Introduction of non-native species of Kappaphycus (Rhodophyta ...
pre_385.fm Page 183 Thursday, August 4, 2005 2:20 PM Blackwell Science, LtdOxford, UKPREPhycological Research1322-08292005 Japanese Society of PhycologySeptember 2005533183188Original ArticleIntroduction of Kappaphycus species in BrazilC. R. Bulboa and E. J. de Paula
Phycological Research 2005; 53: 183–188
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 Cristian R. Bulboa1* and Edison J. de Paula2† 1
Departamento de Biología Marina, Facultad de Ciencias del Mar, Universidad Católica del Norte, Casilla 117, Coquimbo, Chile, and 2Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, Caixa Postal 11.461, Código de endereçamento postal 05422-970, São Paulo, São Paulo, Brazil
SUMMARY 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. Key words: Brazil, exotic species, Kappaphycus alvarezii, Kappaphycus striatum, seaweed.
INTRODUCTION Farmed crops of the seaweeds Kappaphycus alvarezii (Doty) Doty ex P. Silva and, to a lesser extent, Kappaphycus striatum (Schmitz) Doty, are the primary sources of the commercial hydrocolloid kappa-carrageenan in the world (Ask & Azanza 2002). Brazil imports approximately 1000 tons of carrageenan per year (Furtado 1999), in addition to 1000 tons of dried Kappaphycus from the Philippines that is utilized in the production of approximately 500 tons of semirefined k-carrageenan. The local production of refined k-carrageenan is limited to approximately 10 tons year-1 obtained from natural populations of Hypnea musciformis (Wulfen) Lamouroux. Attempts to cultivate H. musciformis, the best native carrageenophytes in Brazil, were unsuccessful because
of technical problems (Oliveira 1990). As a result of these technical problems, Oliveira (1990) suggests that a species of Kappaphycus should be considered for introduction. A program of experimental introduction of K. alvarezii was initiated in 1995 in the subtropical waters of Ubatuba Bay, São Paulo State, Brazil to evaluate the feasibility of commercial cultivation and its environmental impact (Paula et al. 1998; Oliveira & Paula 2003). The program emphasized strain and site selection as key actions to achieve extensive cultivation in the sea (Paula et al. 1999). In addition, the use of proper culture technique and an understanding of environmental factors affecting growth rate were considered (Paula et al. 2002; Paula & Pereira 2003). A second species, K. striatum, was introduced later (E. J. Paula, unpubl. Data, 2000). Although K alvarezii and K. striatum were found to be consistently different in their physiology and morphology (Doty & Norris 1985), there is still some confusion between these species, in the published literature and in the farming industry. According to Doty (1986), the taxonomy of the group becomes more complicated because of the wide range of environmentally induced forms. Strains of K. alvarezii and K. striatum mainly from the Philippines have been introduced into more than 20 tropical countries for the purposes of mariculture over the last 30 years (Russell 1982, 1983; Doty 1986; Glenn & Doty 1990; Areces 1995; Paula et al. 1998, 2002; Oliveira & Paula 2003). On most occasions, however, introductions have been made without
*To whom correspondence should be addressed. Email: [email protected] Communicating editor: J. A. West. †Died in November 2003 Received 17 December 2004; accepted 16 February 2005.
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C. R. Bulboa and E. J. de Paula Fig. 1. Morphology of the green strain of Kappaphycus striatum used in the experimental cultivation, and known as ‘sacol type’. (a) Dried specimen divaricately dichotomous branched, known as ‘sacol type’, originally colleted from a farm in Northern Bohol in the Philippines. (b) Living specimens of the same strain, grown in the sea at Ubatuba, São Paulo, Brazil. Scale bar = 5 cm.
observation of minimum protocols of quarantine (Oliveira & Paula 2003). Moreover, details of the introduction procedures, the strain response to environmental factors, and the environmental impact of the introduction were hardly reported. Here we compare the growth rates of K. alvarezii and K. striatum in vitro under different conditions of light and temperature, and in the sea, as affected by seasonal variation of environmental factors.
