from 97.6% to 98.5%, levels comparable to some intergeneric identities within Gracilariales. In phylogenetic analyses, the two Eucheuma ssu-rDNAs group stably together vis-a-vis the ssu-rDNA of K. alvarezii, and these three ssu-rDNAs form a monophyletic group within a larger grouping of other carrageenophytes. The results demonstrate quantitatively that analysis of nuclear-encoded ssu-rDNA sequences is likely to be useful in resolving taxonomic, phylogenetic and biogeographic questions among tribe Eucheumatoideae Doty.     

Impacts of glyphosate on photosynthetic behaviors in Kappaphycus alvarezii and Neosiphonia savatieri detected by JIP-test

Neosiphonia savatieri, a filamentous red alga, had spread and caused a massive death of its host Kappaphycus alvarezii since March 2009 in China. With an aim to found a specific method to eliminate the N. savatieri efficiently from carrageenan producing K. alvarezii, the effects of glyphosate on the photosynthetic behaviors of K. alvarezii and N. savatieri were comparatively studied by using fast chlorophyll a (Chl a) fluorescence kinetics. A dose- and time-dependent changes of fast Chl a fluorescence kinetics were obtained in N. savatieri treated by glyphosate, meanwhile no significant change was detected in the K. alvarezii under the same treatment conditions. Moreover, the maximum PSII photochemical efficiency for dark-adapted tissues (F _V/F _m) of N. savatieri decreased significantly when the N. savatieri was treated with glyphosate. Above results were further supported by transitory offshore glyphosate soak experiment. The brownish-red N. savatieri turned to be olivine then drew off within 5?days after soaking in >1?g?L^?1 of glyphosate for more than 1?min, meanwhile, no visible harmful effects were detected on K. alvarezii. Based on above results, glyphosate is suggested to be an effective chemical to eliminate N. savatieri from K. alvarezii.     

Growth rate and carrageenan analyses in four strains of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Rhodophyta,Gigartinales) farmed in the subtropical waters of São Paulo State,Brazil

Growth rate, semi-refined and refined carrageenan yields, 3,6-anhydrogalactose and sulphate contents, and gel strength were investigated in four strains of Kappaphycus alvarezii (brown, green and red tetrasporophytic strains, and one strain derived from tetraspores progeny, called G11) farmed in subtropical waters of São Paulo State, Brazil. All studied strains showed higher growth rates from February to May, decreasing from July to December. The G11 strain exhibited lower growth rates, but had semi-refined and refined carrageenan yields significantly higher than the others and similar to a commercial sample. A negative correlation between growth rate and semi-refined carrageenan yield was verified for all strains. The brown strain had the lowest content of 3,6-anhydrogalactose, while G11 and green strains showed higher values. No clear pattern of variation and no relationship were observed for sulphate and gel strength. However, all strains presented gel strength values near the one obtained from commercial sample. These results show that the carrageenan extracted from brown, green, red, and G11 strains of K. alvarezii cultured in subtropical waters of São Paulo State, Brazil with commercial potentials.     

Laboratory and field growth studies of commercial strains ofEucheuma denticulatum andKappaphycus alvarezii in the Philippines

Daily growth rates of 0.1 to 8.4% d^-1 for the brown form and 0.2 to 6.3% d^-1 of the green form were measured for 3 to 5-cm long branches of the tropical red seaweedKappaphycus alvarezii cultured in the laboratory. Highest growth rates were found using inexpensive enrichments such as soil water and coconut water supplemented with 0.7 mM N and 13 µM P and with a liquid fertilizer, Algafer, produced from seaweeds in the Philippines. Laboratory grown branches of bothK. alvarezii andEucheuma denticulatum transplanted to rafts in the field showed daily growth rates of 4.4 to 8.9% d^-1, as high or higher than other reported growth rates. The studies, carried out in the Philippines, demonstrate the viability and high yield of laboratory cultivars and methods to keep laboratory culture costs low.     

Ploidy determination of three Kappaphycus alvarezii strains (Rhodophyta, Gigartinales) by confocal fluorescence microscopy

Kappaphycus alvarezii is a red alga that is commercially important as a source of carrageenan. Since K. alvarezii presents large phenotypic plasticity and rarely develops reproductive structures in culture, identification of gametophytic and tetrasporophytic phases in cultivation systems are difficult. The aim of this study was to determine the ploidy of three K. alvarezii strains previously identified as brown “tetrasporophyte”, brown “gametophyte” and “Edison de Paula” (EP). Nuclei from these strains were stained with DAPI, and analyzed using confocal fluorescence microscopy and ImageJ software. The brown “tetrasporophyte” had the highest nuclear fluorescence intensity, consistent with a diploid tetrasporophyte (2N). The brown “gametophyte” and “EP” strains had nuclear fluorescence intensities of 55.78% and 57.10% in relation to the tetrasporophyte, respectively, consistent with haploid gametophytes (N). The present study demonstrated that this technique can be used as a rapid and effective tool to distinguish between haploid (gametophytic) and diploid (tetrasporophytic) plants of K. alvarezii, in addition to help identify new strains developed through alterations of ploidy level.     