MATERIALS AND METHODS The strains of both species were introduced from a ‘Eucheuma’ farm located in Northern Bohol, in the Philippines (see details in Paula et al. 1999, 2001). The strain of K. striatum was green and originally dichotomously branched (Fig. 1), similar to the dichotomous form illustrated by Doty (1988), and named ‘sacol type’ by Azanza-Corrales (1990).
Laboratory experiments Unialgal cultures were established from approximately 10 mm apical segments, as described by Paula et al. (2001). The cultures were maintained and propagated for 3 months prior to the experiments at 25 ± 2∞C under 100 mmol photons m-2 s-1 (40 W white cold fluorescent light), 14:10 h LD photoperiod, and aerated for alternating periods of 30 min. The culture medium was sterile seawater enriched with F/2 culture medium (Guillard & Ryther 1962) diluted to 50%. The growth of apical portions was evaluated in an experimental matrix at temperatures of 15, 18, 21, 24, 27, and 30∞C and photon flux densities of 50, 100, and 150 mmol photons m-2 s-1. Each treatment included one culture flask of each species with three apical segments of 10 mm in 150 mL of culture medium. The culture medium was renewed and the apical segments were cleaned, drained on paper towels,
and weighed weekly. The experimental culture period was 4 weeks, and the experiment was repeated four times. The growth rate (GR) was estimated from increases in wet weight and presented as a percentage of daily growth, GR: [(Wt/Wo)1/t - 1] ¥ 100, where Wo = initial wet weight and Wt = wet weight after t days (28 days). Growth rates were analyzed using a multifactorial analysis of variance (Sokal & Rohlf 1981). Homogeneity of the variances was reviewed for all results. Tukey’s a posteriori test was used when the treatments showed significant differences.
Sea experiments Cuttings of both species obtained in vitro were transferred to the sea at Ubatuba, São Paulo, Brazil (23∞26¢9≤S, 45∞0¢3≤W), as described for K. alvarezii (Paula et al. 2002; Paula & Pereira 2003). The experiments were carried out for 13 months starting in December 1998. Cultivation was made on a floating raft, on polypropylene ropes, placed horizontally at a constant depth of 30–40 cm from the surface of the water. Each rope supported 20 (100–150 g) cuttings that were kept in the sea for 30 days. The plants were drained and weighed individually. After weighing, the harvested plants were replaced with new cuttings. Growth rates were calculated from the equation presented above. Water temperature and salinity were measured 5 days per week on site at 09:00hours, using a mercury thermometer and a refractometer (American Optical Corp.). Total rainfall and sunlight hours per day were recorded at the weather station of Ubatuba (Instituto Agronômico de Campinas). Correlation coefficients between growth rate and environmental factors were calculated using simple linear models (Pearson’s r). Statistical analyses were performed using the Statistica software package (Release 5.0).
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Introduction of Kappaphycus species in Brazil
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RESULTS
21 °C
7.0 6.0
Laboratory experiments
5.0 4.0 3.0 2.0 1.0 0.0 50
100
150 24 °C
7.0 6.0 5.0 4.0 3.0 2.0
Growth Rate (% day-1)
Both K. alvarezii and K. striatum exhibited a healthy appearance and grew well for the experimental period of 28 days at 21, 24, 27 and 30∞C. At 15 and 18∞C dead tips (white and soft tissue) were observed after 14 days for K. striatum. For K. alvarezii dead tips were observed only at 15∞C Significant differences in the growth rates were observed for both species (P < 0.05, Table 1) at 21, 24, 27 and 30∞C (P < 0.01, Table 2), but no significant differences were observed under the photon flux densities tested (P > 0.05, Table 1). Figure 2 shows that growth rates increased with increasing temperatures from 21 to 30∞C for both species. K. alvarezii showed higher growth rates than K. striatum at 21, 24 and 30∞C. At 27∞C the differences between both species were not statistically significant (P < 0.05, Table 2). The highest growth rate for K. alvarezii (5.7% day-1) was observed at 30∞C and 100 mmol photons m-2 s-1, whereas for K. striatum the highest growth rate (4.8% day-1) occurred at 30∞C and 150 mmol photons m-2 s-1 (Fig. 2).
1.0 0.0 50
100
150 27 °C
7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
Sea experiments
50
Figure 1 shows the morphological variation of K. striatum, from its originally divaricately dichotomously
100
150
30 °C
7.0 6.0 5.0
Table 1. Multifactorial analysis of variance, of the effect of temperature (T) and photon flux density (P.F.D.) on the growth rates of the species (E), Kappaphycus alvarezii and Kappaphycus striatum Source
Degrees of Freedom
F
P-value
T P.F.D. E T ¥ P.F.D. T¥E P:F.D. ¥ E T ¥ P.F.D. ¥ E
5 2 1 10 5 2 10
364.7681 0.4907 119.6780 0.9980 6.6905 1.1398 0.1878
0.05 0.05 0.05 >0.05
4.0 3.0 2.0 1.0 0.0 50
100
Photon Flux Density (mmol photons
150 m–2 s–2)
Fig. 2. Mean growth rate (% day-1) of Kappaphycus alvarezii (white bar) and Kappaphycus striatum (black bar) in relation to temperature (21, 24, 27 and 30∞C) and photon flux density (50, 100, 150 mmol photons m-2 s-1), after 28 days in culture. Error bar = 1 standard error of mean.
*Significant P-values.
Table 2. Results of the Tukey test on the Kappaphycus alvarezii and Kappaphycus striatum growth rates at different temperatures (21, 24, 27 and 30∞C). Significant P-values shown in bold type
Kappaphycus alvarezii (Doty) Doty ex P. Silva Kappaphycus striatum (Schmitz) Doty P-value *Significant P-values.
21∞C
24∞C
27∞C
30∞C
3.8097 1.4894
Phycological Research 2005; 53: 183–188
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 Cristian R. Bulboa1* and Edison J. de Paula2† 1
Departamento de Biología Marina, Facultad de Ciencias del Mar, Universidad Católica del Norte, Casilla 117, Coquimbo, Chile, and 2Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, Caixa Postal 11.461, Código de endereçamento postal 05422-970, São Paulo, São Paulo, Brazil
SUMMARY 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. Key words: Brazil, exotic species, Kappaphycus alvarezii, Kappaphycus striatum, seaweed.
INTRODUCTION Farmed crops of the seaweeds Kappaphycus alvarezii (Doty) Doty ex P. Silva and, to a lesser extent, Kappaphycus striatum (Schmitz) Doty, are the primary sources of the commercial hydrocolloid kappa-carrageenan in the world (Ask & Azanza 2002). Brazil imports approximately 1000 tons of carrageenan per year (Furtado 1999), in addition to 1000 tons of dried Kappaphycus from the Philippines that is utilized in the production of approximately 500 tons of semirefined k-carrageenan. The local production of refined k-carrageenan is limited to approximately 10 tons year-1 obtained from natural populations of Hypnea musciformis (Wulfen) Lamouroux. Attempts to cultivate H. musciformis, the best native carrageenophytes in Brazil, were unsuccessful because
of technical problems (Oliveira 1990). As a result of these technical problems, Oliveira (1990) suggests that a species of Kappaphycus should be considered for introduction. A program of experimental introduction of K. alvarezii was initiated in 1995 in the subtropical waters of Ubatuba Bay, São Paulo State, Brazil to evaluate the feasibility of commercial cultivation and its environmental impact (Paula et al. 1998; Oliveira & Paula 2003). The program emphasized strain and site selection as key actions to achieve extensive cultivation in the sea (Paula et al. 1999). In addition, the use of proper culture technique and an understanding of environmental factors affecting growth rate were considered (Paula et al. 2002; Paula & Pereira 2003). A second species, K. striatum, was introduced later (E. J. Paula, unpubl. Data, 2000). Although K alvarezii and K. striatum were found to be consistently different in their physiology and morphology (Doty & Norris 1985), there is still some confusion between these species, in the published literature and in the farming industry. According to Doty (1986), the taxonomy of the group becomes more complicated because of the wide range of environmentally induced forms. Strains of K. alvarezii and K. striatum mainly from the Philippines have been introduced into more than 20 tropical countries for the purposes of mariculture over the last 30 years (Russell 1982, 1983; Doty 1986; Glenn & Doty 1990; Areces 1995; Paula et al. 1998, 2002; Oliveira & Paula 2003). On most occasions, however, introductions have been made without
*To whom correspondence should be addressed. Email: [email protected] Communicating editor: J. A. West. †Died in November 2003 Received 17 December 2004; accepted 16 February 2005.