Ultrastructural,morphological, and molecular characterization of Colaconema infestans (Colaconematales,Rhodophyta) and its host Kappaphycus alvarezii (Gigartinales,Rhodophyta) cultivated in the Brazilian tropical region

Endophytic and epiphytic infections have caused serious problems for Kappaphycus farmers, such as reduction in biomass production and decrease in the yield and quality of carrageenan. During environmental monitoring from January 2011 to December 2012, along Pitimbu Beach, Paraíba State, northeastern Brazil, drifting thalli of Kappaphycus alvarezii (Gigartinales, Rhodophyta) were detected with red spots, apparently caused by epiphytic/endophytic infections. Therefore, drifting thalli of K. alvarezii farmed along the northeastern Brazilian coast were cultured in the laboratory and submitted to molecular and morphological analyses to identify and characterize the causative agent and its effects on the cellular structure and ultrastructure of the host alga K. alvarezii that was found to be infected by the endophyte Colaconema infestans (Colaconematales) identified through morphological and rbcL molecular evidence. Infected thalli of K. alvarezii were processed and analyzed through light, transmission electron, and scanning electron microscopy. Alterations were observed in morphology and cellular organization, including structural changes of chloroplasts and decrease in floridean starch grains, along with increased cell wall thickness. Therefore, while no outbreak has been reported, the discovery of C. infestans infection in drifting thallus of K. alvarezii suggests a potential threat to its cultivation that should be monitored.     

Osmotic dehydration of Kappaphycus alvarezii

The red alga Kappaphycus alvarezii has been reported to be a potential raw material for functional food due to its high content of soluble dietary fibre, mineral, omega-3 fatty acids as well as a substantial amount of essential amino acids. In order to benefit from these excellent nutritional properties, this project aimed to develop a high-value dehydrated snack from K. alvarezii using osmotic dehydration (OD) treatment prior to hot air-drying. A 3?×?3 factorial design with 50°, 60° and 70°Brix sucrose concentration as well as treatment temperatures of 30, 35 and 40 °C were used. In general, an increase in sucrose concentration and temperature promoted mass transfer. OD treatment using 70°Brix sucrose concentration at 40 °C caused case hardening of the seaweed that reduced the solid gain (p?<?0.05). Firmness of the seaweed increased with sucrose concentration and was not altered by temperature (p?<?0.05). The colour of the seaweed was not affected by OD treatment (p?>?0.05), but dehydrated seaweed became darker at high sugar concentration. Interaction effect between sucrose concentration and temperature was found to affect the water loss and solid gain of the OD treatment (p?<?0.05). The best sensory acceptable dehydrated seaweed was successfully identified. The final product contained high dietary fibre and very low Na/K ratio.     

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.     

In vitro cultivation of three <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Rhodophyta,Areschougiaceae) variants (green,red and brown) exposed to a commercial extract of the brown alga <Emphasis Type="Italic">Ascophyllum nodosum</Emphasis> (Fucaceae,Ochrophyta)

Diseases such as “ice-ice” and “goose bumps” are responsible for loss of almost 70% of infected Kappaphycus production. To improve the growth of K. alvarezii and reduce the impact of diseases, the effect of the commercial Ascophyllum nodosum extract was tested. Five grams of apical branches of each K. alvarezii variant (n = 6) were bathed at different extract concentrations (e.g., 5, 10, 15, 20, 25, and 30 g L⁻¹) for 1 hour. To test the effect of the extract on epiphytes apical tips were not cleaned. After 2 weeks, the epiphytes such as Cladophora sp. and Ulva sp. disappeared. Polysiphonia subtilissima survived but its appearance was weak. The highest K. alvarezii growth rates were observed at extract concentrations of 15 and 20 g L⁻¹ (p < 0.001), almost double the expected for a 45-day production cycle used at Sepetiba Bay, Brazil. These results show that the extract was efficient at improving the growth of K. alvarezii and reducing the presence of some epiphytes.