pre_385.fm Page 184 Thursday, August 4, 2005 2:20 PM
184
C. R. Bulboa and E. J. de Paula Fig. 1. Morphology of the green strain of Kappaphycus striatum used in the experimental cultivation, and known as ‘sacol type’. (a) Dried specimen divaricately dichotomous branched, known as ‘sacol type’, originally colleted from a farm in Northern Bohol in the Philippines. (b) Living specimens of the same strain, grown in the sea at Ubatuba, São Paulo, Brazil. Scale bar = 5 cm.
observation of minimum protocols of quarantine (Oliveira & Paula 2003). Moreover, details of the introduction procedures, the strain response to environmental factors, and the environmental impact of the introduction were hardly reported. Here we compare the growth rates of K. alvarezii and K. striatum in vitro under different conditions of light and temperature, and in the sea, as affected by seasonal variation of environmental factors.
MATERIALS AND METHODS The strains of both species were introduced from a ‘Eucheuma’ farm located in Northern Bohol, in the Philippines (see details in Paula et al. 1999, 2001). The strain of K. striatum was green and originally dichotomously branched (Fig. 1), similar to the dichotomous form illustrated by Doty (1988), and named ‘sacol type’ by Azanza-Corrales (1990).
Laboratory experiments Unialgal cultures were established from approximately 10 mm apical segments, as described by Paula et al. (2001). The cultures were maintained and propagated for 3 months prior to the experiments at 25 ± 2∞C under 100 mmol photons m-2 s-1 (40 W white cold fluorescent light), 14:10 h LD photoperiod, and aerated for alternating periods of 30 min. The culture medium was sterile seawater enriched with F/2 culture medium (Guillard & Ryther 1962) diluted to 50%. The growth of apical portions was evaluated in an experimental matrix at temperatures of 15, 18, 21, 24, 27, and 30∞C and photon flux densities of 50, 100, and 150 mmol photons m-2 s-1. Each treatment included one culture flask of each species with three apical segments of 10 mm in 150 mL of culture medium. The culture medium was renewed and the apical segments were cleaned, drained on paper towels,
and weighed weekly. The experimental culture period was 4 weeks, and the experiment was repeated four times. The growth rate (GR) was estimated from increases in wet weight and presented as a percentage of daily growth, GR: [(Wt/Wo)1/t - 1] ¥ 100, where Wo = initial wet weight and Wt = wet weight after t days (28 days). Growth rates were analyzed using a multifactorial analysis of variance (Sokal & Rohlf 1981). Homogeneity of the variances was reviewed for all results. Tukey’s a posteriori test was used when the treatments showed significant differences.
Sea experiments Cuttings of both species obtained in vitro were transferred to the sea at Ubatuba, São Paulo, Brazil (23∞26¢9≤S, 45∞0¢3≤W), as described for K. alvarezii (Paula et al. 2002; Paula & Pereira 2003). The experiments were carried out for 13 months starting in December 1998. Cultivation was made on a floating raft, on polypropylene ropes, placed horizontally at a constant depth of 30–40 cm from the surface of the water. Each rope supported 20 (100–150 g) cuttings that were kept in the sea for 30 days. The plants were drained and weighed individually. After weighing, the harvested plants were replaced with new cuttings. Growth rates were calculated from the equation presented above. Water temperature and salinity were measured 5 days per week on site at 09:00hours, using a mercury thermometer and a refractometer (American Optical Corp.). Total rainfall and sunlight hours per day were recorded at the weather station of Ubatuba (Instituto Agronômico de Campinas). Correlation coefficients between growth rate and environmental factors were calculated using simple linear models (Pearson’s r). Statistical analyses were performed using the Statistica software package (Release 5.0).
pre_385.fm Page 185 Thursday, August 4, 2005 2:20 PM
Introduction of Kappaphycus species in Brazil
185
RESULTS
21 °C
7.0 6.0
Laboratory experiments
5.0 4.0 3.0 2.0 1.0 0.0 50
100
150 24 °C
7.0 6.0 5.0 4.0 3.0 2.0
Growth Rate (% day-1)
Both K. alvarezii and K. striatum exhibited a healthy appearance and grew well for the experimental period of 28 days at 21, 24, 27 and 30∞C. At 15 and 18∞C dead tips (white and soft tissue) were observed after 14 days for K. striatum. For K. alvarezii dead tips were observed only at 15∞C Significant differences in the growth rates were observed for both species (P < 0.05, Table 1) at 21, 24, 27 and 30∞C (P < 0.01, Table 2), but no significant differences were observed under the photon flux densities tested (P > 0.05, Table 1). Figure 2 shows that growth rates increased with increasing temperatures from 21 to 30∞C for both species. K. alvarezii showed higher growth rates than K. striatum at 21, 24 and 30∞C. At 27∞C the differences between both species were not statistically significant (P < 0.05, Table 2). The highest growth rate for K. alvarezii (5.7% day-1) was observed at 30∞C and 100 mmol photons m-2 s-1, whereas for K. striatum the highest growth rate (4.8% day-1) occurred at 30∞C and 150 mmol photons m-2 s-1 (Fig. 2).
1.0 0.0 50
100
150 27 °C
7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
Sea experiments
50
Figure 1 shows the morphological variation of K. striatum, from its originally divaricately dichotomously
100
150
30 °C
7.0 6.0 5.0
Table 1. Multifactorial analysis of variance, of the effect of temperature (T) and photon flux density (P.F.D.) on the growth rates of the species (E), Kappaphycus alvarezii and Kappaphycus striatum Source
Degrees of Freedom
F
P-value
T P.F.D. E T ¥ P.F.D. T¥E P:F.D. ¥ E T ¥ P.F.D. ¥ E
5 2 1 10 5 2 10
364.7681 0.4907 119.6780 0.9980 6.6905 1.1398 0.1878
0.05 0.05 0.05 >0.05
4.0 3.0 2.0 1.0 0.0 50
100
Photon Flux Density (mmol photons
150 m–2 s–2)
Fig. 2. Mean growth rate (% day-1) of Kappaphycus alvarezii (white bar) and Kappaphycus striatum (black bar) in relation to temperature (21, 24, 27 and 30∞C) and photon flux density (50, 100, 150 mmol photons m-2 s-1), after 28 days in culture. Error bar = 1 standard error of mean.
*Significant P-values.
Table 2. Results of the Tukey test on the Kappaphycus alvarezii and Kappaphycus striatum growth rates at different temperatures (21, 24, 27 and 30∞C). Significant P-values shown in bold type
Kappaphycus alvarezii (Doty) Doty ex P. Silva Kappaphycus striatum (Schmitz) Doty P-value *Significant P-values.
21∞C
24∞C
27∞C
30∞C
3.8097 1.4894
